University of South FloridaScholar Commons
Graduate Theses and Dissertations Graduate School
January 2013
Partial Characterization of PF13_0027: A PutativePhosphatase of Plasmodium falciparumChristopher CampbellUniversity of South Florida, [email protected]
Follow this and additional works at: http://scholarcommons.usf.edu/etd
Part of the Biology Commons
This Dissertation is brought to you for free and open access by the Graduate School at Scholar Commons. It has been accepted for inclusion inGraduate Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please [email protected].
Scholar Commons CitationCampbell, Christopher, "Partial Characterization of PF13_0027: A Putative Phosphatase of Plasmodium falciparum" (2013).Graduate Theses and Dissertations.http://scholarcommons.usf.edu/etd/4451
Partial Characterization of PF13_0027: A Putative Phosphatase of
Plasmodium falciparum
by
Christopher Oliver Campbell
A dissertation submitted in partial fulfillment of the requirements for the degree of
Doctor of Philosophy Department of Global Health
College of Public Health University of South Florida
Major Professor: John H. Adams, Ph.D. Dennis E. Kyle, Ph.D.
Andreas Seyfang, Ph.D. Roman Manetsch, Ph.D.
Date of Approval: April 8, 2013
Keywords: Malaria, Drug, piggyBac, Phosphorylation, Signal Transduction
Copyright © 2013, Christopher Oliver Campbell
Dedication
This dissertation is dedicated to my family for their love and support.
Acknowledgments
The completion of this dissertation was made possible by the support of
my doctoral committee, the members of the Adams laboratory and our
collaborators. I would first like to thank my research mentor Dr. John Adams for
helping me develop the skills necessary for conducting research. He has been
an excellent advisor and was instrumental in the organization, and completion of
the work necessary for this dissertation. I would also like to thank the members
of my doctoral committee, Dr Kyle, Dr. Seyfang and Dr. Manetsch , for their
valuable input that helped be develop the multidisciplinary experience necessary
to bring the various parts of this project together. Their input also provided a
solid foundation that is necessary for advancing in this field. I would also like to
thank Dr. Wayne Guida and Daniel Santiago for allowing me to use their facilities
for the computational analysis required for the in silico docking portion of this
project. This project also benefited greatly from the research expertise of Dr.
Bharath Balu, Dr. Naresh Singh, Min Zhang, Siddharth Kamath, and Steven
Maher. Additionally, I greatly appreciate the administrative assistance of Judy
Sommers, Katherine Johnson and Samantha Barnes, Melissa Bayley, and the
Department of Academic and Student Affairs.
i
Table of Contents
List of Tables ........................................................................................................ iv
List of Figures ....................................................................................................... v
List of Abbreviations ........................................................................................... viii
Abstract ................................................................................................................ ix
Chapter 1: Background and Introduction ............................................................. 1
Malaria: A Global View............................................................................... 1 Forward Genetics with piggyBac Transposon-mediated
Mutagenesis ......................................................................................... 2 Global Impact of Malaria ................................................................. 3
Pathogenesis ............................................................................................. 6 Plasmodium Life Cycle .............................................................................. 8
Mosquito (Definitive Host) ............................................................... 8
Human (Intermediate Host) ............................................................. 9
Antimalarials ............................................................................................ 10
Endoperoxides .............................................................................. 10 Quinolines ..................................................................................... 13
4-Aminoquinolines .............................................................. 13
8-Aminoquinolines .............................................................. 14 Antifolates ..................................................................................... 15
Sulfonamides ................................................................................ 17 Amino Alcohols ............................................................................. 17
Kinases and Phosphatases of Plasmodium ............................................. 17 Plasmodium Kinases ..................................................................... 19
The ACG Group ................................................................. 20 The CMGC Group .............................................................. 20
The CamK Group ............................................................... 21
The CK1 Group .................................................................. 22 The TKL Group ................................................................... 22 The OPK Group .................................................................. 23
Plasmodium Phosphatases ........................................................... 23
The PPP group ................................................................... 24
The PPM Group ................................................................. 26 The PTP group ................................................................... 26
ii
The NIF group .................................................................... 28
Focus of Study ......................................................................................... 28
Chapter 2: Identification of a Putative Phosphatase in Plasmodium falciparum Regulating Progression from Pre-S Phase Blood Stage Development (Specific Aim 1) ....................................................................... 41
Rationale for Study .................................................................................. 41 Introduction .............................................................................................. 42 Materials and Methods............................................................................. 44
In vitro Parasite Culture Conditions ............................................... 44
Determination of Merozoite Number Per Schizont ........................ 44 RNA Extraction and Analysis by qRT-PCR and RT-PCR .............. 44
Plasmid Constructs and Genetic Complementation ...................... 45 Growth Assay and Cell Cycle Determination ................................ 46 Invasion Assays ............................................................................ 46
Multiple alignments and phylogenetic analysis ............................. 47 Southern Blot Hybridization ........................................................... 47
Results ..................................................................................................... 48 Identification of an Attenuated Growth Mutant in P.
falciparum ................................................................................ 48
Defining Characteristics of PF13_0027 ......................................... 48 PF13_0027 Regulates Transition from Pre-S phase to S/M
Phase ...................................................................................... 50 Phenotype Rescue of Wild-type Growth by Genetic
Complementation ..................................................................... 50
Discussion ............................................................................................... 52
Chapter 3: Identification of Novel Inhibitory Compounds and Evaluations of Plasmodium falciparum Susceptibility (Specific Aim 2) ............................. 69
Rationale for Study .................................................................................. 69
Introduction .............................................................................................. 70 Materials and Methods............................................................................. 72
Identification of Conserved Domains and Evolutionary Lineage .................................................................................... 72
Evaluation of Secondary Structure and Post-translational Modifications ............................................................................ 73
Molecular Modeling and Structure Validation ................................ 73
Identification of the Binding Pocket ............................................... 74 Selection of the Compound Dataset and High-throughput in
silico Docking ........................................................................... 74 In vitro parasite culture conditions ................................................. 75 In vitro Drug Susceptibility Assay Using SYBR Green I ................ 75
Growth Assay and Cell Cycle Determination ................................ 76
iii
Results ..................................................................................................... 76
Evaluation of the Physical Properties of PF13_0027 .................... 76 Molecular Structure of PF13_0027 ................................................ 77
Active Site Prediction .................................................................... 78 Ligand Selection and Drug Susceptibility Assay ........................... 78
Discussion ............................................................................................... 81
Chapter 4: Conclusions and Future Directions ................................................. 111
References ....................................................................................................... 114
Appendices ....................................................................................................... 150
Appendix A: Transfection Plasmids ....................................................... 151
Appendix B: Primers .............................................................................. 151 Appendix C: Flow Cytometry Gating ...................................................... 151 Appendix D: Whole genome sequencing. .............................................. 151 Appendix E: Southern Blot of complemented parasites ......................... 151
Appendix F: PCR Validation of complemented clones ........................... 151 Appendix G: Statistical Analysis Tables ................................................. 151
Appendix H: Bioinformatics .................................................................... 151 Appendix I: Content Permissions ......................................................... 151
iv
List of Tables
Table 1.1 Classifications of phosphatases .................................................... 36 Table 1.2 Phosphatases identified in P. falciparum ...................................... 37 36 Table 1.3 Orthologs of PF13_0027 ............................................................... 40 Table 3.1 Templates identified for homology modeling ................................. 85 Table 3.2 Selected ChEMBL-NTD compounds used for in vitro
screening ...................................................................................... 90 Table 3.3 Comparison of average pre-S phase times in treated
cultures to NF54 and C9 ............................................................. 107 Table 3.4 Comparison of average cycle times in treated cultures to
NF54 and C9 ............................................................................... 108 Table 3.5 Comparison of NF54 and C9 susceptibility to the selected
compounds ................................................................................. 110 Table BI Primer list .................................................................................... 154 Table DI Transposon insertions and SNPs in the genomes of NF54,
C9 and the complemented parasite lines .................................... 156 Table GI Growth assay analysis for NF54, C9, E3 and E8 ........................ 159 Table GII Invasion assay analysis for NF54, C9, E3 and E8 ...................... 160 Table GIII Growth assay analysis for NF54, C9, D345A, C383A and
K388A/ K394A............................................................................. 161 Table GIV Invasion assay analysis for NF54, C9, D345A, C383A, and
K388A/ K394A............................................................................. 162 Table HI Bioinformatics Resources ........................................................... 163
v
List of Figures
Figure 1.1 The piggyBac transposon mutagenesis system. ........................... 30 Figure 1.2 The spatial distribution of P. falciparum malaria endemicity in
2010. ............................................................................................. 31 Figure 1.3 The spatial distribution of P. vivax malaria endemicity in
2010 .............................................................................................. 32 Figure 1.4 Life cycle of the malaria parasite. .................................................. 33 Figure 1.5 A global map of dominant malaria vector species ......................... 34 Figure 1.6 Summary of the MAPK pathway ................................................... 35 Figure 2.1 Growth phenotype of C9 mutant parasite is due to disruption
of PF13_0027................................................................................ 56 Figure 2.2 Growth of mutant C9 parasites as a percent of NF54 ................... 57 Figure 2.3 Morphologic analysis of the C9 mutant compared to NF54 ........... 58 Figure 2.4 Comparison of merozoites produced per schizont in mutant and
wild-type parasites ........................................................................ 59 Figure 2.5 Multiple alignment and phylogenetic analysis of PF13_0027 ........ 60 Figure 2.6 Transcription profile of PF13_0027 .............................................. 61 Figure 2.7 The cell cycle of C9 null MKP mutant is altered ............................ 62 Figure 2.8 Transfection plasmid used for complementation of C9 ................. 63 Figure 2.9 RT-PCR analysis of complemented parasites ............................... 64 Figure 2.10 Genetic complementation of C9 mutant rescues wild-type
growth ........................................................................................... 65
vi
Figure 2.11 Genetic complementation of C9 mutant rescues wild-type invasion ......................................................................................... 66
Figure 2.12 Growth phenotype is not rescued when conserved residues
are mutated ................................................................................... 67 Figure 2.13 Invasion phenotype is not rescued when conserved residues
are mutated ................................................................................... 68 Figure 3.1 Alignment of potential homology modeling templates ................... 86 Figure 3.2 The quality of the model was validated by Ramachandran
plot. ............................................................................................... 87 Figure 3.3 Overall quality assessment of the model evaluated using ERRAT .......................................................................................... 88 Figure 3.4 Homology model of PF13_0027 .................................................... 89 Figure 3.5 Identified binding site of PF13_0027 in the DUSP domain ............ 91 Figure 3.6 Vacuum electrostatics of PF13_0027 DUSP homology model ..... 92 Figure 3.7 Orientation of 390097 in the binding pocket with LIGPLOT .......... 93 Figure 3.8 Orientation of 524725 in the binding pocket with LIGPLOT .......... 94 Figure 3.9 Orientation of 533073 in the binding pocket with LIGPLOT. ......... 95 Figure 3.10 Orientation of 533730 in the binding pocket with LIGPLOT .......... 96 Figure 3.11 Orientation of 525841 in the binding pocket with LIGPLOT .......... 97 Figure 3.12 Orientation of 579624 in the binding pocket with LIGPLOT .......... 98 Figure 3.13 Orientation of 585222 in the binding pocket with LIGPLOT .......... 99 Figure 3.14 NF54 parasites challenged with compound 390097 .................... 100 Figure 3.15 NF54 parasites challenged with compound 524725 .................... 101 Figure 3.16 NF54 parasites challenged with compound 533073 .................... 102 Figure 3.17 NF54 parasites challenged with compound 533730 .................... 103
vii
Figure 3.18 NF54 parasites challenged with compound 525841 .................... 104 Figure 3.19 NF54 parasites challenged with compound 579624 .................... 105 Figure 3.20 NF54 parasites challenged with compound 585222 .................... 106 Figure 3.21 Comparison of cycle times and pre-S phase for treated and
untreated cultures ....................................................................... 109 Figure AI The helper plasmid codes for the piggyBac transposase used in
random insertional mutagenesis ................................................. 151 Figure AII Plasmid used for the initial transfection knocking out PF13_0027 ................................................................................. 152 Figure AIII Plasmid used for complementation of C9 .................................... 153 Figure CI Gating used to sort the different developmental stages of asexual
P. falciparum cultures by flow cytometry ..................................... 155 Figure EI Southern blot analysis of the transfected clones ......................... 157 Figure FI PCRs used to validate the complemented parasite clones ......... 158
viii
List of Abbreviations
ACT Artemisinin Combination Therapy
ANOLEA Atomic Non-local Environment Assessment
API Annual Parasite Index
BLAST Basic Local Alignment Search Tool
CASTp Computed Atlas of Surface Topography of proteins
CDART Conserved Domain Architecture Retrieval Tool
CDD Conserved Domain Database
ChEMBL-NTD European Molecular Biology Laboratories database of bioactive drug-like small molecules for Neglected Tropical Disease
CQ Chloroquine
CSP Circumsporozoite Protein
DHA Dihydroartemisinin
DUSP Dual-specificity Phosphatase
EC50 Half Maximal Effective Concentraion
EIR Entomological Innoculation Rate
ELISA Enzyme-linked Immunosorbent Assay
GROMOS Groningen Molecular Simulation
HTS High-throughput Screening
LDH Lactate Dehydrogenase
MAPK Mitogen-activated Protein Kinase
MEGA Molecular Evolutionary Genetics Analysis
MKP MAPK Phosphatase
pB piggyBac
PR Parsite Rate
PTP Protein Tyrosine Phosphatase
RBC Red Blood Cell
RFU Relative Fluorescence Unit
RMS Root Mean Square
RMSD Root Mean Square Deviation
SMART Simple Modular Architect Research Tool
SR Splenic Rate
WHO World Health Organization
XP Extra Precision
ix
Abstract
Signal transduction and stage-specific gene expression are essential
components of Plasmodium falciparum development. In this study, the putative
phosphatase PF13_0027 is investigated as a critical component of
intraerythrocytic development contributing to maturation of the late trophozoite.
This putative phosphatase was identified during the course of a large-scale
insertional mutagenesis project by insertion of the piggyBac (pB) element,
containing a human dihydrofolate reductase (hDHFR) drug selection cassette
into the open reading frame (ORF) preventing expression and attenuating
parasite development. PF13_0027 codes for a protein with a rhodanese (RHD)
and dual specificity phosphatase (DUSP) in a tandem arrangement typically
identified with mitogen-activated protein kinase (MAPK) phosphatases (MKP).
Despite numerous INDELs, the tertiary structure is conserved when compared to
the solved structures of MKP homologs. The expression profile reveals
transcripts at all stages of the blood cycle with a highest relative abundance in
the late trophozoite. Restoration of the phenotype was achieved through genetic
complementation using the complete PF13_0027 open reading frame (ORF)
under the control of its endogenous promoter. A homology model of PF13_0027
was developed for structural analysis and evaluated using in silico high-
x
throughput screening (HTS) to identify antimalarial compounds with predicted
affinity to the active site and used to challenge parasites in vitro. This study
reveals that PF13_0027 is a vital component of asexual development and a
potential target for a new class of antimalarial compounds targeting
phosphorylation pathways in P. falciparum. Discovery of the functional role of
this unknown ORF provides additional insight into the importance of MAPK
signaling in P. falciparum.
1
Chapter 1: Background and Introduction
Malaria: A Global View
Malaria is a devastating disease responsible for approximately 800,000
deaths and 250 million clinical illnesses annually [10-12]. Clinical disease
resulting from malaria is caused by cyclic intraerythrocytic development of the
parasites in the blood [13-15]. This cycle of development is highly dependent on
a progressive pattern of gene expression, and it is widely believed that the
observed cyclical pattern of malaria parasites is ‘hard wired’ into the genome in
contrast with most eukaryotic organisms that can variably regulate cell cycle
development [16-20]. The protozoan parasites responsible for this disease have
a history stretching back as far as 30 million years and have been mentioned
throughout recorded history [21-25]. These parasites belong to the phylum
apicomplexa and there are five species of Plasmodium known to infect humans;
P. falciparum, P, vivax, P. malariae, P. ovale, and P. knowlesi [26, 27]. Of these
five species, P. falciparum is the most severe and thought to have originated in
Africa about 10 000 years ago [26-34]. To overcome the chronic and widespread
nature of this disease there is an urgent need for vaccines to prevent infection
and new drugs for prophylaxis and curative treatments [29, 35, 36]. Currently,
many of the antimalarial drugs used to control malaria are rapidly losing their
2
efficacy due to the adaptations of the parasite and the common chemical nature
and targets of many current drugs [20, 37-40]. In the effort to develop new
therapies, studies interpreting the genome have been vital to the understanding
of Plasmodium biology [10]. The completed sequencing of the P. falciparum
genome in 2002 has contributed a lot of vital information to this effort, but
converting that information into new therapies has been slow due to the high
quantity of encoded hypothetical genes and unknown ORFs [10].
Forward Genetics with piggyBac Transposon-mediated Mutagenesis
Due to the lack of robust molecular genetic tools for manipulating the
Plasmodium genome, translation of the genomic information to chemotherapeutic
strategies has been an arduous process [41-43]. Of all the developmental
stages in P. falciparum, the blood stages are the only stage that can be
effectively cultured in vitro [44-46]. This stage is also the only one amenable to
transfection with exogenous DNA [44, 47]. To investigate the Plasmodium
genome, our lab uses random insertional mutagenesis with the transposable
element piggyBac (pB) [9, 48]. Through this method, we have been able to
identify genes important for the development of P. falciparum through individual
gene knockouts followed by phenotypic characterization [9]. One of the main
advantages to this method is that it has proven to be quicker and more efficient
than homologous recombination, which is a lengthy process (6-12 months)
requiring individual targeting plasmids for each recombination [9, 45, 46, 49-52].
With the pB mutagenesis method, we have been able to knock out several genes
3
in the genome of P. falciparum, providing new avenues for in depth
characterization of the genome (Figure 1.1). Throughout this process, the
putative phosphatase PF13_0027 was identified as important to intraerythrocytic
development. Further investigation revealed that the structure of PF13_0027 is
conserved with MKPs and may provide insight into a more complex underlying
signal transduction pathway. This novel putative phosphatase could potentially
develop much needed avenues for future antimalarial drug design.
Global Impact of Malaria
Malaria has been a huge problem in endemic regions throughout history;
however, in the past century progress has been made successfully reducing the
incidence of malaria in some endemic regions. In recent times major advances
controlling malaria have occurred in Sub-Saharan Africa, which experiences the
greatest burden due to malaria. The burden of malaria in this region is mainly
fueled by the prevalence of the vector Anopheles gambiae, a long lived mosquito
that feeds predominantly on humans and has been effective in transmitting
malaria from person to person [53]. The most common cause of severe malaria,
and the cause of most cases in Africa are the result of P. falciparum (Figure 1.2)
infection, while P. vivax (Figure 1.3) is the most common cause of malaria
outside of Africa [54-58].
Fortunately some progress has been made in controlling the effects of
malaria in endemic regions. Reported malaria cases in 2000 to 2010 decreased
more the 50% in 43 of the 99 countries with ongoing transmission. Downward
4
trends of 25-50% were observed in 8 other countries while global incidence of
malaria was reduced by 17% since 2000 with mortality rates reduced by 26%
[59]. According to the World Malaria Report released by the World Health
Organization (WHO) in 2011, this target fell short of the international goal of 50%;
however, it is still a major achievement. Conversely, the global reach of malaria
still threatens approximately 40% of the earth’s population in tropical and
subtropical regions [33, 60, 61]. In Sub-Saharan Africa, this disease is both a
cause and consequence of poverty, having an economic impact that slows
growth by 1.3% annually, translating into a gross domestic product cost of $12
billion [38, 62, 63]. Most cases of the most severe form of malaria, P falciparum,
are confined to the continent of Africa accounting for approximately 90% of all
deaths [53, 59]. Globally 86% of all malaria cases are children under the age of
five [38, 64].
To understand the larger picture of malaria’s effect within endemic regions
it is necessary to have an effective method in place to track and record its
impact. Tracking and measuring malaria prevalence is an arduous process
which is undertaken utilizing a variety of methods. These approaches have both
advantages and disadvantages, but they provide critical information supporting
strategies aimed at controlling and eradicating malaria. Four of the methods
used for measuring malaria transmission are; entomological inoculation rate
(EIR), parasite rate (PR), annual parasite index (API), and spleen rate (SR). The
EIR is a measure of infectious bites per unit time (usually one year), and is
considered a direct reflection of the vector control and gametocytocidal drugs
5
[65, 66]. One of the drawbacks to this method is that there are not any standard
protocols, so there is variability in the methodologies, and there are few
specialists trained in the technique. It was found that in some cases false
positives from enzyme-linked immunosorbent assay (ELISA) techniques
detecting circumsporozoite protein (CSP) could lead to an overestimation of EIR
[67]. The PR looks at the proportion of the population carrying asexual stage
parasites in the blood and can assess the gametocyte rates by age group. An
advantage of this method is that it provides a direct reflection of the effectiveness
of inoculations, immunity and treatment in humans. Some of the challenges
imposed by this method are that the results can be affected by environmental
control factors and the accuracy depends on the technical efforts of the
microscopy, which can be inconsistent when comparing a variety of regions [68,
69]. The API looks at the number of parasite infections within a defined
geographical area (typically 1000 persons per year). This method is considered
a direct reflection of all the prevention and control effects on humans, but
depends on active case detection data that often can be poor. SR looks at the
proportion of children 2-9 year of age that have a palpable spleen. This is a non-
invasive way of measuring the impact of malaria on the spleen. However, there
are many causes of splenomegaly that complicate interpretation. Additionally,
rapid changes in point prevalence, and variability in examiner techniques, has
also been an issue contributing to inconsistencies in this method of reporting
[70].
6
Pathogenesis
Manifestation of malaria’s clinical symptoms can range from mild to severe
and even death, although it is curable if diagnosed and treated promptly. Most
infections of adults living in endemic areas are clinically silent due to adaptive
immunity, that is able to prevent disease, while the more clinically overt cases
occur in non-immune individuals [71]. Two major factors influencing the
progression of disease is the age and immune status of the infected individual.
In the endemic regions of Africa, the major burden of malaria is in children under
the age of five and pregnant women [12, 38]. Due to this threat, the fight against
malaria has employed a multifaceted approach to reduce infection in these at-risk
populations.
The pathogenesis of malaria is a complicated process modulated by both
parasite and host factors allowing it to successfully propagate in the various
environments of their hosts. One such mechanism is the ability to express
variant surface antigens such as P. falciparum erythrocyte membrane protein 1
(PfEMP-1) that allows the infected red blood cells (RBCs) to bind to vascular
receptors preventing splenic clearance [72, 73]. The challenge posed by
immune recognition via this surface protein is circumvented by antigenic variation
[38, 71, 74]. However, development of cross-reactive antibodies to
subpopulations of variant antigens has been found to produce semi-protective
immunity [74, 75]. Unfortunately multiple gaps still remain in the most effective
strategies leaving solutions to this challenge out of our reach [76].
7
During development in RBCs several toxic byproducts of metabolism
including hemozoin, accumulate in the infected cell. When the RBC lyses to
release invasive merozoites, toxic byproducts stimulate macrophages to release
cytokines producing acute inflammatory responses [77]. Typically, these
infections are divided into two categories; uncomplicated and complicated (or
severe) malaria. Uncomplicated cases commonly present a combination of
fever, chills, sweats, headaches, nausea, body aches and general malaise [13,
14]. These symptoms can sometimes be associated with other illnesses, such
as a common cold or influenza, however when recognized as malaria, residents
in areas with frequent malaria infection tend to seek diagnostic confirmation or
treat themselves [38, 78]. Diagnosis depends on the observation of parasites in
the blood by microscopy, while other diagnostic observations may include a
decrease in blood platelets or elevation in bilirubin and aminotransferases [77].
More serious symptoms can occur in severe malaria which is accompanied by
organ failures and abnormalities in the patient’s blood and metabolism [79, 80].
Additionally, cerebral malaria and anemia are common occurrences as well as
low blood pressure, respiratory acidosis, and hypoglycemia [38, 74]. Recovery
from infections of P. vivax and P. ovale are sometimes followed by relapses due
to hypnozoites that can lay dormant in the liver for several weeks, months, or
even years [31, 81].
8
Plasmodium Life Cycle
The life cycle of Plasmodium depends on the infection of two distinct
hosts, a mosquito and a vertebrate (Figure 1.4). Asexual development in the
vertebrate host is comprised of an initial single round of exo-erythrocytic
schizogony in infected liver hepatocytes followed by potentially unlimited rounds
of intraerythrocytic schizogony. The intraerythrocytic stage of development (48
hour cycle) is the underlying cause of malaria. A product of intraerythrocytic
development is macro- and microgametocytes, which are ingested by mosquitos
when obtaining a blood meal.
Mosquito (Definitive Host)
Transmission of the malaria parasite from person to person occurs
through the bite of the female Anopheles mosquito. There are approximately
3500 species of mosquitos, of which, 430 are Anopheles [77]. Their geographic
range covers most global regions with the exception of the Antarctic (Figure 1.5)
[82]. Of the known Anopheles, 40 to 50 of these species are capable of
transmitting malaria and the ability of a given species to transmit malaria is
dependent on the region and environment. Since their range is not limited to
endemic regions, and includes areas where malaria has been eliminated, there is
always a looming risk of re-introduction [83]. Efforts have been made to
understand the behavior of mosquitos in an attempt to develop deeper insights
into the methods of malaria transmission. Some key factors that facilitate
mosquito transmission are the choice of host, life span, and susceptibility to
9
Plasmodium. Female mosquitos have a life span that can extend up to a month
while the males will live for approximately one week. Both male and female
mosquitos feed on nectar as an energy source, however, only the female
requires blood for the production of eggs [77]. Following ingestion of
gametocytes, the process of sporogony is activated by a combination of a 5 ºC
drop in temperature and the gametocyte activating factor xanthurenic acid which
stimulates the final maturation of the gametocytes to form gametes [84]. This
process is followed by fusion of the gametes to form a motile oökinete that
travels by intracellular migration through the midgut epithelium towards the BL.
The oökinete differentiates to an oöcyst and matures for approximately 10-15
days before releasing thousands of sporozoites. The sporozoites then migrate to
the salivary glands and are injected into the vertebrate host when the mosquito
feeds.
Human (Intermediate Host)
Humans become infected when mosquitos feed, injecting sporozoites into
skin around the bite area. During the feeding process, a single infected mosquito
can inject anywhere from 1-1297 sporozoites, although infection can occur with
as few as five [85, 86]. Sporozoites can remain in the skin for several hours
before migrating out into the blood vessels. Additionally up to 20% of the
sporozoite inoculum can enter the lymphatic system [87, 88]. Upon entering the
blood vessels, some sporozoites eventually arrive at the liver where they traverse
Küpffer cells prior to invading hepatocytes [14]. Development in the liver
10
hepatocytes produces large packages of merozoites (merosomes), which are
released from the liver into the bloodstream [14, 89]. Each merozoite invades a
RBC where they produce several invasive merozoites [13, 90, 91]. Lysis of the
RBC releases the merozoites that invade more uninfected RBCs. Continuation
of this cyclical process in the blood results in the fever and chills commonly
associated with malaria. In a process branching from this cycle, some of the
merozoites develop into gametocytes which are the stage ingested by the
mosquito during blood feeding.
Antimalarials
Over the years, numerous effective antimalarial drugs have been
developed, however these efforts have been threatened by emerging resistance.
With the decreasing effectiveness of some of the more common frontline drugs
such as chloroquine, atovaquone, sulfadoxine-pyrimethamine and mefloquine,
the effort to develop new drugs has been intensified [36, 40, 92-98].
Furthermore, recent emergence of artemisinin resistance has increased the
urgency of this effort [36, 40, 99, 100]. Notably, resistance to a certain
compound is manifest throughout the entire chemotype class, so efforts cannot
simply focus on modifying current drugs but rather aim to find new
pharmacophores as platforms for new malaria therapy [100-102].
Endoperoxides
The endoperoxides are comprised of artemisinin and its derivatives.
Artemisinin is extracted from the annual wormwood Artemisia annua and has
11
been used in traditional Chinese medicine for more than 2000 years in the
treatment of febrile illnesses [40]. Since its introduction and use in several
African and Asian countries the burden of malaria has successfully been
reduced. Use of these drugs as artemisinin combination therapies (ACTs),
combining two or more antimalarials with different modes of action together, is
encouraged due to the potential emergence of drug resistance in areas of high
drug pressure [103]. Though a consensus has not been reached on the
mechanism of action, the antimalarial activity is thought to arise from the
peroxide bridge present in all the compounds [64, 104]. Endoperoxides have
been safe and active against a wide range of Plasmodium stages including
immature and developing gametocytes [105].
Derivatives that are used in combination therapies vary in their activity
profiles and have been utilized in diverse situations. Dihydroartemisinin (DHA)
has been used in combination with piperaquine, and registered for distribution
under the name Artekin™ [93]. It has been very effective and was evaluated in
clinical trials in Thailand, Vietnam, Cambodia, and China [106]. Derivatives of
DHA are artemether, artesunate and artemotil. These derivatives have better
biavailability than artemisinin and are used as a once or twice a day dosing
regimen which is effective at reducing parasite biomass by four orders of
magnitude over a 48 hour period [36]. Artemether is used for the treatment of
severe malaria and in combination with lumefantrine for the treatment of
uncomplicated P. falciparum malaria. Artemether-lumefantrine is taken as a six-
dose regimen and has been highly effective against multidrug resistant P.
12
falciparum [107-109]. Following the 1999 registration of the drug in Switzerland,
resulting from a collaboration of Novartis and the Chinese developers, it was
dually branded and marketed as Riamet® in developed non-endemic countries
and as Coartem® in malaria endemic countries [106]. Artesunate is the treatment
of choice for severe malaria and has been used in combination with
amodiaquine, mefloquine and pyronaridine [36, 106]. Artesunate-mefloquine has
been widely used in South East Asia and is effective against multidrug resistant
P. falciparum. Artesunate-amodiaquine has variable effectiveness and is
acceptable for use in areas where amodiaquine resistance is low. Artesunate-
pyronidine has been used for the treatment of malaria since the 1980s and has
been effective against malaria in Africa, but less effective in Thailand [106].
Artemisone is a promising second-generation derivative with a longer half-life,
lower curative dose, greater bioavailabily compared to the other derivatives, and
did not display neurotoxicity in preclinical testing [105, 110]. Another
endoperoxide drug, the synthetic peroxide OZ439, was designed to provide a
single dose oral cure in humans. It is fast acting against all asexual erythrocytic
stages of P. falciparum [104]. The peroxide, OZ277 was the first synthetic
ozonide to be evaluated clinically as a combination therapy with piperaquine
phosphate. It exhibits activity against all asexual development stages and is
currently in Phase III trials [104, 111].
13
Quinolines
The quinolines target heme polymerization in the erythrocytic stages of
Plasmodium development. Blocking this process promotes the accumulation of
toxic free heme that eventually leads to the death of the parasite [38, 95, 112-
114]. Two subgroups of these compounds, the 4-aminoquinolines and the 8-
aminoquinolines differ in the position of the amine group.
4-Aminoquinolines
Compounds within this group have been effective against erythrocytic and
sexual stages of Plasmodium in vivo and in vitro. These compounds have a
history of being safe and economical, but emerging drug resistance has
increased the need for alternative analogues [95, 98]. Chloroquine (CQ) is one
of the most widely used drugs for treating malaria. It was mainly affective against
erythrocytic stages of each of the human strains of Plasmodium and
gametocytes of P. vivax [95]. Emerging resistance in P. vivax to CQ is beginning
to limit its effectiveness, but it is still effective in most P. vivax endemic areas.
Hydroxychloroquine is a variation of CQ with a hydroxyl group added to the side-
chain. The activity and mechanism is similar to CQ and it is prescribed to treat
the same types of infections [64]. Amodiaquine has been in use for more than 40
years, and is similar to CQ in both structure and activity [101]. This drug has
been useful due to its effectiveness against some CQ resistant strains [115].
Another drug, naphthoquine, is effective against schizonts when used as a
monotherapy [116]. Though naphthoquine has proven effective against some
14
CQ resistant strains, other recent applications combine it with artemisinin [96,
107, 116, 117]. Piperaquine is an orally active option that is more effective than
CQ against P. falciparum [106]. It was first synthesized in the 1960s in China
and has been just as effective as CQ against P. falciparum and P. vivax. This
drug has also been combined with DHA, and following approval by the European
Commission in 2011, it was marketed as Euratesim®. This ACT is administered
once a day for 3 days, and provides longer protection against new infections than
other ACTs [93, 109, 118, 119]. Pyronidine, in combination with artesunate is
administered in a similar 3-day fixed-dose regimen. As a monotherapy,
pyronidine is effective against drug-resistant P. falciparum malaria, and is also
used on combination with arteminins [120]. Tert-butyl isoquine was developed
as a drug candidate as part of a public-private partnership and demonstrates
excellent activity against P. falciparum both in vitro and in vivo. It is still under
investigation as a potential future antimalarial [121]. Amodiaquine-13 is another
analogue of CQ that is active against CQ-resistant strains [122].
8-Aminoquinolines
One of the most commonly used drugs within this group is primaquine,
which is effective against gametocytes with low activity against erythrocytic
stages, and is the only drug that can clear dormant (hypnozoite) liver stage
infections of P. vivax [113, 123]. Monotherapy using primaquine has been
permitted in some endemic regions and it is also recommended as a follow-up
treatment to ACTs [38]. A variation of primaquine, diethylprimaquine is effective
15
preventing exflagellation, however, its activity is similar to another derivative
bulaquine with lower efficacy than primaquine against drug resistant strains [64].
Tafenoquine is also effective against hypnozoites and can be taken as a 2-3 day
treatment course [124]. However, use of these drugs has been known to be
associated with increased risks of hemolysis in glucose-6-phosphate
dehydrogenase (G6PD) deficient patients [81]. This is due to the formation of a
reactive quinone imine and peroxy radical that would be toxic in the absence of
G6PD [38]. Due to its major toxicity in humans, development of NPC-1161B
provided a new lead compound, which has reduced hematological toxicity and
promising efficacy against P. vivax [124, 125].
Antifolates
Development of antimalarials from this group was based on the
understanding of folate derivatives in humans [126]. In the early 1930s folic acid
was identified as a factor able to reverse some forms of anemia. Later,
development of antifolate agents was carried out to treat leukemia. All the
antifolates have a greater affinity to P. falciparum dihydrofolate reductase
(PfDHFR), accounting for the favorable therapeutic index [126]. One study
suggested that the difference in affinity is due to the different methods of
regulation where the parasite form is less readily replenished when targeted by
inhibitors [3]. However, the hypothesis requires further investigation since it
could not be confirmed in a subsequent study [127]. For the treatment of
malaria, this group is subdivided into two categories, class I (dihydropteroate
16
synthase inhibitors) and class II (dihydrofolate reductase inhibitors) antifolates,
and when used in combination, both classes work synergistically in treating
malaria infections [126]. Proguanil was the first antifolate developed in 1945, and
was found to be more effective than quinine in animal models [126, 128]. It also
has a derivative cycloguanil, which has been effective in treating cases of drug
resistant infections [129]. Proguanil has been used both as a monotherapy, and
more recently, in combination with atovaquone (Malarone®), as a prophylactic, to
inhibit electron transport to the cytochrome bc1 complex [126, 130]. There is
also a chlorinated variant of proguanil, chlorproguanil, that is used less
frequently. Due to higher potency, chlorproguanil is recommended as a
prophylactic at a lower dose. It was developed through a collaboration of
GlaxoSmithKline (GSK), Liverpool University and WHO/TDR in East Africa, and
is more effective than sulfadoxine-pyrimethamine [106]. When used in
combination with Dapsone (LapDap), chlorproguanil was found to be more potent
and retain activity against sulfadoxin-pyrimethamine resistant strains [97, 126,
131-133]. Pyrimethamine (Daraprim®) was identified when antifolate analogues
were being tested in the treatment of tumors. Because of a structure similar to
that of proguanil, pyrimethamine was hypothesized to have antimalarial activity
and was later screened against the parasites [134, 135]. In practice it is mostly
used in combination with sulfadoxine with limited use as a monotherapy [126].
Eventual emerging resistance to antifolates prompted the development of
additional effective compounds, and this led to development of P218 that was
deemed suitable for initial studies in humans [136].
17
Sulfonamides
Sulfonamides, also known as sulfa drugs, completely inhibit folic acid
synthesis in microorganisms. As class I antifolate inhibitors, these compounds
block dihydropteroate synthase. Low efficacy and toxicity discouraged their use
as monotherapy, but interest in this group was maintained due to synergistic
effects when combined with anti-DHFR compounds. Dapsone, in addition to
being combined with chlorproguanil, has also been combined with pyrimethamine
(Maloprim®). Pyrimethamine is also combined with sulfadoxine (Fansidar®),
sulphalene (Metakelfin®) as well as sulfamethoxazole and sulfadiazine [137-
139].
Amino Alcohols
Amino alcohols are widely used in combination with artemisinin derivatives
and have been affective against resistant parasite strains. One of the prominent
amino alcohols lumefantrine is typically administered in combination with
artemether (Coartem®) [36, 140].
Kinases and Phosphatases of Plasmodium
Bioinformatic characterization of PF13_0027 in PlasmoDB identifies it as a
putative phosphatase due to presence of a conserved DUSP. Tandem
arrangement of the DUSP and the RHD suggest that it is homologous with a
class of phosphatases known as MKPs that are typically characterized with a
similar domain arrangement. The unique phenotype resulting from the disruption
of PF13_0027’s open reading frame led us to investigate the P. falciparum
18
pathways involving proteins with similar characteristics. Interestingly, the MAPK
signal transduction pathway is a well characterized pathway in several
eukaryotes responsible for multiple intracellular processes when disrupted might
result in an attenuated phenotype similar to the one observed in the C9 mutant
parasite. Functions affected by MAPK pathways are typically utilized to regulate
critical cellular process such as stress response, osmoregulation, cell cycle
regulation, signal transduction, or transcription factor interaction (Figure 1.4)
[141-145]. It is our current understanding that in P. falciparum these pathways
could be utilized in a similar fashion [146-154]. It is likely that the phenotype of
C9 parasites results from disrupting one or more of these pathways, however
further investigation would be necessary to confirm this assertion. Furthermore,
since protein phosphatases are integral to cell survival, this putative phosphatase
provided a novel subject for investigation. Therefore, my hypothesis is that
PF13_0027 functions as an important component of intraerythrocytic
development.
Protein kinases occupy an important role in eukaryotic cell development
by regulating the activity of various proteins through additive phosphorylation.
Phosphatases serve an antagonistic function, removing phosphate groups,
throughout cellular development and response to external stimuli, which when
knocked out, can result in aberrant or deleterious effects on development.
Phosphorylation cascades have become the focus of many studies in
Plasmodium because of their deduced importance for development. Much of the
literature investigates their involvement in cascades regulating the progression
19
from gametocyte to the formation of the zygote and oöcyst in the mosquito
midgut [142, 154-158]. Given the level of importance that phosphorylation and
dephosphorylation serve in cellular development, further investigation of this
process is vital to expanding our knowledge of this malaria parasite’s biology. In
studying these pathways it is also important to consider the relationships of both
kinases and phosphatases.
Plasmodium Kinases
Exhaustive evaluation of the kinome in model organisms such as C.
elegans, D. melanogaster, S. cerevisiae and H. sapiens resulted in the
identification of 7 groups of protein kinases. These groups are the cyclic-
nucleotide and calcium/phospholipid-dependent kinases (ACG group); the
CMGC group, comprised of cyclin-dependent kinases (CDK), mitogen-activated
kinases (MAPK), glycogen-synthase kinases (GSK) and CDK-like kinases;
calmodulin-dependent kinases (CamK) group; tyrosine kinases (TyrK) group
which is absent from Plasmodium; casein kinase 1 (CK1) group; the sterile (STE)
group, also absent from Plasmodium; and the tyrosine kinase-like (TKL) group
[159-161]. Another important group to acknowledge in Plasmodium is the
orphan protein kinases (OPKs) that do not share any homology with mammalian
kinases and possess atypical enzymes with features from more than one family
of kinase [162].
20
The ACG Group
There are five Plasmodium phosphatases from this group; cAMP-
dependent kinase (PfPKA, PFI1685w), cGMP-dependent kinase (PfPKG,
PF14_0346), PKB-like serine/ threonine kinase (PfPKB, PFL2250c) that
functions in the phosphoinositide-3-kinase (PI3K)-dependent pathway. Aurora
related kinase (ARK2, PFC0385c) a putative serine/ threonine kinase involved in
kinetochore organization, and PF11_0464 a putative serine/ threonine kinase
[159, 163-165].
The CMGC Group
Kinases within the CMGC group [cyclin-dependent kinases (CDK),
mitogen-activated kinases (MAPK), glycogen-synthase kinases (GSK) and CDK-
like kinases] control cell proliferation and development and in the Plasmodium
kinome, they make up the most prominent kinase group. This group includes the
CDKs which regulate cell-cycle progression, several of which have been
identified in Plasmodium [162, 166]. These kinases include; PfPK5
(PF14_0605), PfPK6 (MAL13P1.185), Pfmrk (PF14_0605), Pfcrk-3 (PFD0740w),
Pfcrk-4 (PFC0755c) and Pfcrk-5 (PFF0750w) [159, 166]. Pfcrk-4 and PfPK6
both have features of CDKs and MAPKs and form a cluster at an intermediate
position between both groups in phylogenetic analysis [159].
The two MAPKs, Pfmap-1 (PF14_0294) and Pfmap-2 (PF11_0147) serve
a critical function as transducers of intra- and extracellular signals to cell cycle
control elements and transcription factors [162]. Studies involving the MAPKs
21
have been carried out in both P. falciparum and P. berghei resulting in numerous
insights into the biology of malaria parasites even though the roles of these two
MAPKs appear to be reversed in these Plasmodium species. During
microgamete formation in P. berghei, MAPKs are involved in control of
cytokinesis and flagellar motility [152]. Expression profiles in P. berghei have
shown that Pfmap-2 is not essential to asexual growth and gametocytogenesis,
but it is essential to microgametogenesis [142]. Contrasting results were
discovered when investigating MAPK function in P. falciparum when it was
discovered that Pfmap-2 is critical for asexual development [167]. Furthermore,
they identified that levels of Pfmap-2 were elevated in Pfmap-1-KO parasites
suggesting that not only Pfmap-2, but also Pfmap-1 have relevance to asexual
development. The CMGC group also has two GSK3-related kinases, PfPK1
(PF08_0044) and PfGSK3 (PFC0525c), as well as a LAMMER-related kinase
Clk1 (PF14_0431) [168-170]. The GSKs have a critical function in regulation of
cell proliferation and the Clks are important for RNA metabolism [162].
The CamK Group
This group occupies an important role in the development of the oökinete
in the mosquito vector. Calcium dependent protein kinases (CDPKs) make up a
family of serine/ threonine kinases found only in protozoa and plants and are
distinct from all other animal protein kinases [148, 154]. The P. falciparum
genome encodes 6-7 CDPKs that are developmentally restricted to Ca2+
signaling. This group is characterized by the presence of a kinase catalytic
22
domain located adjacent to four EF-hand calcium binding domains, an overall
structure that is shared with CDPKs of plants and ciliates [159]. PfCDPKs 1-3
and 5 (PFB0815w, PFF0520w, PFC0420w and PF13_0211 respectively) are
expressed throughout the asexual stages while PfCDPK4 (PF07_0072) is
expressed in the sexual stages [171-174]. PfCDPK4 is essential for the
development of parasites in the mosquito during male gametocyte exflagellation
[154, 159]. CPDK4 specifically is one of the kinases responsible for the
transduction of the Ca2+ signals within the parasite prior to the differentiation of
the gametocyte to the microgamete. When CDPK4 is knocked out in P. berghei,
parasites fail to produce oöcysts supporting the understanding that its expression
is essential to reproduction [154]. PfCDPK7 (PF11_0242) is similar to the other
CDPKs with the exception that it contains one EF-hand motif. An additional
branch of the CDPK group is PfPK2 (PFL1885c) and PfCDPK6 (PF11_0239) that
do not have the EF-hand motif [175, 176].
The CK1 Group
Plasmodium only has one characterized kinase in this group, PfCK1
(PF3D7_1136500.1) [159, 177]. Though it is able to phosphorylate several
proteins in vitro, the role of has not been determined [162].
The TKL Group
In this group, of the four malarial enzymes, three kinases, PfTKL4
(PFF1145c) and PfTKL1 (PFB0520w), and PfTKL2 (PF11_0220) share
homology with MAPKKK-related enzymes [159, 161, 178]. PfTKL3 (PF13_0258)
23
has a sterile α-motif (SAM) domain dependent kinase expressed in both asexual
development and gametogenesis and is being studied as a viable drug target
[178].
The OPK Group
The NIMA-related kinases (Nek) family is responsible for centrosome
replication during eukaryotic cell division [162, 179]. Pfnek-1 (PFL1370w), which
has been shown to phosphorylate Pfmap-2, is predominantly expressed in both
the asexual and sexual stages. Interestingly, the P. berghei ortholog of Pfnek-1
is only expressed in the microgametocyte which is consistent with the suggestion
that Pfnek-1 is important for male gametogenesis [162, 180, 181]. The
remaining, Pfnek 2-4 (PFE1290w, PFL0080c, and MAL7P1.100 respectively) are
expressed in gametocytes [162, 182]. Studies with the Pfnek-4 ortholog in P.
berghei have revealed that Pbnek-4 is essential for oökinete maturation,
revealing another possible role of the Neks [183].
Plasmodium Phosphatases
Phosphatases dephosphorylate proteins reversing and controlling the
actions of protein kinases. This function is critical to cell viability since
unregulated kinase activity can have detrimental effects [184]. Earlier research
had characterized two main functional groups of protein phosphatases; protein
tyrosine phosphatases (PTP) that are typically membrane associated and protein
serine/threonine phosphatases (PP) that are cytosolic [185, 186]. The members
of these phosphatase families have high sequence conservation within the active
24
site and participate in regulation of cell cycle progression, protein synthesis,
carbohydrate metabolism, transcription, and neuronal signaling in eukaryotic
cells which underscores their importance to survival [185]. Furthermore, within
the PPs, there are two distinct sub-families; Mg2+-dependent phosphatases
(PPM) and Mg2+-independent phosphatases (PPP) [186]. This group is
subsequently divided into more specific groups, PP1 (PF14_0142), PP2A, PP2B
and PP2C. With respect to PP2C, studies carried out in Toxoplasma gondii
determined it to have a significant effect on the host cell following invasion [187].
One study revealed this using GFP fusion tags to localize PP2C and determine
that the nuclei of host cells were the targets, and that the protein was being
released from the bulbous region of the rhoptries of the invading parasites [188].
In recent years, advances in our understanding of phosphatases have led to the
identification of additional classifications. Within the genome of Plasmodium,
four groups of phosphatases are represented: The PPP group; the PPM group;
the PTP group; and the NLI-interacting factor like (NIF) group (Table 1.1) [2].
The PPP group
The phosphatases of this group are highly conserved and among the most
extensively studied type of protein phosphatases [189, 190]. These enzymes
target a large variety of substrates that are not limited to phosphoproteins, since
they are similar to the metallophosphatases in their dependency on Mn2+, Ca2+,
and/ or Co2+. Classification of subgroups has been recently extended to include
as many as eight distinct subtypes of serine/threonine phosphatases (PP1,
25
PP2A, PP2B, PP4, PP5, PP6, PP7 and BSU (plant-specific) [191, 192]. There
are also three conserved motifs that have been considered as the signature motif
of the PPP family (GDXHG, GDXXDRG and GNH [E/D]) [191-194].
Within Plasmodium, several PPs have been identified. PF3D7_1466100
(BSU subfamily) is a PP1-related enzyme with closely related orthologs in plants
encoding Kelch motifs in the N-terminal domain. These Kelch motifs form distinct
tertiary structures that are thought interact with regulatory subunits [195, 196].
Another such phosphatase, PF14_0142 (PP1 subgroup), is expressed in all
stages of the developmental cycle with a slight reduction during the late
trophozoite stage [2]. PF14_0224 (PP7 subgroup) has metal (Mn2+) and
phosphate and water binding motifs, except there are substantial differences
from the other phosphatase subgroups. PFC0595c, similar to the PP2/4/6 type
phosphatases, is expressed with a similar profile; however, microarray data
suggests additional activity in the sexual stages [182, 197]. PF08_0129 shares
characteristics with the PP3, PP2B and calcineurin subgroups. It is
characterized as a calcineurin-type enzyme with a calmodulin-binding domain.
PFI1245c is part of the PP2 subfamily and is homologous to the mammalian
phosphatase PP2A. These phosphatases are asparate-rich proteins with the
ability to inhibit the phosphatase PFI1245c. Subgroup PP5 phosphatases are
represented in the Plasmodium phosphatome by MAL13P1.274 that contains a
nuclear targeting sequence in the N-terminus as well as TPR (tetratricopeptide)
repeats [2]. Phylogenetic analysis of PFI1360c identifies it as closely related to
the PP2/4/6 subgroups. Previous data has suggested that it may be involved in
26
centrosome maturation, spliceosome assembly, chromatin modification and
regulation of the NF-κB and mTOR signaling pathways [2, 193].
The PPM Group
This group is made up of a diverse set of enzymes with Mg2+ and Mn2+
dependent phosphatase activity that typically function in modulating stress
responses. They typically have regulatory domains in the N- or C-terminal
extensions [2]. Despite structural similarities between this group and the PPP
group, they do not share sequence homology [198, 199]. Also a PP2C-type
phosphatase PF11_0396 has been reported for Plasmodium in literature
suggesting that it regulates translation factor 1B [2, 187]. Two other PPM-related
phosphatases are PFE1010w and MAL8P1.109, but they do not have any
experimental data to support a suggested function.
The PTP group
The group is subdivided into three main subfamilies; the PTPs, the
DUSPs, and the low molecular weight phosphatases [200-204]. Eukaryotic
phosphatases of the PTP and DUSP subgroups are required for signaling, cell
growth, differentiation and control of the cell cycle [205]. Since the P. falciparum
cell cycle is also driven by sequential activation of CDKs [146], the phosphatase
CDC25 is an essential regulator of the cell cycle that works by activating the
CDKs through dephosphorylation at the G2-M transition [151, 206, 207]. In
contrast, humans have three CDC25s (CDC25A, CDC25B and CDC25C) that
dephosphoryate the threonine and tyrosine residues in order to trigger activation
27
of CDK/cyclin activity. The CDC25 phosphatases form a distinct group which
has little sequence similarity with the other PTPs except for the signature motif.
They appear to have evolved from RHDs that have been known to catalyze
sulfur-transferase reactions [147]. These types of phosphatases are essential for
differentiation of the gametocyte to the microgamete since this transition is
dependent on the three rounds of division that producing eight motile
microgametes [208]. The microgametes, when fused with the macrogamete,
form the zygote that further differentiates to the oökinete, which migrates through
the midgut wall [152].
The conserved catalytic mechanism of these enzymes is mediated by
cysteine, arginine and aspartic acid residues comprising the Cx5R signature
motif [2]. Within P. falciparum some PTPs have been investigated biochemically
[209, 210]. The first PFC0380w (YVH1) contains a Zn2+-binding domain,
exhibiting activity against phosphorylated serine and threonine residues. The
second, PF11_0139 (PRL, “Protein of Regerating Liver”)) is a protein tyrosine
phosphatase with the CaaX motif in the C-terminal motif used for farnesylation. It
has also been identified as a target of farnesyl-transferase. In merozoites, it co-
localizes with AMA-1 and may be involved in invasion [2, 210]. Within this group,
the phosphatase MAL13P1.168, which as similar structure to the PTP family, is
characterized as having a PTP-like motif. The catalytic site contains a
substitution of a proline in place of arginine in the catalytic site [2, 211]. Similar
to this substitution, PF13_0027 has an isoleucine substitution in place of the
conserved arginine in the catalytic site.
28
The NIF group
Phosphatases in this group are responsible for the dephosphorylation of
the carboxy-terminal domain of RNA polymerase II. These phosphatases are
also known to interact with the transcription factor TFIIF. They are believed to
dephosphorylate serine in the C-terminal to reactivate the polymerase after
transcription termination [212-216]. P. falciparum has four identified genes within
this group. The NIF phosphatases have a distinct DxDx(T/V) motif in the active
site that is conserved in two of the sequences, PFE0795c, and MAL13P1.275.
The third sequence PF10_0124 is closely related, but does not have the intact
motif, and therefore is considered to be inactive based on preliminary
bioinformatics analysis. The fourth sequence PF07_0110 falls within a distinct
clade of NIF-type domains with a disrupted DxDx(T/V) motif (Table 1.2) [2].
Focus of Study
This dissertation research project investigates the involvement of
PF13_0027 during intraerythrocytic development of P. falciparum. PF13_0027
has a DUSP domain that maintains structural homology to DUSP domains and it
is conserved across Plasmodium spp. with orthologs in other species (Table 1.3).
Attenuation of blood-stage parasite growth following functional knockout
suggests that it has an important regulatory mechanism in the developmental
cycle, specifically during the late trophozoite when it is maximally expressed.
Phosphatases have been the focus of several recent drug development
studies and it is reasonable to investigate this as a novel target since new
29
mechanisms of action for antimalarial drugs are a critical priority. This priority is
emphasized by decreased drug efficacy due to increasing drug resistance. It has
also been demonstrated that cascades involving protein phosphorylation are
required for with successful development of Plasmodium parasites as well as
eukaryotic cells. Expansion of our knowledge of Plasmodium biology through the
study of PF13_0027 will augment our understanding of the regulatory processes
used by these parasites.
In addition to partially characterizing the function of PF13_0027, this
phosphatase was also evaluated as a possible drug target through high-
throughput in silico techniques. Utilizing the available compound libraries in the
ChEMBL-NTD, we were able to screen a preliminary model of PF13_0027
against compounds with antimalarial activity and determine if any of these
compounds interact with the predicted active site. Several compounds were
identified and obtained for further in vitro assays and assessed for their effect on
the asexual cycle. Through this study we investigate the role of PF13_0027 in P.
falciparum and suggest a function explaining its contribution to parasite
development and evaluate it as a drug target.
30
Figure 1.1: The piggyBac transposon mutagenesis system [9]. (A) The helper plasmid carries a selectable marker for human dihydrofolate reductase (hDHFR) under the control of a 5' calmodulin promoter and 3' calmodulin terminator. (B) The helper plasmid codes for the piggyBac transposase which excises the selectable marker allowing it to randomly insert into the genome. (C) Late stage parasites are separated from mixed cultures using a magnetic column and mixed with red blood cells (RBCs) containing the transposon and helper plasmids. Transfected parasites are then selected by applying drug pressure and cloned to identify parasites with single insertions.
31
Figure 1.2: The spatial distribution of P. falciparum malaria endemicity in 2010 [7].
32
Figure 1.3: The spatial distribution of P. vivax malaria endemicity in 2010 [6].
33
Figure 1.4: Life cycle of the malaria parasite [5].
34
Figure 1.5: A global map of dominant malaria vector species [4].
35
Figure 1.6: Summary of the MAPK pathway.
36
Table 1.1: Classifications of phosphatases [1-3]. .
Subgroup Examples Signature motif
Metal ions Function
Phosphoprotein phosphatases (PPP, PPM, PP2C)
Metallophosphatases GDxHG, GDx2GRD GNH[E/D]
Mn2+
, Mg2+
, Ca2+
, Co2+
Modulate stress responses (ex. PP1, PP2A, PP2B, PP4, PP5, PP6, PP7, BSU)
Protein Tyrosine Phosphatases (PTP, DUSP)
Tyrosine specific and dual specificity phosphatases
DXnCX5R - Cell cycle regulation and signal transduction (ex. Cdc25, MKP)
NLI interacting factor-like phosphatase (NIF)
TFIIF-associating C-terminal domain phosphatase 1 and Small CTD phosphatases
DxDx(T/V) -
Interaction with transcription factor TFIIF, dephosphorylation of the carboxy domain of RNA polymerase II
37
Table 1.2: Phosphatases identified in P. falciparum
ID Annotation
PPP group (Phospho-Protein Phosphatases)
PF14_0630 Protein serine/threonine phosphatase
PF14_0142 Serine/threonine protein phosphatase, putative
PF14_0224 PP1-like protein serine/threonine phosphatase
PF10_0320a Protein phosphatase 1 regulatory subunit 7
PFC0595c Serine/threonine protein phosphatase, putative
PF10_0177 Erythrocyte membrane-associated antigen HT; SP; API
PF08_0129 Protein phosphatase, putative
PFI1245c Protein phosphatase-beta
PFI1360c Serine/threonine protein phosphatase, putative
MAL13P1.274 Serine/threonine protein phosphatase pfPp5
PF13_0222 RNA lariat debranching enzyme, putative
PFL0980w RNA lariat debranching enzyme, putative
PFA0390w DNA repair exonuclease, putative
PF14_0064 Vacuolar protein sorting 29, putative
PF14_0036 Acid phosphatase, putative
PF14_0282 Acid phosphatase, putative
PF14_0660 Hypothetical protein; Protein phosphatase (PPP group, Shelphs bacterial-like subgroup), putative SP; API
PFL0300c Phosphoesterase, putative SP
PF14_0614 Hypothetical protein; metallo-dependent phosphatase SP
PF10_0177a Serine/threonine protein phosphatase, putative
38
Table 1.2: Continued
ID Annotation
PPM group (Mn2+
or Mg2+
dependent protein serine/threonine phosphatases)
MAL13P1.44 Protein phosphatase 2c-like protein, putative
PFL2365w Hypothetical protein, conserved; Protein phosphatase (PP2C/PPM group), putative
PF14_0523 Protein phosphatase 2C, putative
PFD0505c Protein phosphatase 2C
PFE1010w Protein phosphatase 2c, putative
PF11_0362 Protein phosphatase, putative
PF11_0396 Protein phosphatase 2C
MAL8P1.109 Protein phosphatase 2C, putative
PFL0445w Conserved Plasmodium protein, unknown function; protein phosphatase
PF10_0093 Protein phosphatase, putative
MAL8P1.108 Protein phosphatase, putative
PF10_0093 Hypothetical protein; Protein phosphatase (PP2C/PPM group), putative
PTP group (Protein Tyrosine Phosphatases)
PF14_0524 Protein phosphatase 7 homolog, putative API
PFC0380w Dual-specificity protein phosphatase, putative
PF11_0139 Protein tyrosine phosphatase, putative
PF11_0281 Hypothetical protein: weak similarity to dual specificity protein phosphatase
MAL13P1.168 Hypothetical protein, conserved; Protein tyrosine phosphatase
PF13_0027 Protein phosphatase, putative
39
Table 1.2: Continued
ID Annotation
NIF group (NLI interacting factor-like phosphatases)
PFE0795c Nif-like protein, putative
PF07_0110 Hypothetical protein, conserved; CTD phosphatase
PF10_0124 Hypothetical protein; CTD phosphatase
MAL13P1.275 NLI interacting factor-like phosphatase, putative; CTD phosphatase
Others
PF14_0492 Protein phosphatase 2b regulatory subunit, putative
40
Table 1.3: Orthologs of PF13_0027 [8].
Accession Taxon Description
PBANKA_140400 Plasmodium berghei str. ANKA conserved Plasmodium protein, unknown function
PCHAS_140590 Plasmodium chabaudi chabaudi conserved Plasmodium protein, unknown function
PFIT_1304700 Plasmodium falciparum IT protein phosphatase, putative
PKH_140400 Plasmodium knowlesi strain H conserved Plasmodium protein, unknown function
PVX_122110 Plasmodium vivax SaI-1 hypothetical protein, conserved
PY00561 Plasmodium yoelii yoelii str. 17XNL hypothetical protein
41
Chapter 2: Identification of a Putative Phosphatase in Plasmodium
falciparum Regulating Progression from Pre-S Phase Blood Stage
Development (Specific Aim 1)
Rationale for Study
Regulation and developmental checkpoints in blood-stage P. falciparum
are complex and critical components of malaria transmission. Regulation of this
important developmental phase depends on the functions of kinases and
phosphatases [143, 151, 211, 217-223]. Kinases and phosphatases modulate
the active-to-inactive state of substrates through phosphorylation-to-
dephosphorylation, respectively. Much of the current research in Plasmodium
has focused on mechanisms controlling transcription regulation and cell
proliferation, as a target for novel antimalarials, directed at the asexual blood-
stage cycle [224]. Other studies have investigated phosphorylation cascades
during the gametocyte-oökinete-oöcyst transition in the mosquito midgut [154,
225]. PF13_0027 is conserved in Plasmodium species and expressed
throughout the intraerythrocitic cycle, suggesting a conserved role throughout
parasite development. In this study, we use a PF13_0027 mutant (C9), created
by disruption of the gene’s ORF during the course of a large-scale transposon
mutagenesis project of P. falciparum, to evaluate the function and significance
during intraerythrocytic development.
42
Introduction
P. falciparum is the most deadly of the five known human malaria parasite
species. It causes deaths in the hundreds of thousands each year, and millions
of clinical illnesses [10]. It is the major cause of severe malaria and grows
rapidly within the blood of infected individuals through successive cycles of
asexual growth and proliferation. Active entry into erythrocytes is followed by a
pre-S growth phase consuming the host proteins until a switch to S/M
proliferation and cytokinesis in the last third of the cycle. This process
culminates in a dynamic release of erythrocyte-invading merozoites. The rate of
growth and extent of proliferation varies among isolates and is implicitly of
importance for disease progression during an infection. Other malaria parasites
of the phylum Apicomplexa, such as T. gondii or Eimeria tenella, share this
pattern of asexual development suggesting the importance of this pattern of
development throughout the phylum [226].
In eukaryotic systems, phosphorylation cascades are critical to cellular
development and depend on the coordinated activity of kinases, which are in turn
modulated by the activity of phosphatases. There is growing evidence that
kinases are critical regulators of cell growth and development in Plasmodium
species [227-229]. Plasmodium kinases, for example, have been found to be
involved with the initial invasion of host cells in addition to egress and
differentiation [226, 230]. Additional studies have identified kinases in
phosphorylation cascades of the gametocyte-oökinete-oöcyst transition in the
mosquito midgut [154, 225, 231-235]. In contrast, few studies have described
43
the phosphatases involved in these processes, which would understandably
function with kinases to co-regulate cell cycle progression at key checkpoints [2,
188, 236-238]. This dearth of information about phosphatases may be due to a
smaller relative number of identifiable phosphatases in the Plasmodium genome
compared to kinases [2, 239]. However, it is not uncommon for phosphatases to
be fewer in number due to their non-specific mechanism of targeting
phosphorylated substrates [240].
The protein tyrosine phosphatase (PTP) superfamily is defined by a
conserved CX5R motif located in a phosphate-binding pocket. Dual-specificity
phosphatases (DUSPs) are a subset of this superfamily that includes the
mitogen-activated protein kinase (MAPK) phosphatases (MKPs). MKPs are
frequently involved in regulation of cell cycle progression, growth, and
proliferation [222]. One class of MKP is characterized by the presence of a non-
catalytic N-terminal rhodanese (RHD)-like domain utilized for substrate
recognition upstream from a catalytic DUSP domain [143, 153, 219]. In P.
falciparum, PF13_0027 is the only gene encoding a product with these
characteristics [2]. In this study, we hypothesize that PF13_0027 is an atypical
MAPK phosphatase of P. falciparum expressed during intraerythrocytic
development.
44
Materials and Methods
In vitro Parasite Culture Conditions
P. falciparum NF54 and mutant C9 clones were cultured according to
standard methods at 37°C (5% O2 and 5% CO2, nitrogen balanced) in 5%
hematocrit (O+ blood) and RPMI 1640 medium with and 0.5% Albumax II, 0.25%
sodium bicarbonate and 0.01 mg/ml gentamicin [241]. The C9 mutant parasite
line was created by random insertional mutagenesis using the piggyBac
transposon pXL-BACII-HDGH (Appendix A). The location of the insertion in the
PF13_0027 ORF was confirmed by thermal asymmetric interlaced (TAIL)
sequence analysis.
Determination of Merozoite Number Per Schizont
Parasite cultures were synchronized with 5% sorbitol [241]. Merozoite
numbers were counted in 300 segmented schizonts in Giemsa-stained thin
smears from NF54 and C9 parasites to determine average number of merozoites
produced per schizont.
RNA Extraction and Analysis by qRT-PCR and RT-PCR
NF54 RNA was collected from six intraerythrocytic developmental stages;
early rings, late rings, early trophozoites, late trophozoites, early schizonts and
late schizonts (ER, LR, ET, LT, ES and LS, respectively) followed by saponin
lysis and suspended in TRIzol® reagent (Life Technologies™), RNA purified, and
treated with DNase I. Purity was confirmed by PCR carried out without the
addition of reverse transcriptase. PF13_0027 transcripts were amplified using
45
primers 5′-TCGATTTTGAGGAGCTGAA-3′ and 5′-
GGGTAAAACATCCTTTTTGTT-3′ with SuperScript® III Platinum® SYBR® Green
One-Step qRT-PCR Kit (Life Technologies™) following the manufacturer’s
protocol. For RT-PCR analysis, 100 ng DNAse1-treated total RNA was amplified
using primers 5′-CACCATGGAATATAAAAGCATCGATTTTG-3′ and 5′-
GTTTATGTAATTATTTATTACTATAAATGGTC-3′, and analyzed by horizontal
agarose gel electrophoresis.
Plasmid Constructs and Genetic Complementation
The plasmid was developed with the full-length PF13_0027 ORF and its
native 5ʹ untranslated region (UTR), a C-terminal hemagglutinin (HA) tag and the
3ʹ UTR calmodulin (CAM) termination sequence. For drug selection, the plasmid
carried a blasticidin S deaminase (BSD) drug selection cassette under control of
5′UTR of histidine rich protein (HRP3) and 3′ UTR histidine-rich protein-2
(HRP2). Mutant variations of PF13_0027 were generated using the
complementation plasmid by site-directed mutagenesis of the conserved cysteine
(C383A), aspartic acid (D345A), and the two conserved lysines (K388A and
K394A).
Schizonts were isolated from a 20 mL culture with 3-5% parasitemia using
a VarioMACS™ Separator (Miltenyi Biotec) and counted with a hemacytometer.
Fresh 50% hematocrit blood was washed and combined with Cytomix [241] in a
1:1 v/v ratio and aliquot into chilled 2 mm cuvettes, and electroporated using a
Gene Pulser Xcell CE™ (Bio-Rad) to load RBCs with transposon and helper
46
plasmids purified using methods described previously [9]. Positive transfected
clones were selected using blasticidin, diluted, then transferred to 96-well culture
plates and maintained for 17 days to select individual clones. Clones were
validated by PCR following transfection and drug selection to verify presence of
the correct drug selection cassette (Appendix B).
Growth Assay and Cell Cycle Determination
Growth assays for cell cycle determination were performed by maintaining
tightly synchronized cultures of P. falciparum NF54 and C9 clones at 0.5–2%
parasitemia. Time points were collected every two hours and then fixed in 0.05%
glutaraldehyde after removal of culture medium. Parasitemia was estimated
using flow cytometry as described previously [242] by staining parasites with
ethidium bromide and analyzed using an Accuri C6 flow cytometry system (BD
Accuri™). A total of 100,000 cells were counted for each sample and the data
was analyzed using CFlow Plus software (Appendix C) (BD Accuri™). The cell
cycle was determined by comparing the relative abundance of each
developmental stage at each time point according to methods developed
previously [243]. Relative fold change was determined by calculating the fold
increase in parasitemia between time zero and the endpoint. Each sample was
then plotted as a percent relative to NF54.
Invasion Assays
Schizonts were isolated from a 20 mL culture with 3-5% parasitemia using
a VarioMACS™ Separator (Miltenyi Biotec) and counted with a hemacytometer.
47
Purified late-stage schizonts were used to initiate cultures in 96-well plates in
triplicate and cultured for 30 hours to allow segmentation and invasion of fresh
RBCs. Parasites were fixed and labeled for flow cytometry as stated in the
growth assay protocol and the fold increase in parasitemia over the 30-hour
assay was calculated as a representation of the percent of parasites successfully
invading and continuing through development.
Multiple alignments and phylogenetic analysis
The sequence of PF13_0027 and orthologous Plasmodium sequences
were retrieved from PlasmoDB v9.2 (www.plasmodb.org). Outlier species were
identified through BLAST searches with the DUSP domain using NCBI BLASTP.
Sequences with greatest homology to PF13_0027 were retrieved and used to
build the multiple alignments using ClustalW [244, 245]. Phylogenetic trees were
created using the Neighbor-joining method with 1000 bootstrap in MEGA5 [246-
248].
Southern Blot Hybridization
Genomic DNA (4 μg) extracted from transformed parasites was digested
with 10 units of SalI and DraIII (New England Biolabs®) overnight and separated
on a 0.8% TAE agarose gel. The DNA was depurinated in 0.25 M HCl,
denatured in 0.5 M NaOH/1.5 M NaCl, neutralized in 0.02N NaOH, 1M
C2H3O2NH4 and blotted overnight to a nylon membrane. A 711 bp probe was
developed against the bsd drug selection cassette using primers 5′-
ATAAGAAGAAGTATATAATGAATTATATATAATGCT-3′ and 5′-
48
CATATGTATTTTTTTTGTAATTTCTGTGTTTAT-3′. The blot was then
hybridized to 32P-labeled probe washed three times in 2X SSC/0.5% SDS (1X
SSC/0.15 M NaCl/ 0.015 M NaC6H5O7, pH 7.0) for 15 min, and exposed to a
Kodak photographic film at -80°C to visualize the hybridized fragments (Appendix
D).
Results
Identification of an Attenuated Growth Mutant in P. falciparum
A collection of unique mutant clones was created from a laboratory line of
P. falciparum NF54 using random insertional mutagenesis with a piggyBac (pB)
transposon [9]. The C9 parasite line carried one copy of a pB transposon (pXL-
BACII-HGDH) inserted near the 5′ end of the single ORF of PF13_0027 (Fig.
2.1). Intraerythrocytic growth for clone C9 was analyzed and determined to be
severely attenuated with a net growth rate consistently 50% of the NF54 parent
(Fig. 2.2). During development, intraerythrocytic stages did not demonstrate any
obvious differences in morphological characteristics (Fig. 2.3), and mean
numbers of merozoites produced did not vary significantly from the NF54 parent
(Fig. 2.4).
Defining Characteristics of PF13_0027
The protein encoded by PF13_0027 was determined to have two key
structural features, a RHD domain followed by a DUSP-like domain.
Bioinformatics analysis defined this tandem arrangement as characteristic of
certain MKPs conserved within humans and other model organisms such as fruit
49
fly and yeast [145, 221, 249, 250]. It is for this reason that we refer to the
PF13_0027 product as P. falciparum MKP. The conserved signature motif of
CX5R, which is typically definitive of DUSP domains in MKPs, is only partially
conserved in PF13_0027 (Fig. 2.5A). The putative binding pocket of PF13_0027
was identified through BLAST searches and multiple sequence alignments with
MKPs that have 3-D crystal structures (Plasmodb version 9.2; NCBI GenBank
Flat files release 193.0). Within this binding pocket the amino acid residue
cysteine-383 (C383) along with another residue of the catalytic triad aspartate-
345 (D345) are conserved and align with conserved cysteine and aspartic acid of
the other identified DUSPs. However, the third conserved catalytic residue,
arginine, aligns with isoleucine-398 (I398) in PF13_0027. During
dephosphorylation, the conserved arginine is critical for dephosphorylation
activity, since active DUSP domains often depend on arginine to maintain the
transition state with the phosphorylated substrate [251]. Absence of arginine is
expected to drastically reduce the catalytic activity of the DUSP; therefore, this is
an important departure from the consensus motif defined for catalytically active
DUSP domain orthologs and would be expected to reduce phosphatase activity
[251, 252].
Prior mutagenesis studies and analyses of catalytic domains in the
DUSPs of model organisms suggest that unique characteristics, such as the
ones identified in PF13_0027, may be characteristic of a pseudophosphatase or
a low activity phosphatase [253-255]. Additionally, there is an insertion of nine
residues disrupting the spacing within the CX5R signature motif. Though it
50
cannot be determined if this insertion changes the three-dimensional structure
within the putative binding pocket, this unique stretch of residues is conserved in
each of the Plasmodium orthologs. Conservation of these unique characteristics
in Plasmodium species supports the formation of an individual clade (Fig. 2.5B).
PF13_0027 Regulates Transition from Pre-S phase to S/M Phase
In wild-type parasites, the highest relative abundance of PF13_0027
transcripts is at the end of the pre-S development phase (i.e., late trophozoite)
during intraerythrocytic development [256]. This expression profile coincides with
the stage when the C9 mutant cycle deviates from the wild-type cell cycle (Fig.
2.6). Utilizing the detailed time course experimental protocol developed
previously [243], the timing for NF54 was determined to be 46 hours compared to
52 hours in C9 null MKP mutant. The difference resulted from a prolonged pre-S
trophozoite stage causing late entry into the S/M schizont phase. The length of
schizont development (S/M - C) was similar in C9 and NF54 making pre-S phase
the only abnormal growth phase of the intraerythrocytic cycle (Fig. 2.7).
Phenotype Rescue of Wild-type Growth by Genetic Complementation
Attenuated growth of the C9 MKP null mutant remained stable over
multiple subsequent generations suggesting that survival was not due to
phenotype reversion. This is consistent with general experience using the
piggyBac system as it is now extensively used in a number of organisms and the
transposable elements remain integrated in the genome in the absence of
transposase [9, 257-262]. However, the extended maintenance of P. falciparum
51
intraerythrocytic cultures required for transfection and the selection process in
the experimental studies increases the possibility for secondary mutations to alter
the cell cycle or cause growth attenuation. Therefore, to validate that the
phenotype was due to disruption of PF13_0027, we genetically complemented
the C9 mutant with a full-length copy of PF13_0027, including its putative
promoter region (Fig. 2.8). The 5′ intergenic region between PF13_0027 and the
upstream gene MAL13P1.28 was added to the ORF to ensure the native
promoter was included. Using the BSD resistance marker on the complement
vector, we were able to select for two independent cloned lines, E3 and E8. RT-
PCR analysis of both E3 and E8 revealed that the complemented parasite lines
transcribed PF13_0027 in contrast to C9 that did not have detectable transcripts
(Fig. 2.9). Through the use of whole genome sequencing (Appendix D),
Southern blot hybridization analysis (Appendix E), and PCR validation (Appendix
F), it was also determined that the complemented parasite lines maintained the
full-length copy of PF13_0027 as stable episomes. Complementation of the C9
mutant with the full-length copy of PF13_0027 rescued the phenotype of both E3
and E8 as evident by their return to normal growth (Fig 2.10). Additionally, the
invasion phenotype was also restored in complemented parasites as well (Fig.
2.11). Results from site-directed mutagenesis confirmed that rescue of the
phenotype was not achieved in the absence of cysteine or aspartic acid for
growth (Fig 2.12) or invasion (Fig. 2.13). Replacement of the conserved lysines
did not attenuate the phenotype and parasites were complemented as seen with
the unaltered ORF (Statistical tables in Appendix G). Considering the roles
52
cysteine and aspartic acid play in the binding and removal of phosphates, it is a
possibility that these residues are critical to the function of PF13_0027.
Discussion
Cell cycle progression, in P. falciparum, and completion of
intraerythrocytic development is highly dependent on a precise pattern of
metabolic events. Disruption of any of the numerous biochemical pathways and
processes is anticipated to have detrimental effects on the efficiency of this
process. In our study, we discovered that normal cell cycle development was
delayed by disruption of PF13_0027, indicating that this atypical phosphatase is
a regulator of the P. falciparum cell cycle. The delayed transition from the pre-S
trophozoite to the S/M schizont suggests this transition phase during the
parasite’s intraerythrocytic growth is a cell cycle checkpoint. Rescuing the
phenotype in the null mutant through genetic complementation validated the
attenuated phenotype.
Considering the attenuated phenotype along with the homology found
between PF13_0027 and the other well characterized MKPs, it can be suggested
that this putative atypical phosphatase fulfills a similar function in Plasmodium.
MKPs of similar structure are often involved in signaling pathways, which could
be a likely function of PF13_0027 [2, 263]. Investigations in yeast demonstrate
MKPs are critical components of various signal transduction pathways that
regulate transcription and maturation, which can also have an influence on the
cell cycle [145, 250]. Disruption of such functions in P. falciparum could produce
53
the phenotype observed in C9. This domain structure is not evident in any other
gene in the P. falciparum genome, but single copy orthologs are evident in all of
the other Plasmodium species with completed genomes, suggesting its function
is conserved among all malaria parasites. The presence of an RHD domain
upstream of the DUSP is consistent of a secondary regulatory function that aids
in substrate recognition and activity of MKPs [211, 219, 222]. Conservation of
this domain in PF13_0027 as determined by bioinformatics analysis implicates
MAPK-like function for this atypical phosphatase.
Identification of PF13_0027 as a putative protein phosphatase is likely due
to the partial conservation of the conserved CX5R signature motif. Each catalytic
residue is critical to optimal function of a phosphatase, and the modifications
suggest that this DUSP may not be highly catalytic or possibly a
pseudophosphatase. Non-catalytic pseudophosphatases typically maintain
structural homology with active phosphatases allowing them to trap
phosphoproteins, thereby regulating cellular functions without dephosphorylation
activity [253, 255]. One such DUSP-homology domain in pseudophosphatases
is referred to as a serine threonine tyrosine interacting (STYX) domain, which
has an endogenous substitution of one, or more, of the catalytic residues [255,
264, 265]. Reports of pseudophosphatase activity in Caenorhabditis elegans
demonstrate this function as an important role fulfilled by Egg-4 and Egg-5 in
controlling oocyte-to-zygote transition [266]. It has also been proposed that
physical access of native phosphatases is blocked by these
pseudophosphatases, which exert a “dominant negative” function, thereby
54
protecting substrates from dephosphorylation [253]. This is not a surprising
regulatory mechanism considering that the DUSP binding pockets generally lack
substrate specificity. Interestingly, the Apicomplexa possess a unique group of
pseudophosphatase with long N-terminal domains and EF-hand motifs termed
“EFPPs” [267]. However a grouping of the variety of STYX domain
pseudophosphatases, which have a substitution of the cysteine residue in the
CX5R motif, has never been characterized in Plasmodium. PF13_0027 does not
have the EF-hand motif or Ca2+ binding sites typically associated with the EFPP
grouping; however, it is missing the conserved arginine that would make it a
unique classification of putative protozoan pseudophosphatase.
The pressing need for new antimalarial drugs and identification of new
targets is critical due to emerging resistance to frontline drugs and the lack of
diverse chemotherapeutic targets [36, 268]. Furthermore, there have not been
any new classes of antimalarial drugs introduced into clinical practice since 1996
[94, 269, 270]. As a result, the preferred methods for use of antimalarial drugs
have been combination therapies due to the foreseeable challenges associated
with monotherapy or highly mutable drug targets [36, 271]. Kinases and other
regulators of phosphorylation pathways of malaria parasites represent potential
high value targets for future antimalarial drugs. However, the complex processes
of phosphorylation cascades in Plasmodium are poorly understood and limit our
ability to identify the highest value targets. Our discovery of PF13_0027 as an
important regulator of the cell cycle helps elucidate the trophozoite to schizont
transition stage as a potentially vulnerable step of the developmental cycle and
55
will help create new avenues into understanding Plasmodium biology. The
phenotype associated with C9 highlights this pathway and the regulated
processes as potential targets. With further delineation of the function of the
PF13_0027 and identification of its interacting partners, additional knowledge
arising from its study will aid future drug discovery.
56
Figure 2.1: Growth phenotype of C9 mutant parasite is due to disruption of PF13_0027. A schematic of PF13_0027 disrupted by a single insertion of the piggyBac transposon. Tandem RHD and DUSP domains are characteristic of MKPs.
57
Figure 2.2: Growth of mutant C9 parasites as a percent of NF54. Calculation of fold change reveals that the knockout of PF13_0027
resulted in a reduced fold change of 50% relative to NF54.
0
10
20
30
40
50
60
70
80
90
100
NF54 C9
% o
f N
F54
58
Figure 2.3: Morphologic analysis of the C9 mutant compared to NF54. Comparison of Giemsa-stained thin blood smears of the wild-type parent NF54 and C9 did not reveal an obvious difference in major developmental stages.
NF54
C9
ER LR ET LT ES LS
59
Figure 2.4: Comparison of merozoites produced per schizont in mutant and wild-type parasites. Average merozoite counts in segmented schizonts of NF54 and C9 were not statistically different.
0
5
10
15
20
NF54 C9
Me
rozo
ite
s/
sc
hiz
on
t
60
Figure 2.5: Multiple alignment and phylogenetic analysis of PF13_0027. (A) Alignment of PF13_0027 with its Plasmodium orthologs and outlier species showing the conservation of catalytic residues (boxes). Cysteine and aspartic acid align with all homologs. Isoleucine aligns with the position of the conserved arginine and is conserved in all species of Plasmodium. A string of residues (bracket) are inserted into the signature motif and is conserved among the Plasmodium orthologs. (B) Phylogenetic analysis using Neighbor-joining method with 1000 bootstrap shows grouping of the Plasmodium sequences independent of the other species suggesting an early divergence in evolutionary lineage.
61
Figure 2.6: Transcription profile of PF13_0027. Analysis by qRT-PCR showed that expression of PF13_0027 has its highest expression relative to actin during late trophozoite 32 hours post invasion. This stage corresponds to late pre-S development where the cell cycle of null mutants deviates from the wild-type development pattern.
0
0.5
1
1.5
2
2.5
8 16 24 32 40 48
Rela
tive E
xp
ressio
n
Time (hrs)
62
Figure 2.7: The cell cycle of C9 null MKP mutant is altered. Cell cycle analysis reveals a prolonged pre-S phase in mutant parasite lines. Late entry into the S/M phase leads to an overall longer cycle time producing a slow growing phenotype. The blue, red and green graph lines represent the relative abundance of rings, trophozoites and schizonts, respectively. The pre-S phase in NF54 is 26 hours followed by a 16 hour S/M and 2 hour cytokinesis (C). In C9 the cycle time is increased by 6 hours due to the longer 32-hour pre-S phase. Late entry into the S/M phase coincides with the timing for peak expression of PF13_0027 suggesting that the deficiency in the mutant cycle can be correlated to the gene expression pattern.
NF54
C9
63
Figure 2.8: Transfection plasmid used for complementation of C9. The plasmid construct used to complement the attenuated C9 parasite line was developed using the full-length PF13_0027 ORF inserted adjacent to a BSD drug selection cassette.
pL-BACII-HBH
PF13_0027
64
Figure 2.9: RT-PCR analysis of complemented parasites. Results detected PF13_0027 transcripts in NF54 and the complemented parasites (E3, E8), but not in C9 null MKP mutant (red arrow). As a positive control, 18S RNA was also included for each sample (blue arrow).
65
Figure 2.10: Genetic complementation of C9 mutant rescues wild-type growth. The growth of the complemented parasites (E3, E8) were graphed as a percent of NF54. Each of the complemented parasite lines were statistically the same as wild type NF54 demonstrating successful rescue of the phenotype.
0
20
40
60
80
100
120
NF54 C9 E3 E8
% o
f N
F54
Growth
66
Figure 2.11: Genetic complementation of C9 mutant rescues wild-type invasion. The invasion phenotype of the complemented parasites (E3, E8) were graphed as a percent of NF54. Each of the complemented parasite lines were statistically the same as wild type NF54 demonstrating successful rescue of the phenotype.
0
20
40
60
80
100
120
NF54 C9 E3 E8
% o
f N
F5
4
Invasion
67
Figure 2.12: Growth phenotype is not rescued when conserved residues are mutated. Mutant constructs of the complementation plasmid were developed. Two single mutants (C383A and D345A respectively) and one double mutant (K388A, K394A) were used to complement C9. The phenotype was not rescued with the C383A and D345A constructs, however restoration of the wild type phenotype was observed in the K388A/ K394A construct suggesting these residues are not as critical to the function of PF13_0027.
0
20
40
60
80
100
120
% o
f N
F5
4
Growth
68
Figure 2.13: Invasion phenotype is not rescued when conserved residues are mutated. The invasion phenotype of the complemented parasites with mutations was compared to NF54. The results were consistent with the growth assays showing phenotypic rescue in only the double lysine mutant.
0
20
40
60
80
100
120
Fo
ld C
ha
ng
e (
%)
Invasion
69
Chapter 3: Identification of Novel Inhibitory Compounds and Evaluations
of Plasmodium falciparum Susceptibility (Specific Aim 2)
Rationale for Study
As the causative agent of malaria, P. falciparum poses a great threat to
global health, and the need for effective drugs is a high priority. Currently,
emerging resistance to several of the most common antimalarials has intensified
the need for drugs with novel mechanisms [20, 36, 38, 40]. A potential new
target is PF13_0027 that was identified through the course of a large-scale
random insertional mutagenesis project. In this current study we were able to
build on this discovery to identify molecular inhibitors from the European
Molecular Biology Laboratories database of bioactive drug-like small molecules
for Neglected Tropical Disease (ChEMBL-NTD) through high-throughput in silico
screening (HTS) with a homology model of the DUSP domain. The homology
model of the tertiary structure of PF13_0027 was generated using the resolved
crystal structure of the human phosphatase MKP3 as a template. The binding
pocket was identified readily by a sequence similarity search and multiple
sequence alignments. Then the pocket was cross-referenced with results from
the computed atlas of surface topography of proteins (CASTp) server for
validation. In silico high-throughput docking screens identified seven antimalarial
compounds from ChEMBL-NTD which were selected for functional analysis. The
70
compounds were evaluated against P. falciparum NF54 for their ability to alter
the parasite’s cell cycle in a fashion similar to the attenuated C9 mutant. At least
one of the compounds extended the normal cell cycle length producing a
phenotype similar to the C9 attenuated mutant. Through this study we
demonstrate the potential for the phenotype of a pB mutant to guide identification
of novel target-specific antimalarials.
Introduction
Malaria primarily affects tropical and subtropical regions worldwide, putting
approximately 40% of the global population at risk [272]. In these endemic
regions, pregnant women and children are at the greatest risk and emergence of
antimalarial drug resistance has intensified efforts in search of new
chemotherapeutic agents [38, 59]. Currently the most effective drug for dealing
with malaria infections is artemisinin. In an effort to decrease further
development of multi-drug resistance, ACTs are the recommended first line
therapy endorsed by the WHO [36, 40, 273]. This strategy has been highly
successful in treating cases of uncomplicated malaria for several years, however
recent emergence of artemisinin resistance has become a concern [36]. Use of
artemisinin derivatives as monotherapies or sub-therapeutic dosages has
contributed to the increase of insensitive Plasmodium strains [36]. With this
spread of antimalarial resistance, identification of new antimalarials is a priority.
Widespread use of computational HTS has allowed large sets of compounds to
be investigated for biological activity [269]. Using this method, suitable lead
71
candidates can be identified from large data sets improving productivity and
lowering cost to a level more favorable than in vitro screening methods,
enhancing structure-based drug design [274, 275].
Previously we identified that PF13_0027 is homologous with the PTP
superfamily, and investigated the structural characteristics further to determine
that it is most closely related to MKPs. Closer investigation of the signature motif
revealed a native substitution (R398I), which is conserved throughout
Plasmodium spp. This single substitution in the conserved MKP homology
domain is non-consensus defining PF13_0027 is an atypical MAPK
phosphatase. We hypothesize that PF13_0027 helps regulate the MAPK
pathway and may therefore be a candidate for drug design.
The MKPs in other eukaryotes are critical for intracellular signaling in
response to numerous types of stimuli [193, 224, 276]. In Plasmodium,
processes utilizing these proteins may be vital as an intracellular response to
various external stimuli to regulate stage specific gene expression. Due to the
ubiquitous nature of this pathway in eukaryotes, MAPK signaling has been
studied extensively in model organisms such as yeast [145]. In a broader
context, increased understanding of this pathway has also contributed
significantly to cancer research [263, 277, 278].
In malaria research, MAPK pathways were determined to be critical
components of sexual proliferation and for the sexual stages [143, 151, 211, 217-
223]. As a potential component of the MAPK signaling pathway, the PF13_0027
MKP provides an avenue for future drug discovery and warrants further research.
72
Conservation of PF13_0027 orthologs in Plasmodium suggests functional
significance in parasite blood-stage development as well. Using the DUSP
domain of PF13_0027, a homology model was generated and used to screen
ChEMBL-NTD. This study utilized the distinct structural features of PF13_0027
to select an effective drug against this P. falciparum target.
Materials and Methods
Identification of Conserved Domains and Evolutionary Lineage
The sequence of PF13_0027 was retrieved from a public database [279].
The physicochemical parameters of the amino acid sequence were determined
using ProtParam [280]. The conserved domains were identified using the
Conserved Domains Database (CDD) [281], Conserved Domain Architecture
Retrieval Tool (CDART) [282], InterProScan [283, 284], Prosite [285, 286],
Superfamily [287], and the Simple Modular Architecture Research Tool (SMART)
[288, 289]. Ortholog searches were done using NCBI protein BLAST (BLASTP)
with the full deduced amino acid sequence and individual domains. Multiple
sequence alignments were constructed from the retrieved sequences with the
lowest E-values to identify conserved regions. The phylogenetic tree was
inferred using the Neighbor-joining method computing the evolutionary distance
using the Poisson correlation method with the Molecular Evolutionary Genetics
Analysis software (MEGA5) [247, 290, 291].
73
Evaluation of Secondary Structure and Post-translational Modifications
The secondary structure of PF13_0027 was evaluated using JPRED [292]
and PSIPRED [293, 294]. Phosphorylation sites were assessed using NetPhos
2.0 [295] which identifies serine, threonine and tyrosine phosphorylation sites,
and NetPhosK 1.0 [296] to identify kinase binding sites. Prediction of a signal
peptide and cleavage site was searched using Signal IP 3.0 [297]. Mitochondrial
and plastid targeting sequences were searched using the prediction servers
Predotar [272] and PATS [298-300]. N-terminal myristoylation was investigated
using the Myristoylator from ExPASy [301].
Molecular Modeling and Structure Validation
The three-dimensional structure of PF13_0027 has not been resolved so a
homology model was built using the automated protein structure homology
modeling server Swiss-Model. Suitable templates for modeling were identified
using PSI-BLAST in the Swiss-Model repository [205, 302-304]. The crystal
structure of MKP3 (NCBI Accession No. 1MKP_A) was the most suitable of the
available templates with greatest sequence coverage and similarity [305]. The
remaining residues of PF13_0027 not showing any significant similarity were not
included for homology modeling. The model was assessed using the atomic
empirical mean force potential with Atomic Non-Local Environment Assessment
(ANOLEA), empirical force field with Gröningen Molecular Simulation program
(GROMOS), and QMEAN6 [303, 306]. The stereochemistry was assessed with
a minimum resolution of 2.5 Å using PROCHECK [307]. The final structure was
74
also compared to the predicted secondary structure represented using DSSP
and PROMOTIF [308]. ERRAT plots were generated to check structure quality
of the template and homology structure using a nine residue sliding window
[309]. This process was repeated in an iterative fashion until all the residues in
the plot were not below 95% as done previously [310]. The quality of the final
structure was also verified using Verify 3D, Procheck and Ramachandran plots
[307, 311, 312].
Identification of the Binding Pocket
The binding pocket was identified using Pocket Finder and Q-site finder
which uses the Ligsite algorithm [76, 313, 314]. The output of the predicted
binding pocket was also compared to the Computed Atlas of Surface Topography
of proteins (CASTp), which uses the alpha shape theory pocket algorithm [315-
317]. The identified pocket was also validated through comparison to the
structural alignment of the resolved homology model and template (MKP3).
Selection of the Compound Dataset and High-throughput in silico Docking
All docking and scoring calculations were performed using the 2012
Schrödinger Suite. The compound library was retrieved from ChEMBL-NTD
(ftp://ftp.ebi.ac.uk/pub/databases/chembl/ChEMBLNTD/) and prepared using
LigPrep (LigPrep v2.5, Schrödinger LLC). The homology model, made from
sequence PF13_0027, was minimized using the OPLS2005 [318] force-field
algorithm and the grid files were generated in GLIDE (Glide v5.8, Schrödinger
LLC) [319-321]. The modeled structure was used to identify small molecular
75
inhibitors with affinity to the predicted active site through in silico docking
experiments using extra precision (XP) mode [321] on a Dell Precision 490
workstation with an Intel Xeon dual quad-core processors running Ubuntu
10.04. From the results obtained, the molecule structures with the highest
predicted affinity; lowest docking scores within GLIDE's error of 2 kcal/mol to the
active site were selected. From this subset, the commercially available
compounds were identified and used for in vitro culture assays.
In vitro parasite culture conditions
P. falciparum NF54 and C9 were cultured according to standard methods
at 37°C (5% O2 and 5% CO2, nitrogen balanced) in 5% hematocrit (O+ blood)
and RPMI 1640 medium with 0.5% Albumax II, 0.25% sodium bicarbonate and
0.01 mg/ml gentamicin [241].
In vitro Drug Susceptibility Assay Using SYBR Green I
Synchronized cultures were seeded into 96-well plates at 0.5%
parasitemia and cultured for 96 hours under the previously stated conditions.
Plates were then frozen overnight at -80C. Plates were thawed for 15 minutes
then mixed by pipetting. Eighty microliters of each well were transferred to a new
96-well plate followed by 100 µL of SYBR Green I (Sigma Aldrich) in lysis buffer
(0.2 µL of SYBR Green I/mL 2X lysis buffer). Plates were covered and incubated
in the dark for 1 hour at room temperature. Fluorescence intensity was
measured with a SpectraMax M2e microplate fluorescence reader (Molecular
Devices) with excitation and emission wavelengths of 485 nm and 525 nm
76
respectively. The sample values were expressed in relative fluorescence units
(RFU). EC50 values were obtained by normalizing the values using control wells
of samples cultured without drug and plotted using a one-phase exponential dose
response curve using GraphPad Prism 6 (GraphPad Software Inc.).
Growth Assay and Cell Cycle Determination
Parasite cultures were maintained in 96-well plates for 96 hours. Parasite
cultures were plated in triplicate with sample collection at six hour intervals.
Relative fold change of parasite growth in treated cultures was estimated by
using ethidium bromide intensity as an index for parasite growth and quantified
using flow cytometry (Appendix C). Parasite cultures were fixed in 0.05%
glutaraldehyde after removal of culture medium and permeabilized with 0.3%
Triton-X 100. Cultures were then treated with 0.5 mg/mL RNAse A then stained
with 0.1mg/mL ethidium bromide. Each sample was then quantified using a C6
Flow Cytometer H System™ (BD Accuri™). The data was analyzed using CFlow
Plus software (BD Accuri™) and GraphPad Prism 6 (GraphPad Software Inc.)
(Summary of statistical analysis in Appendix G).
Results
Evaluation of the Physical Properties of PF13_0027
Initial evaluation of PF13_0027 included an investigation of potential
binding sites, phosphorylation sites, and post translational modifications. These
methods provided additional insight into potential interactions and function of this
putative phosphatase through the use of publicly available bioinformatics tools.
77
Through the use of these tools, it was found that there were not any significant
phosphorylation sites or post translational modifications. PF13_0027 also did not
show presence of a signal peptide. The predicted secondary structure was used
to validate the homology model and supported the final structure.
Molecular Structure of PF13_0027
A crystallographic structure of PF13_0027 has not yet been resolved by
experimental methods and, neither is there a homologous protozoa protein that
could be used for a template. The closest template available in the Swiss Model
repository was the human phosphatase MKP3 with 21% similarity (Table 3.1 and
Figure 3.1). Analysis and validation of the structure using the WHAT-IF web
interface (version 8) revealed that the structure was in agreement with standard
structural conditions. Analysis of the Ramachandran plot gave a Z-score of -
2.972 that was within the expected ranges for well-refined structures with 89.2%
of the amino acid residues in favored regions (Figure 3.2). All bonds were in
agreement with standard bond lengths with a RMS Z-score of 0.669 and RMS
deviation of 0.015. The overall quality of the model predicted by ERRAT was
69.375 compared to 88.235 of the template which was favorable considering the
numerous INDELs in primary sequence (Figure 3.3). Additionally, the RMSD
score from DaliLite of Cα trace between 141 aligned residues of 1MKP and the
homology model of PF13_0027 DUSP was 0.5 Å with a Z-score of 26.7 and 21%
sequence identity (Figure 3.4). The combined results from these various
78
analyses suggest that the homology model of PF13_0027 DUSP is reasonable
and reliable.
Active Site Prediction
The predicted binding pockets for PF13_0027 were identified and
validated using Qsite-Finder, Pocket Finder and CASTp. A total of 10 binding
pockets were found and compared to the active site of the template protein
(Figure 3.5). The analysis revealed that the identified pocket in the region of the
signature motif was highly conserved with the template active site as predicted
through multiple sequence alignments. Sequence identity of the catalytic site
was greater (78%) between the template and homology model than in any other
region. This comparison also suggests functional conservation between the
template and model. The residues within the binding pocket include the
signature motif residues of previously characterized active phosphatases. For
example, the residues C383, D345 and I398 of PF13_0027 align with the
conserved C293, D262 and R299 of MKP3. Conservation of the predicted site
in the homology model and the validated site in the template suggests that the
selected pocket was the most favorable for HTS (Figure 3.6).
Ligand Selection and Drug Susceptibility Assay
From the ChEMBL-NTD dataset, 10001 compounds were docked and
ranked according to glide score. These molecular inhibitors were previously
screened with P. falciparum 3D7 and have a minimum inhibitory potential of 80%
validated using LDH activity assays as an index of growth (GSK TCAMS
79
Dataset) or erythrocyte-based proliferation assays (Novartis-GNF Malaria Box
Dataset) [269, 322]. The bioactive drug-like small molecules in the database all
adhere to the Lipinski rule-of-five and provide abstracted bioactivities [323].
Cytotoxicity against human hepatocytoma HepG2 cells was observed in 1982 of
the compounds tested at 10 µM [269]. These compounds were evaluated on the
basis of their quality of interaction represented by the GLIDE calculation.
Commercially available compounds were identified from the results and 7
compounds (Table 3.2) were obtained (390097; 7,8-Dihydroxy-2H-chromen-2-
one: 524725; 1-(4-Chlorophenyl)-5-oxo-3-pyrrolidinecarboxylic acid: 533073; 2-
((N-[(4-Fluorophenyl)(2-thienyl)methyl]glycyl)amino)-3-thiophenecarboxamide:
533730; 2-([N-(Diphenylmethyl)glycyl]amino)-3-thiophenecarboxamide: 525841;
3-[(E)-(1H-Benzimidazol-2-ylhydrazono)methyl]-2-chloro-7-methoxyquinoline:
579624; 2-[(2E)-2-(1,3-Benzodioxol-5-ylmethylene)hydrazino]-1H-benzimidazole:
585222; 2-[(2E)-2-(3,4-Dimethoxybenzylidene)hydrazino]-1H-benzimidazole).
Poses of each compound were resolved to show the interaction of each molecule
with the active site residues (Figures 3.7-13).
Growth assays were performed with NF54 parasites while under drug
pressure with the selected compounds in order to evaluate and contrast the drug
attenuated phenotype to the insertional mutant phenotype observed with C9
(Figure 3.14-20). Since each of the selected compounds had published activity
against P. falciparum, it was expected that the parasites would be attenuated
over the course of the assay [269, 322]. The attenuated phenotype of C9, as
stated previously, presents a prolonged pre-S phase contributing to late entry
80
into S/M development and a fold decrease in parasitemia 50% of the wild type.
As a starting point, the EC50 drug concentration of each of the selected inhibitory
compounds was used to challenge the parasites over the course of the
experiment. To assess the cell cycle phenotype, the relative abundance of each
developmental stage was quantified at each time point and plotted using a six-
order polynomial standard curve in order to visualize the function of the
developmental cycle. Overlaying the developmental stage growth curves for
each parasite culture enabled us to determine the course and timing of the cell
cycle. The initial published dose response assessment of each compound was
determined over the course of a 72 hour assay [269]. However, to develop a
clearer picture of the effect on the cell cycle, it was necessary to observe the
course of parasite development over two consecutive cycles. Therefore,
parasites in this study were cultured and monitored under drug pressure for 96
hours. Due to the extended length of the assay, it was possible to observe
attenuation greater than 50% in most treated cultures. Each compound
successfully attenuated pre-S development greater than 30 hours as in the C9
mutant extending the overall cycle time beyond that of the wild-type (Table 3.3).
Using Kruskal-Wallis multiple comparison with the Dunn’s post-test, we
determined that pre-S attenuation was similar to that of the C9 mutant (=0.05).
In addition to the pre-S phase, the overall timing of the development cycle was
compared and tested using the same statistical test (=0.05) (Table 3.4). Both
cycle time and pre-S phase timing are summarized in figure 3.20. Each drug
treated culture also showed a strong similarity to C9 in cycle timing.
81
To evaluate the effects of each of the inhibitors on cultures without the
proposed target, we set up dose-response assays with both NF54 and C9 to
compare the efficacy of the compounds in both parasite lines. In vitro drug
susceptibility assays also revealed that C9 had reduced susceptibility to 533073,
533730 and 579624 when compared with NF54 (Table 3.5).
Discussion
As an important regulatory process, phosphorylation cascades exert a
critical influence on cellular development through signal transduction, and as a
result, have been investigated extensively to elucidate methods of
chemotherapeutic intervention [224, 324-328]. These pathways have not yet
been fully characterized in Plasmodium, and as a result, there have not been any
classes of drugs developed to target phosphorylation-dependent signal
transduction cascades. Through preliminary characterizations of PF13_0027 in
the first part of this study, it was suggested that the pathway utilizing this putative
phosphatase may fulfill an important role in parasite development. Building on
these findings with homology modeling and in silico high-throughput screening,
our results support the idea that PF13_0027 might be suitable drug target. In the
past, computational methods have been successful in identifying potential
inhibitory compounds using critical components of the developmental cycle [329].
PF13_0027 presented as a critical component suitable for investigation through
computational methods. In P. falciparum, PF13_0027 is the only putative
phosphatase of its kind, and it is well conserved with single copy orthologs
82
present in P. berghei (PBANKA_140400), P. c. chabaudi (PCHAS_140590), P.
knowlesi (PKH_140400), P. vivax (PVX_12110), P. cynomolgi (PCYB_141500),
and P. y. yoelii (PYYM_1407600). Additionally, PF13_0027 has low homology
with the closest mammalian orthologs. These characteristics are often indicators
of favorable drug targets since unique and conserved genes are typically under
negative selection making their products essential, and low homology to
mammalian genes limits adverse effects when targeted with drugs [10, 329].
Conservation among the Plasmodium parasites, especially P. falciparum
and P. vivax provides the ability to target both of these parasites, which would be
a great benefit to antimalarial drug discovery [31, 64]. The conserved binding
pocket of the homology modeled DUSP domain maintained the necessary
features for activity and demonstrated an ability to accommodate an inhibitor in
high-throughput in silico screening. Considering the structural characteristics, it
is likely that PF13_0027 interacts with the MAPK signaling pathway which is
critical and indispensable to eukaryote development, and it would be a major
contribution to the antimalarial research effort [143, 145, 157, 167, 201, 219, 330,
331].
The ChEMBL-NTD contains thousands of compounds with validated
antimalarial activity [269, 332, 333]. In light of their inhibitory actions, the targets
and mechanisms of action for most of them in P. falciparum are yet to be
determined. Hypothetical malarial modes of action were developed for a few
compounds to help facilitate their application against Plasmodium through
historical GSK data regarding biochemical activity with human and microbial
83
targets [269]. In those previous studies, possible targets were inferred.
Unfortunately, none of the hypothetical targets were associated with the
compounds identified through the in silico screen in this current study. This
computational study, however, enabled us to sort through the database to isolate
the likely inhibitory compounds and allow us to correlate the proposed binding
interaction to a phenotype of an attenuated parasite line.
In order to determine if our protein of interest was being targeted by the
identified inhibitory compounds, it was necessary to follow up the preliminary
characterization of PF13_0027 with in silico methods of analysis using the
homology modeled DUSP. Replicating the phenotype observed in C9 with
selected inhibitors would suggest a potential mechanism of action. Additionally,
comparing the susceptibility of NF54 and C9 would give further insight into the
potential targeting of PF13_0027. This method would help facilitate lead
identification and optimization progressing toward structure-based drug
discovery. Though further studies would be required to determine the precise
inhibitory mechanism, this study demonstrates a novel translational method
bridging the gap between insertional mutagenesis and drug discovery. In vitro
attenuated growth analysis led to replication of the attenuated mutant phenotype
with the identified compounds and revealed that 524725, 533073 and 533730
have lowered efficacy against C9 parasites compared to the wild-type, validating
this approach and enabling us to postulate a therapeutic target of this inhibitor.
In addition to replicating the phenotype, observation of increased NF54
susceptibility compared to C9 makes it possible to suggest PF13_0027 may be
84
involved in the drug response phenotype in the assay. One of the main
challenges to post genomic biology is translating a pathogen’s genome to new
drugs and combinatorial methods with HTS allow us to identify suitable lead
candidates from a chemical database contributing to drug development efforts
and accelerating structure based design. In practice, experimentally determined
structures are preferred for in silico studies; however the number of
pharmaceutical targets of interest has far outpaced the ability to experimentally
develop protein structures [274]. As a result, homology modeling has become
the popular method of investigation for the growing number of interesting
pharmaceutical targets. Comparisons of docking results from both homology
models and experimentally validated structures have also produced comparable
results [181, 334]. Utilization of multiple strategies is necessary in order to
advance the base of knowledge in this field. In this dissertation research study,
we proposed a method to translate the information obtained from random
insertional mutagenesis into a drug discovery strategy using in silico methods.
The technique employs both experimental and computational methods to identify
drug compounds, which would be vital to the search for new antimalarials.
85
Table 3.1: Templates identified for homology modeling.
Chain Z rmsd lali nres %id Description
1mkp-A 26.7 0.5 141 144 21 MKP3/ PYST1
3lj8-A 20.5 1.8 140 146 24 Tyrosine-protein Phosphatase
2hxp-A 18.1 2.1 136 144 21 DUSP 9
1zzw-B 17.9 2.1 134 147 21 DUSP10
86
Figure 3.1: Alignment of potential homology modeling templates. Potential templates were identified in Swiss Model and aligned in JalView. The regions of greatest identity are marked with green. Below, the alignment of the sequences shows the conserved regions in both the primary and secondary structure.
87
Figure 3.2: The quality of the model was validated by Ramachandran plot. The homology model was found to have 89.2% of the residues in favorable positions.
88
Figure 3.3: Overall quality assessment of the model evaluated using ERRAT. The comparison of the template crystal structure and homology model. (A) PF13_0027 (B) MKP3
89
Figure 3.4: Homology model of PF13_0027. The structure of PF13_0027 (brown) was developed using the crystal structure of MKP3 as a template (PDB 1MKP). Alignment of the model with MKP3 (green) revealed that the final structure showed the catalytic residues aligned to the proposed positions in the active site. The presence of the signature motif insertion does not affect the shape of the active site and forms an alpha-helix adjacent to the binding pocket without obstructing the site.
D345
C383
I398
90
Table 3.2: Selected ChEMBL-NTD compounds used for in vitro screening.
91
Figure 3.5: Identified binding site of PF13_0027 in the DUSP domain. The binding pocket (red mesh) aligned with the predicted active site identified by homology alignments with the template and other phosphatases. The presence of the signature motif insertion does not obstruct the binding site.
92
Figure 3.6: Vacuum electrostatics of the PF13_0027 DUSP homology model. The blue signifies slight positive charge while the red shows the negatively charged regions of the domain. The slight positive charge of the binding pocket (circled) shows that it would be favorable for binding of a negatively charged phosphate group. The predicted surface charges of the homology model help validate the quality of the structure for docking.
93
Figure 3.7: Orientation of 390097 in the binding pocket with LIGPLOT.
390097
C383
I398
D345
N389
Q343
390097
94
Figure 3.8: Orientation of 524725 in the binding pocket with LIGPLOT.
524725
C383
D345
I398 Q343
52472
5
95
Figure 3.9: Orientation of 533073 in the binding pocket with LIGPLOT.
533073
C383 D345
K394
I398
Q343533073
96
Figure 3.10: Orientation of 533730 in the binding pocket with LIGPLOT.
533730
C383 D345
N389K394
I398
Q343533730
97
Figure 3.11: Orientation of 525841 in the binding pocket with LIGPLOT.
525841
N389
C383
D345
I398
525841
98
Figure 3.12: Orientation of 579624 in the binding pocket with LIGPLOT.
579624
N389
D345
C383
I398
579624
99
Figure 3.13: Orientation of 585222 in the binding pocket with LIGPLOT.
585222
N389
D345
C383
I398
585222
100
Figure 3.14: NF54 parasites challenged with compound 390097. The plots represent the asexual growth cycle of parasites treated with compound 390097 and the comparison to untreated NF54 and C9. The cycle was determined by overlaying the relative abundance of rings (blue), trophozoites (red) and schizonts (green).
101
Figure 3.15: NF54 parasites were challenged with compound 524725. The plots represent the asexual growth cycle of parasites treated with compound 524725 and the comparison to untreated NF54 and C9. The cycle was determined by overlaying the relative abundance of rings (blue), trophozoites (red) and schizonts (green).
102
Figure 3.16: NF54 parasites were challenged with compound 533073. The plots represent the asexual growth cycle of parasites treated with compound 533073 and the comparison to untreated NF54 and C9. The cycle was determined by overlaying the relative abundance of rings (blue), trophozoites (red) and schizonts (green).
103
Figure 3.17: NF54 parasites were challenged with compound 533730. The plots represent the asexual growth cycle of parasites treated with compound 533730 and the comparison to untreated NF54 and C9. The cycle was determined by overlaying the relative abundance of rings (blue), trophozoites (red) and schizonts (green).
104
Figure 3.18: NF54 parasites were challenged with compound 525841. The plots represent the asexual growth cycle of parasites treated with compound 525841 and the comparison to untreated NF54 and C9. The cycle was determined by overlaying the relative abundance of rings (blue), trophozoites (red) and schizonts (green).
105
Figure 3.19: NF54 parasites were challenged with compound 579624. The plots represent the asexual growth cycle of parasites treated with compound 579624 and the comparison to untreated NF54 and C9. The cycle was determined by overlaying the relative abundance of rings (blue), trophozoites (red) and schizonts (green).
106
Figure 3.20: NF54 parasites were challenged with compound 585222. The plots represent the asexual growth cycle of parasites treated with compound 585222 and the comparison to untreated NF54 and C9. The cycle was determined by overlaying the relative abundance of rings (blue), trophozoites (red) and schizonts (green).
107
Table 3.3: Comparison of average pre-S phase times in treated cultures to NF54 and C9. P-values represent comparison of each sample to C9.
Sample Pre-S (hrs) p-value
390097 34.332.08 0.676
524725 34.674.16 0.734
533073 35.335.77 0.676
533730 30.671.15 0.071
525841 34.676.49 0.480
579624 35.001.73 0.979
585222 34.002.00 0.638
NF54 26.671.15 0.008
C9 36.004.00 -
108
Table 3.4: Comparison of average cycle times in treated cultures to NF54 and C9. P-values represent comparison of each sample to C9.
Sample Cycle (hrs) p-value
390097 58.672.30 0.338
524725 49.331.15 0.147
533073 55.001.00 0.959
533730 53.333.05 0.698
525841 58.003.46 0.484
579624 47.335.03 0.097
585222 59.337.02 0.484
NF54 47.331.15 0.052
C9 56.331.52 -
109
Figure 3.21: Comparison of cycle times and pre-S phase for treated and untreated cultures. The blue bars represent the pre-S phase of development. Attenuated development is represented by a longer pre-S phase compared to NF54. Each treated culture and C9 show an extended pre-S phase. The total cycle time is represented by the red bars in each sample.
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
Ho
urs
Pre-S
Cycle
110
Table 3.5: Comparison of NF54 and C9 susceptibility to the selected compounds. Compounds 5333073, 533730 and 579624 also show NF54 to have significantly greater susceptibility.
Compound ID EC50 NF54 (nM) EC50 C9 (nM) P-value
390097 340.253.6 248.933.7 0.1124
524725 503.136.5 248.462 0.0240
533073 471.937.4 1227.386.3 0.0013
533730 196.3113.8 510.951.6 0.0073
525841 186.133.8 224.748.1 0.5467
579624 13633.1 58439.86 0.0010
585222 378.897.7 339.328 0.7070
111
Chapter 4: Conclusions and Future Directions
The studies described in this dissertation provide novel insights into a
putative phosphatase of P. falciparum. The presented data not only provides an
understanding of basic parasite biology, but provides a foundation for new
methods to reduce malaria transmission. Similar to the strategies utilized to
target signaling pathways in cancer research, the manipulation of signaling
pathways in Plasmodium can potentially support the development of
antimalarials capable of attenuating parasite development. The major findings of
this dissertation are that PF13_0027 is critical for development of the P.
falciparum trophozoite, and can be targeted for drug discovery. My results also
demonstrate that molecular modeling and in silico HTS can be used to translate
discoveries of critical genes identified through transposon-mediated random
insertional mutagenesis with pB into discoveries of new classes of antimalarial
drugs.
The first part of this dissertation focuses on an attenuated parasite line
(C9) that has an insertional knockout of PF13_0027 and investigates the
phenotype to determine the functional role. My data demonstrates that this gene
is important for pre-S development of the asexual trophozoite and that insertional
knockout attenuates blood stage cycling by delaying entry into the S/M schizont.
112
Through complementation using the ORF with the 700 bp 5’ UTR promoter
region, the WT phenotype can be rescued, validating the critical nature of
PF13_0027 to development. Structural assessment of the domain architecture
and conserved regions reveals that not only does PF13_0027 have MKP
homology, but that it is missing a conserved residue in the signature motif, a
characteristic that is also common in STYX domains. The unique characteristics
in this putative phosphatase support that it could be a pseudophosphatase that
modulates the activity of the MAPK pathway through a dominant-negative
approach, or a low activity phosphatase.
The second part of this dissertation investigates the potential of targeting
PF13_0027 for drug discovery through in silico HTS using a homology model of
the DUSP domain. Using this procedure we attempted to chemically reproduce
the attenuated phenotype observed in C9 using molecular inhibitors from
ChEMBL-NTD. This procedure utilized the combined data sets of GSK-TCAMS,
Novartis-GNF malaria box and St. Jude Children’s Research hospital datasets.
Assessment of the attenuated phenotype of parasites treated with each of the
inhibitors reveals that the cell cycle of NF54 parasites is attenuated similar to that
of C9 when treated with the selected compounds suggesting that an inhibitor
identified through in silico methods could be used to replicate the attenuated
phenotype in vitro. This conclusion is important since this experiment, as a
proof-of-concept, shows that in silico methods may be used to rapidly sort and
translate the large library of data developed through large-scale insertion
mutagenesis into advanced insights in drug discovery. It also allows us to sort
113
through large databases of small molecules to find valuable structures for future
structure-based drug design.
Taken together, the data presented in this dissertation show that
PF13_0027 is a very important component in asexual development and
proliferation. It also provides new insight into the importance of MAPK signaling
cascades to Plasmodium development. Future studies will focus on identification
of downstream signaling pathways and potential substrates. Additionally in depth
proteomic studies involving structural characterization, recombinant expression,
and functional in vitro assays will be able to generate a more specific
understanding of interacting substrates and the effects of potential inhibitors.
Current advancements in molecular and computational biology techniques
enable accurate representations of molecular interactions between potential drug
targets and molecular inhibitors. This study demonstrates that these methods
will augment current efforts expanding our knowledge of parasite biology and
help identify new drugs.
114
References
1. Barford D: Molecular mechanisms of the protein serine/threonine phosphatases. Trends in biochemical sciences 1996, 21:407-412.
2. Wilkes JM, Doerig C: The Protein-Phosphatome of the human malaria parasite Plasmodium falciparum. BMC genomics 2008, 9:412.
3. Zhang K, Rathod PK: Divergent regulation of dihydrofolate reductase between malaria parasite and human host. Science 2002, 296(5567):545-547.
4. Project MA: A Global Map of Dominant Malaria Vector Species.
5. Malaria Life Cycle [http://www.dpd.cdc.gov/dpdx/images/ParasiteImages/M-R/Malaria/malaria_LifeCycle.gif]
6. Project MA: The spatial distribution of P. vivax malaria endemicity in 2010. 2013.
7. The spatial distribution of P. falciparum malaria endemicity in 2010 [http://www.map.ox.ac.uk/]
8. Chen F, Mackey AJ, Stoeckert CJ, Jr., Roos DS: OrthoMCL-DB: querying a comprehensive multi-species collection of ortholog groups. Nucleic Acids Res 2006, 34(Database issue):D363-368.
9. Balu B, Shoue DA, Fraser MJ, Jr., Adams JH: High-efficiency transformation of Plasmodium falciparum by the lepidopteran transposable element piggyBac. Proceedings of the National Academy of Sciences of the United States of America 2005, 102(45):16391-16396.
115
10. Gardner MJ, Hall N, Fung E, White O, Berriman M, Hyman RW, Carlton JM, Pain A, Nelson KE, Bowman S et al: Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 2002, 419(6906):498-511.
11. Hoffman SL, Subramanian GM, Collins FH, Venter JC: Plasmodium, human and Anopheles genomics and malaria. Nature 2002, 415(6872):702-709.
12. World Malaria Report 2010 [http://www.who.int/malaria/world_malaria_report_2010/worldmalariareport2010.pdf]
13. Lindner SE, Miller JL, Kappe SH: Malaria parasite pre-erythrocytic infection: preparation meets opportunity. Cell Microbiol 2012, 14(3):316-324.
14. Sturm A, Amino R, van de Sand C, Regen T, Retzlaff S, Rennenberg A, Krueger A, Pollok JM, Menard R, Heussler VT: Manipulation of host hepatocytes by the malaria parasite for delivery into liver sinusoids. Science 2006, 313(5791):1287-1290.
15. Healer J, Crawford S, Ralph S, McFadden G, Cowman AF: Independent translocation of two micronemal proteins in developing Plasmodium falciparum merozoites. Infection and immunity 2002, 70(10):5751-5758.
16. Tahar R, Boudin C, Thiery I, Bourgouin C: Immune response of Anopheles gambiae to the early sporogonic stages of the human malaria parasite Plasmodium falciparum. The EMBO journal 2002, 21(24):6673-6680.
17. Li J, Gutell RR, Damberger SH, Wirtz RA, Kissinger JC, Rogers MJ, Sattabongkot J, McCutchan TF: Regulation and trafficking of three distinct 18 S ribosomal RNAs during development of the malaria parasite. Journal of molecular biology 1997, 269(2):203-213.
18. Rooney AP: Mechanisms underlying the evolution and maintenance of functionally heterogeneous 18S rRNA genes in Apicomplexans. Mol Biol Evol 2004, 21(9):1704-1711.
116
19. Singh N, Preiser P, Renia L, Balu B, Barnwell J, Blair P, Jarra W, Voza T, Landau I, Adams JH: Conservation and developmental control of alternative splicing in maebl among malaria parasites. Journal of molecular biology 2004, 343(3):589-599.
20. Ganesan K, Ponmee N, Jiang L, Fowble JW, White J, Kamchonwongpaisan S, Yuthavong Y, Wilairat P, Rathod PK: A genetically hard-wired metabolic transcriptome in Plasmodium falciparum fails to mount protective responses to lethal antifolates. PLoS pathogens 2008, 4(11):e1000214.
21. Michon P, Stevens JR, Kaneko O, Adams JH: Evolutionary relationships of conserved cysteine-rich motifs in adhesive molecules of malaria parasites. Mol Biol Evol 2002, 19(7):1128-1142.
22. Hartl DL, Volkman SK, Nielsen KM, Barry AE, Day KP, Wirth DF, Winzeler EA: The paradoxical population genetics of Plasmodium falciparum. Trends Parasitol 2002, 18(6):266-272.
23. Poinar G, Jr.: Plasmodium dominicana n. sp. (Plasmodiidae: Haemospororida) from Tertiary Dominican amber. Systematic parasitology 2005, 61(1):47-52.
24. Joy DA, Feng X, Mu J, Furuya T, Chotivanich K, Krettli AU, Ho M, Wang A, White NJ, Suh E et al: Early origin and recent expansion of Plasmodium falciparum. Science 2003, 300(5617):318-321.
25. Liu W, Li Y, Learn GH, Rudicell RS, Robertson JD, Keele BF, Ndjango JB, Sanz CM, Morgan DB, Locatelli S et al: Origin of the human malaria parasite Plasmodium falciparum in gorillas. Nature 2010, 467(7314):420-425.
26. Cox-Singh J, Davis TM, Lee KS, Shamsul SS, Matusop A, Ratnam S, Rahman HA, Conway DJ, Singh B: Plasmodium knowlesi malaria in humans is widely distributed and potentially life threatening. Clin Infect Dis 2008, 46(2):165-171.
27. White NJ: Plasmodium knowlesi: the fifth human malaria parasite. Clin Infect Dis 2008, 46(2):172-173.
117
28. Rasti N, Wahlgren M, Chen Q: Molecular aspects of malaria pathogenesis. FEMS immunology and medical microbiology 2004, 41(1):9-26.
29. Oyelade J, Ewejobi I, Brors B, Eils R, Adebiyi E: Computational identification of signalling pathways in Plasmodium falciparum. Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases 2011, 11(4):755-764.
30. LaCount DJ, Vignali M, Chettier R, Phansalkar A, Bell R, Hesselberth JR, Schoenfeld LW, Ota I, Sahasrabudhe S, Kurschner C et al: A protein interaction network of the malaria parasite Plasmodium falciparum. Nature 2005, 438(7064):103-107.
31. Enayati A, Hemingway J: Malaria management: past, present, and future. Annual review of entomology 2010, 55:569-591.
32. Martinsen ES, Perkins SL, Schall JJ: A three-genome phylogeny of malaria parasites (Plasmodium and closely related genera): evolution of life-history traits and host switches. Molecular phylogenetics and evolution 2008, 47(1):261-273.
33. Hay SI, Guerra CA, Tatem AJ, Noor AM, Snow RW: The global distribution and population at risk of malaria: past, present, and future. The Lancet infectious diseases 2004, 4(6):327-336.
34. Hume JC, Lyons EJ, Day KP: Human migration, mosquitoes and the evolution of Plasmodium falciparum. Trends Parasitol 2003, 19(3):144-149.
35. Baniecki ML, Wirth DF, Clardy J: High-throughput Plasmodium falciparum growth assay for malaria drug discovery. Antimicrob Agents Chemother 2007, 51(2):716-723.
36. Dondorp AM, Yeung S, White L, Nguon C, Day NP, Socheat D, von Seidlein L: Artemisinin resistance: current status and scenarios for containment. Nat Rev Microbiol 2010, 8(4):272-280.
37. Rieckmann KH, Campbell GH, Sax LJ, Mrema JE: Drug sensitivity of Plasmodium falciparum. An in-vitro microtechnique. Lancet 1978, 1(8054):22-23.
118
38. Wells TN, Alonso PL, Gutteridge WE: New medicines to improve control and contribute to the eradication of malaria. Nat Rev Drug Discov 2009, 8(11):879-891.
39. Chen Q, Schlichtherle M, Wahlgren M: Molecular aspects of severe malaria. Clin Microbiol Rev 2000, 13(3):439-450.
40. Dondorp AM, Nosten F, Yi P, Das D, Phyo AP, Tarning J, Lwin KM, Ariey F, Hanpithakpong W, Lee SJ et al: Artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med 2009, 361(5):455-467.
41. Pink R, Hudson A, Mouries MA, Bendig M: Opportunities and challenges in antiparasitic drug discovery. Nat Rev Drug Discov 2005, 4(9):727-740.
42. Kissinger JC, Brunk BP, Crabtree J, Fraunholz MJ, Gajria B, Milgram AJ, Pearson DS, Schug J, Bahl A, Diskin SJ et al: The Plasmodium genome database. Nature 2002, 419(6906):490-492.
43. Craft JC: Challenges facing drug development for malaria. Curr Opin Microbiol 2008, 11(5):428-433.
44. Balu B: Moving "Forward" in Plasmodium Genetics through a Transposon-Based Approach. Journal of tropical medicine 2012, 2012:829210.
45. O'Donnell RA, Freitas-Junior LH, Preiser PR, Williamson DH, Duraisingh M, McElwain TF, Scherf A, Cowman AF, Crabb BS: A genetic screen for improved plasmid segregation reveals a role for Rep20 in the interaction of Plasmodium falciparum chromosomes. The EMBO journal 2002, 21(5):1231-1239.
46. Wu Y, Kirkman LA, Wellems TE: Transformation of Plasmodium falciparum malaria parasites by homologous integration of plasmids that confer resistance to pyrimethamine. Proc Natl Acad Sci U S A 1996, 93(3):1130-1134.
47. Deitsch K, Driskill C, Wellems T: Transformation of malaria parasites by the spontaneous uptake and expression of DNA from human erythrocytes. Nucleic Acids Res 2001, 29(3):850-853.
119
48. Balu B, Adams JH: Functional genomics of Plasmodium falciparum through transposon-mediated mutagenesis. Cellular microbiology 2006, 8(10):1529-1536.
49. Crabb BS, Cowman AF: Characterization of promoters and stable transfection by homologous and nonhomologous recombination in Plasmodium falciparum. Proc Natl Acad Sci U S A 1996, 93(14):7289-7294.
50. Waters AP, Thomas AW, van Dijk MR, Janse CJ: Transfection of malaria parasites. Methods 1997, 13(2):134-147.
51. Kadekoppala M, Cheresh P, Catron D, Ji DD, Deitsch K, Wellems TE, Seifert HS, Haldar K: Rapid recombination among transfected plasmids, chimeric episome formation and trans gene expression in Plasmodium falciparum. Molecular and biochemical parasitology 2001, 112(2):211-218.
52. Sarkar A, Sim C, Hong YS, Hogan JR, Fraser MJ, Robertson HM, Collins FH: Molecular evolutionary analysis of the widespread piggyBac transposon family and related "domesticated" sequences. Mol Genet Genomics 2003, 270(2):173-180.
53. Greenwood B, Mutabingwa T: Malaria in 2002. Nature 2002, 415(6872):670-672.
54. Chootong P, Ntumngia FB, VanBuskirk KM, Xainli J, Cole-Tobian JL, Campbell CO, Fraser TS, King CL, Adams JH: Mapping epitopes of the Plasmodium vivax Duffy binding protein with naturally acquired inhibitory antibodies. Infection and immunity 2010, 78(3):1089-1095.
55. Ntumngia FB, McHenry AM, Barnwell JW, Cole-Tobian J, King CL, Adams JH: Genetic variation among Plasmodium vivax isolates adapted to non-human primates and the implication for vaccine development. Am J Trop Med Hyg 2009, 80(2):218-227.
56. VanBuskirk KM, Sevova E, Adams JH: Conserved residues in the Plasmodium vivax Duffy-binding protein ligand domain are critical for erythrocyte receptor recognition. Proc Natl Acad Sci U S A 2004, 101(44):15754-15759.
120
57. Gething PW, Elyazar IR, Moyes CL, Smith DL, Battle KE, Guerra CA, Patil AP, Tatem AJ, Howes RE, Myers MF et al: A long neglected world malaria map: Plasmodium vivax endemicity in 2010. PLoS neglected tropical diseases 2012, 6(9):e1814.
58. Gething PW, Patil AP, Smith DL, Guerra CA, Elyazar IR, Johnston GL, Tatem AJ, Hay SI: A new world malaria map: Plasmodium falciparum endemicity in 2010. Malar J 2011, 10:378.
59. World Malaria Report 2011 [http://www.who.int/malaria/world_malaria_report_2011/9789241564403_eng.pdf]
60. Hay SI, Okiro EA, Gething PW, Patil AP, Tatem AJ, Guerra CA, Snow RW: Estimating the global clinical burden of Plasmodium falciparum malaria in 2007. PLoS Med 2010, 7(6):e1000290.
61. Tsuboi T, Takeo S, Arumugam TU, Otsuki H, Torii M: The wheat germ cell-free protein synthesis system: a key tool for novel malaria vaccine candidate discovery. Acta tropica 2010, 114(3):171-176.
62. Gallup JL, Sachs JD: The economic burden of malaria. Am J Trop Med Hyg 2001, 64(1-2 Suppl):85-96.
63. WorldBank: The World Bank Booster Program for Malaria Control in AfricaIn.: The World Bank; 2011.
64. Delves M, Plouffe D, Scheurer C, Meister S, Wittlin S, Winzeler EA,
Sinden RE, Leroy D: The activities of current antimalarial drugs on the life cycle stages of Plasmodium: a comparative study with human and rodent parasites. PLoS Med 2012, 9(2):e1001169.
65. MacDonald G: The epidemiology and control of malaria. Oxford University Press 1957.
66. Kelly-Hope LA, McKenzie FE: The multiplicity of malaria transmission: a review of entomological inoculation rate measurements and methods across sub-Saharan Africa. Malar J 2009, 8:19.
121
67. Durnez L, Van Bortel W, Denis L, Roelants P, Veracx A, Trung HD, Sochantha T, Coosemans M: False positive circumsporozoite protein ELISA: a challenge for the estimation of the entomological inoculation rate of malaria and for vector incrimination. Malar J 2011, 10:195.
68. Coleman RE, Maneechai N, Rachaphaew N, Kumpitak C, Miller RS, Soyseng V, Thimasarn K, Sattabongkot J: Comparison of field and expert laboratory microscopy for active surveillance for asymptomatic Plasmodium falciparum and Plasmodium vivax in western Thailand. Am J Trop Med Hyg 2002, 67(2):141-144.
69. Kasehagen LJ, Mueller I, McNamara DT, Bockarie MJ, Kiniboro B, Rare L, Lorry K, Kastens W, Reeder JC, Kazura JW et al: Changing patterns of Plasmodium blood-stage infections in the Wosera region of Papua New Guinea monitored by light microscopy and high throughput PCR diagnosis. Am J Trop Med Hyg 2006, 75(4):588-596.
70. Shaukat AM, Breman JG, McKenzie FE: Using the entomological inoculation rate to assess the impact of vector control on malaria parasite transmission and elimination. Malar J 2010, 9:122.
71. Schofield L, Grau GE: Immunological processes in malaria pathogenesis. Nature reviews Immunology 2005, 5(9):722-735.
72. Suwanarusk R, Cooke BM, Dondorp AM, Silamut K, Sattabongkot J, White NJ, Udomsangpetch R: The deformability of red blood cells parasitized by Plasmodium falciparum and P. vivax. J Infect Dis 2004, 189(2):190-194.
73. Fairhurst RM, Baruch DI, Brittain NJ, Ostera GR, Wallach JS, Hoang HL, Hayton K, Guindo A, Makobongo MO, Schwartz OM et al: Abnormal display of PfEMP-1 on erythrocytes carrying haemoglobin C may protect against malaria. Nature 2005, 435(7045):1117-1121.
74. Rasti N, Wahlgren M, Chen Q: Molecular aspects of malaria pathogenesis. FEMS Immunology & Medical Microbiology 2006, 41(1):9-26.
75. Chattopadhyay R, Sharma A, Srivastava VK, Pati SS, Sharma SK, Das BS, Chitnis CE: Plasmodium falciparum infection elicits both variant-specific and cross-reactive antibodies against variant surface antigens. Infection and immunity 2003, 71(2):597-604.
122
76. Laurie AT, Jackson RM: Q-SiteFinder: an energy-based method for the prediction of protein-ligand binding sites. Bioinformatics 2005, 21(9):1908-1916.
77. Malaria [http://www.cdc.gov/malaria/]
78. Lillie PJ, Duncan CJ, Sheehy SH, Meyer J, O'Hara GA, Gilbert SC, Hill AV: Distinguishing malaria and influenza: early clinical features in controlled human experimental infection studies. Travel Med Infect Dis 2012, 10(4):192-196.
79. Heddini A: Malaria pathogenesis: a jigsaw with an increasing number of pieces. Int J Parasitol 2002, 32(13):1587-1598.
80. Zhu J, Wu X, Goel S, Gowda NM, Kumar S, Krishnegowda G, Mishra G, Weinberg R, Li G, Gaestel M et al: MAPK-activated protein kinase 2 differentially regulates Plasmodium falciparum glycosylphosphatidylinositol-induced production of tumor necrosis factor-{alpha} and interleukin-12 in macrophages. J Biol Chem 2009, 284(23):15750-15761.
81. Singh AP, Surolia N, Surolia A: Triclosan inhibit the growth of the late liver-stage of Plasmodium. IUBMB life 2009, 61(9):923-928.
82. Sinka ME, Bangs MJ, Manguin S, Rubio-Palis Y, Chareonviriyaphap T, Coetzee M, Mbogo CM, Hemingway J, Patil AP, Temperley WH et al: A global map of dominant malaria vectors. Parasites & vectors 2012, 5:69.
83. Kiszewski A, Mellinger A, Spielman A, Malaney P, Sachs SE, Sachs J: A global index representing the stability of malaria transmission. Am J Trop Med Hyg 2004, 70(5):486-498.
84. Arai M, Billker O, Morris HR, Panico M, Delcroix M, Dixon D, Ley SV, Sinden RE: Both mosquito-derived xanthurenic acid and a host blood-derived factor regulate gametogenesis of Plasmodium in the midgut of the mosquito. Molecular and biochemical parasitology 2001, 116:17-24.
85. Moorthy VS, Good MF, Hill AV: Malaria vaccine developments. Lancet 2004, 363(9403):150-156.
123
86. Medica DL, Sinnis P: Quantitative dynamics of Plasmodium yoelii sporozoite transmission by infected Anopheline mosquitoes. Infection and immunity 2005, 73(7):4363-4369.
87. Yamauchi LM, Coppi A, Snounou G, Sinnis P: Plasmodium sporozoites trickle out of the injection site. Cellular microbiology 2007, 9(5):1215-1222.
88. Kebaier C, Voza T, Vanderberg J: Kinetics of mosquito-injected Plasmodium sporozoites in mice: fewer sporozoites are injected into sporozoite-immunized mice. PLoS pathogens 2009, 5(4):e1000399.
89. Gerald N, Mahajan B, Kumar S: Mitosis in the human malaria parasite Plasmodium falciparum. Eukaryot Cell 2011, 10(4):474-482.
90. Gilson PR, Crabb BS: Morphology and kinetics of the three distinct phases of red blood cell invasion by Plasmodium falciparum merozoites. Int J Parasitol 2009, 39(1):91-96.
91. Mazier D, Renia L, Snounou G: The Plasmodium life cycle. In: Nature Reviews Drug Discovery. Nature Reviews Drug Discovery; 2009.
92. Anderson TJ, Nair S, Nkhoma S, Williams JT, Imwong M, Yi P, Socheat D, Das D, Chotivanich K, Day NP et al: High heritability of malaria parasite clearance rate indicates a genetic basis for artemisinin resistance in western Cambodia. J Infect Dis 2010, 201(9):1326-1330.
93. Davis TM, Hung TY, Sim IK, Karunajeewa HA, Ilett KF: Piperaquine: a resurgent antimalarial drug. Drugs 2005, 65(1):75-87.
94. Ekland EH, Fidock DA: In vitro evaluations of antimalarial drugs and their relevance to clinical outcomes. International journal for parasitology 2008, 38(7):743-747.
95. Hocart SJ, Liu H, Deng H, De D, Krogstad FM, Krogstad DJ: 4-aminoquinolines active against chloroquine-resistant Plasmodium falciparum: basis of antiparasite activity and quantitative structure-activity relationship analyses. Antimicrob Agents Chemother 2011, 55(5):2233-2244.
124
96. Hombhanje FW, Linge D, Saweri A, Kuanch C, Jones R, Toraso S, Geita J, Masta A, Kevau I, Hiawalyer G et al: Artemisinin-naphthoquine combination (ARCO) therapy for uncomplicated falciparum malaria in adults of Papua New Guinea: a preliminary report on safety and efficacy. Malaria Journal 2009, 8:196.
97. Mutabingwa T, Nzila A, Mberu E, Nduati E, Winstanley P, Hills E, Watkins W: Chlorproguanil-dapsone for treatment of drug-resistant falciparum malaria in Tanzania. Lancet 2001, 358(9289):1218-1223.
98. Saenz FE, Mutka T, Udenze K, Oduola AM, Kyle DE: Novel 4-aminoquinoline analogs highly active against the blood and sexual stages of Plasmodium in vivo and in vitro. Antimicrob Agents Chemother 2012.
99. Noedl H, Se Y, Schaecher K, Smith BL, Socheat D, Fukuda MM, Artemisinin Resistance in Cambodia 1 Study C: Evidence of artemisinin-resistant malaria in western Cambodia. N Engl J Med 2008, 359(24):2619-2620.
100. Cross RM, Maignan JR, Mutka TS, Luong L, Sargent J, Kyle DE, Manetsch R: Optimization of 1,2,3,4-tetrahydroacridin-9(10H)-ones as antimalarials utilizing structure-activity and structure-property relationships. J Med Chem 2011, 54(13):4399-4426.
101. O'Neill PM, Ward SA, Berry NG, Jeyadevan JP, Biagini GA, Asadollaly E, Park BK, Bray PG: A medicinal chemistry perspective on 4-aminoquinoline antimalarial drugs. Current topics in medicinal chemistry 2006, 6(5):479-507.
102. Stocks PA, Raynes KJ, Ward SA: Novel Quinoline Antimalarials: Humana Press; 2001.
103. WHO: Guidelines for the Treatment of Malaria, Second Edition edn. Geneva, Switzerland: World Health Organization; 2011.
104. Charman SA, Arbe-Barnes S, Bathurst IC, Brun R, Campbell M, Charman WN, Chiu FC, Chollet J, Craft JC, Creek DJ et al: Synthetic ozonide drug candidate OZ439 offers new hope for a single-dose cure of uncomplicated malaria. Proceedings of the National Academy of Sciences of the United States of America 2011, 108(11):4400-4405.
125
105. Pooley S, Fatih FA, Krishna S, Gerisch M, Haynes RK, Wong HN, Staines HM: Artemisone uptake in Plasmodium falciparum-infected erythrocytes. Antimicrob Agents Chemother 2011, 55(2):550-556.
106. Olliaro PL, Taylor WR: Developing artemisinin based drug combinations for the treatment of drug resistant falciparum malaria: A review. J Postgrad Med 2004, 50(1):40-44.
107. Benjamin J, Moore B, Lee ST, Senn M, Griffin S, Lautu D, Salman S, Siba P, Mueller I, Davis TM: Artemisinin-naphthoquine combination therapy for uncomplicated pediatric malaria: a tolerability, safety, and preliminary efficacy study. Antimicrob Agents Chemother 2012, 56(5):2465-2471.
108. Meshnick SR: Artemisinin: mechanisms of action, resistance and toxicity. Int J Parasitol 2002, 32(13):1655-1660.
109. Zwang J, Ashley EA, Karema C, D'Alessandro U, Smithuis F, Dorsey G, Janssens B, Mayxay M, Newton P, Singhasivanon P et al: Safety and efficacy of dihydroartemisinin-piperaquine in falciparum malaria: a prospective multi-centre individual patient data analysis. PloS one 2009, 4(7):e6358.
110. Haynes RK, Fugmann B, Stetter J, Rieckmann K, Heilmann HD, Chan HW, Cheung MK, Lam WL, Wong HN, Croft SL et al: Artemisone--a highly active antimalarial drug of the artemisinin class. Angew Chem Int Ed Engl 2006, 45(13):2082-2088.
111. Olliaro P, Wells TN: The global portfolio of new antimalarial medicines under development. Clin Pharmacol Ther 2009, 85(6):584-595.
112. Sullivan DJ, Jr., Matile H, Ridley RG, Goldberg DE: A common mechanism for blockade of heme polymerization by antimalarial quinolines. J Biol Chem 1998, 273(47):31103-31107.
113. Vennerstrom JL, Nuzum EO, Miller RE, Dorn A, Gerena L, Dande PA, Ellis WY, Ridley RG, Milhous WK: 8-Aminoquinolines active against blood stage Plasmodium falciparum in vitro inhibit hematin polymerization. Antimicrob Agents Chemother 1999, 43(3):598-602.
126
114. Francis SE, Sullivan DJ, Jr., Goldberg DE: Hemoglobin metabolism in the malaria parasite Plasmodium falciparum. Annu Rev Microbiol 1997, 51:97-123.
115. Watkins WM, Sixsmith DG, Spencer HC, Boriga DA, Kariuki DM, Kipingor T, Koech DK: Effectiveness of amodiaquine as treatment for chloroquine-resistant Plasmodium falciparum infections in Kenya. Lancet 1984, 1(8373):357-359.
116. Wang JY, Shan CQ, Fu DD, Sun ZW, Ding DB: [Efficacy of naphthoquine, artemisinine and a combination of the two drugs in the treatment of falciparum malaria]. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi 2003, 21(3):131-133.
117. Qu HY, Gao HZ, Hao GT, Li YY, Li HY, Hu JC, Wang XF, Liu WL, Liu ZY: Single-dose safety, pharmacokinetics, and food effects studies of compound naphthoquine phosphate tablets in healthy volunteers. J Clin Pharmacol 2010, 50(11):1310-1318.
118. Valecha N, Phyo AP, Mayxay M, Newton PN, Krudsood S, Keomany S, Khanthavong M, Pongvongsa T, Ruangveerayuth R, Uthaisil C et al: An open-label, randomised study of dihydroartemisinin-piperaquine versus artesunate-mefloquine for falciparum malaria in Asia. PloS one 2010, 5(7):e11880.
119. Bassat Q, Mulenga M, Tinto H, Piola P, Borrmann S, Menendez C, Nambozi M, Valea I, Nabasumba C, Sasi P et al: Dihydroartemisinin-piperaquine and artemether-lumefantrine for treating uncomplicated malaria in African children: a randomised, non-inferiority trial. PloS one 2009, 4(11):e7871.
120. Kurth F, Pongratz P, Belard S, Mordmuller B, Kremsner PG, Ramharter M: In vitro activity of pyronaridine against Plasmodium falciparum and comparative evaluation of anti-malarial drug susceptibility assays. Malaria Journal 2009, 8:79.
121. O'Neill PM, Park BK, Shone AE, Maggs JL, Roberts P, Stocks PA, Biagini GA, Bray PG, Gibbons P, Berry N et al: Candidate selection and preclinical evaluation of N-tert-butyl isoquine (GSK369796), an affordable and effective 4-aminoquinoline antimalarial for the 21st century. Journal of medicinal chemistry 2009, 52(5):1408-1415.
127
122. Ramanathan-Girish S, Catz P, Creek MR, Wu B, Thomas D, Krogstad DJ, De D, Mirsalis JC, Green CE: Pharmacokinetics of the antimalarial drug, AQ-13, in rats and cynomolgus macaques. Int J Toxicol 2004, 23(3):179-189.
123. Baird JK, Wiady I, Sutanihardja A, Suradi, Purnomo, Basri H, Sekartuti, Ayomi E, Fryauff DJ, Hoffman SL: Short report: therapeutic efficacy of chloroquine combined with primaquine against Plasmodium falciparum in northeastern Papua, Indonesia. Am J Trop Med Hyg 2002, 66(6):659-660.
124. Nanayakkara NP, Ager AL, Jr., Bartlett MS, Yardley V, Croft SL, Khan IA, McChesney JD, Walker LA: Antiparasitic activities and toxicities of individual enantiomers of the 8-aminoquinoline 8-[(4-amino-1-methylbutyl)amino]-6-methoxy-4-methyl-5-[3,4-dichlorophenoxy]quinol ine succinate. Antimicrob Agents Chemother 2008, 52(6):2130-2137.
125. University of Mississippi, MMV, and DNDi to collaborate on development of anti-parasitic drugs [http://www.mmv.org/newsroom/press-releases/university-mississippi-mmv-and-dndi-collaborate-development-anti-parasitic-d]
126. Nzila A: The past, present and future of antifolates in the treatment of Plasmodium falciparum infection. J Antimicrob Chemother 2006, 57(6):1043-1054.
127. Nirmalan N, Sims PF, Hyde JE: Translational up-regulation of antifolate drug targets in the human malaria parasite Plasmodium falciparum upon challenge with inhibitors. Molecular and Biochemical Parasitology 2004, 136(1):63-70.
128. Curd FH, Davey DG, Rose FL: Studies on synthetic antimalarial drugs; some biguanide derivatives as new types of antimalarial substances with both therapeutic and causal prophylactic activity. Ann Trop Med Parasitol 1945, 39:208-216.
129. Fidock DA, Nomura T, Wellems TE: Cycloguanil and its parent compound proguanil demonstrate distinct activities against Plasmodium falciparum malaria parasites transformed with human dihydrofolate reductase. Molecular pharmacology 1998, 54(6):1140-1147.
128
130. Kain KC: Current status and replies to frequently posed questions on atovaquone plus proguanil (Malarone) for the prevention of malaria. BioDrugs 2003, 17 Suppl 1:23-28.
131. Winstanley PA, Mberu EK, Szwandt IS, Breckenridge AM, Watkins WM: In vitro activities of novel antifolate drug combinations against Plasmodium falciparum and human granulocyte CFUs. Antimicrob Agents Chemother 1995, 39(4):948-952.
132. Nzila-Mounda A, Mberu EK, Sibley CH, Plowe CV, Winstanley PA, Watkins WM: Kenyan Plasmodium falciparum field isolates: correlation between pyrimethamine and chlorcycloguanil activity in vitro and point mutations in the dihydrofolate reductase domain. Antimicrob Agents Chemother 1998, 42(1):164-169.
133. Bukirwa H, Garner P, Critchley J: Chlorproguanil-dapsone for treating uncomplicated malaria. Cochrane Database Syst Rev 2004(4):CD004387.
134. Hitchings GH, Elion GB, Falco EA: Antagonists of nucleic acid derivatives. II. Reversal studies with substances structurally related to thymine. The Journal of biological chemistry 1950, 185(2):643-649.
135. Falco EA, Goodwin LG, Hitchings GH, Rollo IM, Russell PB: 2:4-diaminopyrimidines- a new series of antimalarials. Br J Pharmacol Chemother 1951, 6(2):185-200.
136. Dihydrofolate reductase (P218 DHFR) [http://www.mmv.org/research-development/project-portfolio/dihydrofolate-reductase-p218-dhfr]
137. Watkins WM, Mosobo M: Treatment of Plasmodium falciparum malaria with pyrimethamine-sulfadoxine: selective pressure for resistance is a function of long elimination half-life. Trans R Soc Trop Med Hyg 1993, 87(1):75-78.
138. Winstanley P, Watkins W, Muhia D, Szwandt S, Amukoye E, Marsh K: Chlorproguanil/dapsone for uncomplicated Plasmodium falciparum malaria in young children: pharmacokinetics and therapeutic range. Trans R Soc Trop Med Hyg 1997, 91(3):322-327.
129
139. Segal HE, Chinvanthananond P, Laixuthai B, Pearlman EJ, Hall AP, Phintuyothin P, Na-Nakorn A, Castaneda BF: Comparison of diaminodiphenylsulphonepyrimethamine and sulfadoxine-pyrimethamine combinations in the treatment of falciparum malaria in Thailand. Trans R Soc Trop Med Hyg 1975, 69(1):139-142.
140. Toovey S, Jamieson A, Nettleton G: Successful co-artemether (artemether-lumefantrine) clearance of falciparum malaria in a patient with severe cholera in Mozambique. Travel Med Infect Dis 2003, 1(3):177-179.
141. Boutros T, Chevet E, Metrakos P: Mitogen-Activated Protein ( MAP ) Kinase / MAP Kinase Phosphatase Regulation : Roles in Cell Growth , Death , and Cancer. Pharmacological Reviews 2008, 60:261-310.
142. Dorin-Semblat D, Quashie N, Halbert J, Sicard A, Doerig C, Peat E, Ranford-Cartwright L: Functional characterization of both MAP kinases of the human malaria parasite Plasmodium falciparum by reverse genetics. Molecular microbiology 2007, 65(5):1170-1180.
143. Farooq a, Chaturvedi G, Mujtaba S, Plotnikova O, Zeng L, Dhalluin C, Ashton R, Zhou MM: Solution structure of ERK2 binding domain of MAPK phosphatase MKP-3: structural insights into MKP-3 activation by ERK2. Molecular cell 2001, 7:387-399.
144. Ferrer I, Blanco R, Carmona M, Ribera R: Phosphorylated Map Kinase ( ERK1 , ERK2 ) Expression is Associated with Early Tau Deposition in Neurones and Glial Cells , but not with Increased Nuclear DNA Vulnerability and Cell Death , in Alzheimer Disease , Pick ' s Disease , Progressive Supranuclear. Brain Pathology 2001, 158:144-158.
145. Gustin MC, Albertyn J, Alexander M, Davenport K: MAP kinase pathways in the yeast Saccharomyces cerevisiae. Microbiology and molecular biology reviews : MMBR 1998, 62(4):1264-1300.
146. Bonnet J, Mayonove P, Morris MC: Differential phosphorylation of Cdc25C phosphatase in mitosis. Biochemical and Biophysical Research Communications 2008, 370(3):483-488.
147. Hofmann K, Bucher P, Kajava AV: A model of Cdc25 phosphatase catalytic domain and Cdk-interaction surface based on the presence of a rhodanese homology domain. Journal of molecular biology 1998, 282(1):195-208.
130
148. Kato N, Sakata T, Breton G, Le Roch KG, Nagle A, Andersen C, Bursulaya B, Henson K, Johnson J, Kumar KA et al: Gene expression signatures and small-molecule compounds link a protein kinase to Plasmodium falciparum motility. Nat Chem Biol 2008, 4(6):347-356.
149. Peyregne VP, Kar S, Ham SW, Wang M, Wang Z, Carr BI: Novel hydroxyl naphthoquinones with potent Cdc25 antagonizing and growth inhibitory properties. Molecular cancer therapeutics 2005, 4(4):595-602.
150. Roshak AK, Capper EA, Imburgia C, Fornwald J, Scott G, Marshall LA: The human polo-like kinase, PLK, regulates cdc2/cyclin B through phosphorylation and activation of the cdc25C phosphatase. Cellular Signalling 2000, 12(6):405-411.
151. Rudolph J: Cdc25 phosphatases: structure, specificity, and mechanism. Biochemistry 2007, 46(12):3595-3604.
152. Tewari R, Dorin D, Moon R, Doerig C, Billker O: An atypical mitogen-activated protein kinase controls cytokinesis and flagellar motility during male gamete formation in a malaria parasite. Molecular microbiology 2005, 58(5):1253-1263.
153. Theodosiou A, Ashworth A: MAP kinase phosphatases. Genome Biol 2002, 3(7):REVIEWS3009.
154. Billker O, Dechamps S, Tewari R, Wenig G, Franke-Fayard B, Brinkmann V: Calcium and a calcium-dependent protein kinase regulate gamete formation and mosquito transmission in a malaria parasite. Cell 2004, 117:503-514.
155. Brumlik MJ, Nkhoma S, Kious MJ, Thompson GR, Patterson TF, Siekierka JJ, Anderson TJC, Curiel TJ: Human p38 mitogen-activated protein kinase inhibitor drugs inhibit Plasmodium falciparum replication. Experimental parasitology 2011, 128:170-175.
156. Low H, Lye YM, Sim T-S: Pfnek3 functions as an atypical MAPKK in Plasmodium falciparum. Biochemical and biophysical research communications 2007, 361:439-444.
131
157. Rangarajan R, Bei AK, Jethwaney D, Maldonado P, Dorin D, Sultan AA, Doerig C: A Mitogen-activated Protein Kinase Regulates Male Gametogenesis and Transmission of the Malaria Parasite Plasmodium berghei. EMBO reports 2005, 6:464-469.
158. Zhu J, Wu X, Goel S, Gowda NM, Kumar S, Krishnegowda G, Mishra G, Weinberg R, Li G, Gaestel M et al: MAPK-activated protein kinase 2 differentially regulates Plasmodium falciparum glycosylphosphatidylinositol-induced production of tumor necrosis factor-{alpha} and interleukin-12 in macrophages. The Journal of biological chemistry 2009, 284:15750-15761.
159. Ward P, Equinet L, Packer J, Doerig C: Protein kinases of the human malaria parasite Plasmodium falciparum : the kinome of a divergent eukaryote. BMC Genomics 2004, 19:1-19.
160. Doerig C: The protein-phosphatome of the human malaria parasite Plasmodium falciparum. BMC Genomics 2008.
161. Solyakov L, Halbert J, Alam MM, Semblat JP, Dorin-Semblat D, Reininger L, Bottrill AR, Mistry S, Abdi A, Fennell C et al: Global kinomic and phospho-proteomic analyses of the human malaria parasite Plasmodium falciparum. Nat Commun 2011, 2:565.
162. Doerig C, Billker O, Haystead T, Sharma P, Tobin AB, Waters NC: Protein kinases of malaria parasites: an update. Trends in parasitology 2008, 24(12):570-577.
163. Syin C, Parzy D, Traincard F, Boccaccio I, Joshi MB, Lin DT, Yang XM, Assemat K, Doerig C, Langsley G: The H89 cAMP-dependent protein kinase inhibitor blocks Plasmodium falciparum development in infected erythrocytes. European journal of biochemistry / FEBS 2001, 268(18):4842-4849.
164. Deng W, Baker DA: A novel cyclic GMP-dependent protein kinase is expressed in the ring stage of the Plasmodium falciparum life cycle. Molecular microbiology 2002, 44(5):1141-1151.
165. Kumar A, Vaid A, Syin C, Sharma P: PfPKB, a novel protein kinase B-like enzyme from Plasmodium falciparum: I. Identification, characterization, and possible role in parasite development. The Journal of biological chemistry 2004, 279(23):24255-24264.
132
166. Doerig C, Endicott J, Chakrabarti D: Cyclin-dependent kinase homologues of Plasmodium falciparum. International journal for parasitology 2002, 32(13):1575-1585.
167. Dorin-Semblat D, Quashie N, Halbert J, Sicard A, Doerig C, Peat E, Ranford-Cartwright L, Doerig C: Functional characterization of both MAP kinases of the human malaria parasite Plasmodium falciparum by reverse genetics. Molecular microbiology 2007, 65:1170-1180.
168. Kappes B, Yang J, Suetterlin BW, Rathgeb-Szabo K, Lindt MJ, Franklin RM: A Plasmodium falciparum protein kinase with two unusually large kinase inserts. Molecular and biochemical parasitology 1995, 72(1-2):163-178.
169. Droucheau E, Primot A, Thomas V, Mattei D, Knockaert M, Richardson C, Sallicandro P, Alano P, Jafarshad A, Baratte B et al: Plasmodium falciparum glycogen synthase kinase-3: molecular model, expression, intracellular localisation and selective inhibitors. Biochimica et biophysica acta 2004, 1697(1-2):181-196.
170. Li JL, Targett GA, Baker DA: Primary structure and sexual stage-specific expression of a LAMMER protein kinase of Plasmodium falciparum. International journal for parasitology 2001, 31(4):387-392.
171. Garcia CR: Calcium homeostasis and signaling in the blood-stage malaria parasite. Parasitol Today 1999, 15(12):488-491.
172. Zhao Y, Pokutta S, Maurer P, Lindt M, Franklin RM, Kappes B: Calcium-binding properties of a calcium-dependent protein kinase from Plasmodium falciparum and the significance of individual calcium-binding sites for kinase activation. Biochemistry 1994, 33(12):3714-3721.
173. Farber PM, Graeser R, Franklin RM, Kappes B: Molecular cloning and characterization of a second calcium-dependent protein kinase of Plasmodium falciparum. Molecular and biochemical parasitology 1997, 87(2):211-216.
174. Li JL, Baker DA, Cox LS: Sexual stage-specific expression of a third calcium-dependent protein kinase from Plasmodium falciparum. Biochimica et biophysica acta 2000, 1491(1-3):341-349.
133
175. Zhao Y, Kappes B, Yang J, Franklin RM: Molecular cloning, stage-specific expression and cellular distribution of a putative protein kinase from Plasmodium falciparum. European journal of biochemistry / FEBS 1992, 207(1):305-313.
176. Silva-Neto MA, Atella GC, Shahabuddin M: Inhibition of Ca2+/calmodulin-dependent protein kinase blocks morphological differentiation of Plasmodium gallinaceum zygotes to ookinetes. The Journal of biological chemistry 2002, 277(16):14085-14091.
177. Barik S, Taylor RE, Chakrabarti D: Identification, cloning, and mutational analysis of the casein kinase 1 cDNA of the malaria parasite, Plasmodium falciparum. Stage-specific expression of the gene. The Journal of biological chemistry 1997, 272(42):26132-26138.
178. Abdi A, Eschenlauer S, Reininger L, Doerig C: SAM domain-dependent activity of PfTKL3, an essential tyrosine kinase-like kinase of the human malaria parasite Plasmodium falciparum. Cellular and molecular life sciences : CMLS 2010, 67:3355-3369.
179. O'Regan L, Blot J, Fry AM: Mitotic regulation by NIMA-related kinases. Cell Div 2007, 2:25.
180. Khan SM, Franke-Fayard B, Mair GR, Lasonder E, Janse CJ, Mann M, Waters AP: Proteome analysis of separated male and female gametocytes reveals novel sex-specific Plasmodium biology. Cell 2005, 121(5):675-687.
181. Lye YM, Chan M, Sim TS: Pfnek3: an atypical activator of a MAP kinase in Plasmodium falciparum. FEBS Lett 2006, 580(26):6083-6092.
182. Le Roch KG, Zhou Y, Blair PL, Grainger M, Moch JK, Haynes JD, De La Vega P, Holder AA, Batalov S, Carucci DJ et al: Discovery of gene function by expression profiling of the malaria parasite life cycle. Science 2003, 301(5639):1503-1508.
183. Reininger L, Billker O, Tewari R, Mukhopadhyay A, Fennell C, Dorin-Semblat D, Doerig C, Goldring D, Harmse L, Ranford-Cartwright L et al: A NIMA-related protein kinase is essential for completion of the sexual cycle of malaria parasites. The Journal of biological chemistry 2005, 280(36):31957-31964.
134
184. Kapiloff MS, Chandrasekhar KD: A-kinase anchoring proteins: temporal and spatial regulation of intracellular signal transduction in the cardiovascular system. J Cardiovasc Pharmacol 2011, 58(4):337-338.
185. Kumar R, Adams B, Oldenburg A, Musiyenko A, Barik S: Characterisation and expression of a PP1 serine/threonine protein phosphatase (PfPP1) from the malaria parasite, Plasmodium falciparum: demonstration of its essential role using RNA interference. Malaria Journal 2002, 1:5.
186. Lindenthal C, Klinkert MQ: Identification and biochemical characterisation of a protein phosphatase 5 homologue from Plasmodium falciparum. Molecular and Biochemical Parasitology 2002, 120(2):257-268.
187. Mamoun CB, Sullivan DJ, Jr., Banerjee R, Goldberg DE: Identification and characterization of an unusual double serine/threonine protein phosphatase 2C in the malaria parasite Plasmodium falciparum. The Journal of biological chemistry 1998, 273(18):11241-11247.
188. Mamoun CB, Goldberg DE: Plasmodium protein phosphatase 2C dephosphorylates translation elongation factor 1beta and inhibits its PKC-mediated nucleotide exchange activity in vitro. Molecular microbiology 2001, 39:973-981.
189. Orgad S, Brewis ND, Alphey L, Axton JM, Dudai Y, Cohen PT: The structure of protein phosphatase 2A is as highly conserved as that of protein phosphatase 1. FEBS Lett 1990, 275(1-2):44-48.
190. Barton GJ, Cohen PT, Barford D: Conservation analysis and structure prediction of the protein serine/threonine phosphatases. Sequence similarity with diadenosine tetraphosphatase from Escherichia coli suggests homology to the protein phosphatases. European journal of biochemistry / FEBS 1994, 220(1):225-237.
191. Wera S, Hemmings BA: Serine/threonine protein phosphatases. The Biochemical journal 1995, 311 ( Pt 1):17-29.
192. Andreeva AV, Kutuzov MA: PPP family of protein Ser/Thr phosphatases: two distinct branches? Mol Biol Evol 2001, 18(3):448-452.
135
193. Cohen PT, Philp A, Vazquez-Martin C: Protein phosphatase 4--from obscurity to vital functions. FEBS Lett 2005, 579(15):3278-3286.
194. Kutuzov MA, Andreeva AV: Protein Ser/Thr phosphatases with kelch-like repeat domains. Cellular Signalling 2002, 14(9):745-750.
195. Li JL, Baker DA: A putative protein serine/threonine phosphatase from Plasmodium falciparum contains a large N-terminal extension and five unique inserts in the catalytic domain. Molecular and Biochemical Parasitology 1998, 95(2):287-295.
196. Adams J, Kelso R, Cooley L: The kelch repeat superfamily of proteins: propellers of cell function. Trends Cell Biol 2000, 10(1):17-24.
197. Bastians H, Ponstingl H: The novel human protein serine/threonine phosphatase 6 is a functional homologue of budding yeast Sit4p and fission yeast ppe1, which are involved in cell cycle regulation. J Cell Sci 1996, 109 ( Pt 12):2865-2874.
198. Das AK, Helps NR, Cohen PT, Barford D: Crystal structure of the protein serine/threonine phosphatase 2C at 2.0 A resolution. The EMBO journal 1996, 15(24):6798-6809.
199. Schweighofer A, Hirt H, Meskiene I: Plant PP2C phosphatases: emerging functions in stress signaling. Trends Plant Sci 2004, 9(5):236-243.
200. Boutros R, Dozier C, Ducommun B: The when and wheres of CDC25 phosphatases. Current opinion in cell biology 2006, 18(2):185-191.
201. Owens DM, Keyse SM: Differential regulation of MAP kinase signalling by dual-specificity protein phosphatases. Oncogene 2007, 26(22):3203-3213.
202. Trinkle-Mulcahy L, Lamond AI: Mitotic phosphatases: no longer silent partners. Current opinion in cell biology 2006, 18(6):623-631.
203. Raugei G, Ramponi G, Chiarugi P: Low molecular weight protein tyrosine phosphatases: small, but smart. Cellular and molecular life sciences : CMLS 2002, 59(6):941-949.
136
204. Fauman EB, Saper MA: Structure and function of the protein tyrosine phosphatases. Trends in biochemical sciences 1996, 21(11):413-417.
205. Guex N, Peitsch MC: SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 1997, 18(15):2714-2723.
206. Contour-Galcera MO, Sidhu A, Prevost G, Bigg D, Ducommun B: What's new on CDC25 phosphatase inhibitors. Pharmacology & therapeutics 2007, 115(1):1-12.
207. Rudolph J: Inhibiting transient protein-protein interactions: lessons from the Cdc25 protein tyrosine phosphatases. Nat Rev Cancer 2007, 7(3):202-211.
208. Guttery DS, Ferguson DJ, Poulin B, Xu Z, Straschil U, Klop O, Solyakov L, Sandrini SM, Brady D, Nieduszynski CA et al: A putative homologue of CDC20/CDH1 in the malaria parasite is essential for male gamete development. PLoS Pathog 2012, 8(2):e1002554.
209. Kumar R, Musiyenko A, Cioffi E, Oldenburg A, Adams B, Bitko V, Krishna SS, Barik S: A zinc-binding dual-specificity YVH1 phosphatase in the malaria parasite, Plasmodium falciparum, and its interaction with the nuclear protein, pescadillo. Molecular and Biochemical Parasitology 2004, 133(2):297-310.
210. Pendyala PR, Ayong L, Eatrides J, Schreiber M, Pham C, Chakrabarti R, Fidock DA, Allen CM, Chakrabarti D: Characterization of a PRL protein tyrosine phosphatase from Plasmodium falciparum. Molecular and Biochemical Parasitology 2008, 158(1):1-10.
211. Andreeva AV, Kutuzov MA: Protozoan protein tyrosine phosphatases. International journal for parasitology 2008, 38(11):1279-1295.
212. Yeo M, Lin PS: Functional characterization of small CTD phosphatases. Methods Mol Biol 2007, 365:335-346.
213. Yeo M, Lin PS, Dahmus ME, Gill GN: A novel RNA polymerase II C-terminal domain phosphatase that preferentially dephosphorylates serine 5. The Journal of biological chemistry 2003, 278(28):26078-26085.
137
214. Kobor MS, Greenblatt J: Regulation of transcription elongation by phosphorylation. Biochimica et biophysica acta 2002, 1577(2):261-275.
215. Suh MH, Ye P, Zhang M, Hausmann S, Shuman S, Gnatt AL, Fu J: Fcp1 directly recognizes the C-terminal domain (CTD) and interacts with a site on RNA polymerase II distinct from the CTD. Proceedings of the National Academy of Sciences of the United States of America 2005, 102(48):17314-17319.
216. Hausmann S, Shuman S: Defining the active site of Schizosaccharomyces pombe C-terminal domain phosphatase Fcp1. The Journal of biological chemistry 2003, 278(16):13627-13632.
217. Camps M, Nichols a, Arkinstall S: Dual specificity phosphatases: a gene family for control of MAP kinase function. The FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2000, 14:6-16.
218. Keyse SM: Protein phosphatases and the regulation of mitogen-activated protein kinase signalling. Current opinion in cell biology 2000, 12:186-192.
219. Farooq A, Zhou M-M: Structure and regulation of MAPK phosphatases. Cellular signalling 2004, 16:769-779.
220. Noordman YE, Jansen PAM, Hendriks W: Tyrosine-specific MAPK Phosphatases and the control of ERK Signalling in PC12 Cells. Journal of Molecular Signaling 2006, 1:4.
221. Kim Y, Gentry MS, Harris TE, Wiley SE, Lawrence JC, Dixon JE: A conserved phosphatase cascade that regulates nuclear membrane biogenesis. Proceedings of the National Academy of Sciences of the United States of America 2007, 104:6596-6601.
222. Kondoh K, Nishida E: Regulation of MAP kinases by MAP kinase phosphatases. Biochimica et biophysica acta 2007, 1773:1227-1237.
223. Szöor B, Ruberto I, Burchmore R, Matthews KR: A novel phosphatase cascade regulates differentiation in Trypanosoma brucei via a glycosomal signaling pathway. Genes & development 2010, 24:1306-1316.
138
224. Barr AJ, Knapp S: MAPK-specific tyrosine phosphatases: new targets for drug discovery? Trends in pharmacological sciences 2006, 27:525-530.
225. Alano P: Plasmodium falciparum gametocytes: still many secrets of a hidden life. Molecular microbiology 2007, 66:291-302.
226. Lim DC, Cooke BM, Doerig C, Saeij JP: Toxoplasma and Plasmodium protein kinases: roles in invasion and host cell remodelling. Int J Parasitol 2012, 42(1):21-32.
227. Lucet IS, Tobin A, Drewry D, Wilks AF, Doerig C: Plasmodium kinases as targets for new-generation antimalarials. Future Med Chem 2012, 4(18):2295-2310.
228. Ma J, Rahlfs S, Jortzik E, Schirmer RH, Przyborski JM, Becker K: Subcellular localization of adenylate kinases in Plasmodium falciparum. FEBS Lett 2012, 586(19):3037-3043.
229. Rached FB, Ndjembo-Ezougou C, Chandran S, Talabani H, Yera H, Dandavate V, Bourdoncle P, Meissner M, Tatu U, Langsley G: Construction of a Plasmodium falciparum Rab-interactome identifies CK1 and PKA as Rab-effector kinases in malaria parasites. Biology of the cell / under the auspices of the European Cell Biology Organization 2012, 104(1):34-47.
230. Dvorin JD, Martyn DC, Patel SD, Grimley JS, Collins CR, Hopp CS, Bright AT, Westenberger S, Winzeler E, Blackman MJ et al: A plant-like kinase in Plasmodium falciparum regulates parasite egress from erythrocytes. Science 2010, 328(5980):910-912.
231. Dorin-Semblat D, Schmitt S, Semblat JP, Sicard A, Reininger L, Goldring D, Patterson S, Quashie N, Chakrabarti D, Meijer L et al: Plasmodium falciparum NIMA-related kinase Pfnek-1: sex specificity and assessment of essentiality for the erythrocytic asexual cycle. Microbiology 2011, 157(Pt 10):2785-2794.
232. Reininger L, Garcia M, Tomlins A, Muller S, Doerig C: The Plasmodium falciparum, Nima-related kinase Pfnek-4: a marker for asexual parasites committed to sexual differentiation. Malar J 2012, 11:250.
139
233. Sebastian S, Brochet M, Collins MO, Schwach F, Jones ML, Goulding D, Rayner JC, Choudhary JS, Billker O: A Plasmodium calcium-dependent protein kinase controls zygote development and transmission by translationally activating repressed mRNAs. Cell host & microbe 2012, 12(1):9-19.
234. Reininger L, Tewari R, Fennell C, Holland Z, Goldring D, Ranford-Cartwright L, Billker O, Doerig C: An essential role for the Plasmodium Nek-2 Nima-related protein kinase in the sexual development of malaria parasites. J Biol Chem 2009, 284(31):20858-20868.
235. Tewari R, Straschil U, Bateman A, Bohme U, Cherevach I, Gong P, Pain A, Billker O: The systematic functional analysis of Plasmodium protein kinases identifies essential regulators of mosquito transmission. Cell host & microbe 2010, 8(4):377-387.
236. Yokoyama D, Saito-Ito A, Asao N, Tanabe K, Yamamoto M, Matsumura T: Modulation of the growth of Plasmodium falciparum in vitro by protein serine/threonine phosphatase inhibitors. Biochemical and biophysical research communications 1998, 247(1):18-23.
237. Hills T, Srivastava A, Ayi K, Wernimont AK, Kain K, Waters AP, Hui R, Pizarro JC: Characterization of a new phosphatase from Plasmodium. Molecular and Biochemical Parasitology 2011, 179(2):69-79.
238. Guttery DS, Poulin B, Ferguson DJ, Szoor B, Wickstead B, Carroll PL, Ramakrishnan C, Brady D, Patzewitz EM, Straschil U et al: A unique protein phosphatase with kelch-like domains (PPKL) in Plasmodium modulates ookinete differentiation, motility and invasion. PLoS pathogens 2012, 8(9):e1002948.
239. Chung DW, Ponts N, Cervantes S, Le Roch KG: Post-translational modifications in Plasmodium: more than you think! Molecular and Biochemical Parasitology 2009, 168(2):123-134.
240. Almo SC, Bonanno JB, Sauder JM, Emtage S, Dilorenzo TP, Malashkevich V, Wasserman SR, Swaminathan S, Eswaramoorthy S, Agarwal R et al: Structural genomics of protein phosphatases. J Struct Funct Genomics 2007, 8(2-3):121-140.
241. MR4: Methods in Malaria Research, vol. 5.2. Paris, France: Malaria Research and Reference Reagent Resourse Center; 2008.
140
242. Balu B, Singh N, Maher SP, Adams JH: A genetic screen for attenuated growth identifies genes crucial for intraerythrocytic development of Plasmodium falciparum. PloS one 2010, 5(10):e13282.
243. Russo I, Oksman A, Vaupel B, Goldberg DE: A calpain unique to alveolates is essential in Plasmodium falciparum and its knockdown reveals an involvement in pre-S-phase development. Proceedings of the National Academy of Sciences of the United States of America 2009, 106(5):1554-1559.
244. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R et al: ClustalW and ClustalX version 2. Bioinformatics 2007, 23(21):2947-2948.
245. Goujon M, McWilliam H, Li W, Valentin F, Squizzato S, Paern J, Lopez R: A new bioinformatics analysis tools framework at EMBL-EBI. Nucleic Acids Res 2010, 38(Web Server issue):W695-699.
246. Tamura K, Dudley J, Nei M, Kumar S: MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 2007, 24(8):1596-1599.
247. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S: MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 2011, 28(10):2731-2739.
248. Kumar S, Stecher G, Peterson D, Tamura K: MEGA-CC: computing core of molecular evolutionary genetics analysis program for automated and iterative data analysis. Bioinformatics 2012, 28(20):2685-2686.
249. Arino J: Novel protein phosphatases in yeast. An update. European Journal of Biochemistry 2002, 269:1072-1077.
250. Martín H, Flández M, Nombela C, Molina M: Protein phosphatases in MAPK signalling: we keep learning from yeast. Molecular microbiology 2005, 58:6-16.
251. Theodosiou A, Ashworth A: Protein family review MAP kinase phosphatases Gene organization and evolutionary history. Genome 2002:1-10.
141
252. Denu JM, Dixon JE: Protein tyrosine phosphatases: mechanisms of catalysis and regulation. Current opinion in chemical biology 1998, 2:633-641.
253. Tonks NK: Previews Pseudophosphatases : Grab and Hold on. October 2009:464-465.
254. Wishart MJ, Denu JM, Williams JA, Dixon JE: Communication A Single Mutation Converts a Novel Phosphotyrosine Binding Domain into a Dual-specificity Phosphatase *. Biochemistry 1995:26782-26785.
255. Wishart MJ, Dixon JE: Gathering STYX: phosphatase-like form predicts functions for unique protein-interaction domains. Trends in biochemical sciences 1998, 23:301-306.
256. Otto TD, Wilinski D, Assefa S, Keane TM, Sarry LR, Bohme U, Lemieux J, Barrell B, Pain A, Berriman M et al: New insights into the blood-stage transcriptome of Plasmodium falciparum using RNA-Seq. Molecular microbiology 2010, 76(1):12-24.
257. Elick TA, Bauser CA, Fraser MJ: Excision of the piggyBac transposable element in vitro is a precise event that is enhanced by the expression of its encoded transposase. Genetica 1996, 98(1):33-41.
258. Fraser MJ, Ciszczon T, Elick T, Bauser C: Precise excision of TTAA-specific lepidopteran transposons piggyBac (IFP2) and tagalong (TFP3) from the baculovirus genome in cell lines from two species of Lepidoptera. Insect Mol Biol 1996, 5(2):141-151.
259. O'Brochta DA, Atkinson PW: Transposable elements and gene transformation in non-drosophilid insects. Insect Biochem Mol Biol 1996, 26(8-9):739-753.
260. Morales ME, Mann VH, Kines KJ, Gobert GN, Fraser MJ, Jr., Kalinna BH, Correnti JM, Pearce EJ, Brindley PJ: piggyBac transposon mediated transgenesis of the human blood fluke, Schistosoma mansoni. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2007, 21(13):3479-3489.
142
261. Sethuraman N, Fraser MJ, Jr., Eggleston P, O'Brochta DA: Post-integration stability of piggyBac in Aedes aegypti. Insect Biochem Mol Biol 2007, 37(9):941-951.
262. Fonager J, Franke-Fayard BM, Adams JH, Ramesar J, Klop O, Khan SM, Janse CJ, Waters AP: Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites. BMC Genomics 2011, 12:155.
263. Kozlov S, Waters NC, Chavchich M: Leveraging cell cycle analysis in anticancer drug discovery to identify novel plasmodial drug targets. Infectious disorders drug targets 2010, 10(3):165-190.
264. Wishart MJ, Dixon JE: The archetype STYX dead-phosphatase complexes with a spermatid mRNA-binding protein and is essential for normal sperm production. PNAS 2002.
265. Hinton SD, Myers MP, Roggero VR, Allison LA, Tonks NK: The pseudophosphatase MK-STYX interacts with G3BP and decreases stress granule formation. The Biochemical journal 2010, 427(3):349-357.
266. Cheng KC, Klancer R, Singson A, Seydoux G: Regulation of MBK-2/DYRK by CDK-1 and the pseudophosphatases EGG-4 and EGG-5 during the oocyte-to-embryo transition. Cell 2009, 139(3):560-572.
267. Kutuzov MA, Andreeva AV: Protein Ser/Thr phosphatases of parasitic protozoa. Molecular and biochemical parasitology 2008, 161(2):81-90.
268. Hyde JE: Drug-resistant malaria. Trends Parasitol 2005, 21(11):494-498.
269. Gamo FJ, Sanz LM, Vidal J, de Cozar C, Alvarez E, Lavandera JL, Vanderwall DE, Green DV, Kumar V, Hasan S et al: Thousands of chemical starting points for antimalarial lead identification. Nature 2010, 465(7296):305-310.
270. Jensen K, Plichta D, Panagiotou G, Kouskoumvekaki I: Mapping the genome of Plasmodium falciparum on the drug-like chemical space reveals novel anti-malarial targets and potential drug leads. Molecular bioSystems 2012, 8(6):1678-1685.
143
271. Stepniewska K, Ashley E, Lee SJ, Anstey N, Barnes KI, Binh TQ, D'Alessandro U, Day NP, de Vries PJ, Dorsey G et al: In vivo parasitological measures of artemisinin susceptibility. J Infect Dis 2010, 201(4):570-579.
272. Small I: Predotar v. 1.03 A prediction service for identifying putative N-terminal targeting sequences. In.: Genoplante; 2003.
273. Maude RJ, Pontavornpinyo W, Saralamba S, Aguas R, Yeung S, Dondorp AM, Day NP, White NJ, White LJ: The last man standing is the most resistant: eliminating artemisinin-resistant malaria in Cambodia. Malaria Journal 2009, 8:31.
274. Kitchen DB, Decornez H, Furr JR, Bajorath J: Docking and scoring in virtual screening for drug discovery: methods and applications. Nat Rev Drug Discov 2004, 3(11):935-949.
275. McInnes C: Virtual screening strategies in drug discovery. Curr Opin Chem Biol 2007, 11(5):494-502.
276. Toth K, Djeha H, Ying B, Tollefson AE, Kuppuswamy M, Doronin K, Krajcsi P, Lipinski K, Wrighton CJ, Wold WS: An oncolytic adenovirus vector combining enhanced cell-to-cell spreading, mediated by the ADP cytolytic protein, with selective replication in cancer cells with deregulated wnt signaling. Cancer research 2004, 64(10):3638-3644.
277. Zheng CH, Yang H, Zhang M, Lu SH, Shi D, Wang J, Chen XH, Ren XH, Liu J, Lv JG et al: Design, synthesis, and activity evaluation of broad-spectrum small-molecule inhibitors of anti-apoptotic Bcl-2 family proteins: characteristics of broad-spectrum protein binding and its effects on anti-tumor activity. Bioorganic & medicinal chemistry letters 2012, 22(1):39-44.
278. Bulut G, Hong SH, Chen K, Beauchamp EM, Rahim S, Kosturko GW, Glasgow E, Dakshanamurthy S, Lee HS, Daar I et al: Small molecule inhibitors of ezrin inhibit the invasive phenotype of osteosarcoma cells. Oncogene 2012, 31(3):269-281.
279. Aurrecoechea C, Brestelli J, Brunk BP, Dommer J, Fischer S, Gajria B, Gao X, Gingle A, Grant G, Harb OS et al: PlasmoDB: a functional genomic database for malaria parasites. Nucleic acids research 2009, 37(Database issue):D539-543.
144
280. Gasteiger E., Hoogland C., Gattiker A., Duvaud S., Wilkins M.R., Appel R.D., A B: Protein Identification and Analysis Tool on the ExPASy Server. In: The Protemics Protocols Handbook. Humana Press; 2005.
281. Marchler-Bauer A, Lu S, Anderson JB, Chitsaz F, Derbyshire MK, DeWeese-Scott C, Fong JH, Geer LY, Geer RC, Gonzales NR et al: CDD: a Conserved Domain Database for the functional annotation of proteins. Nucleic acids research 2011, 39(Database issue):D225-229.
282. Geer LY, Domrachev M, Lipman DJ, Bryant SH: CDART: protein homology by domain architecture. Genome Res 2002, 12(10):1619-1623.
283. Hunter S, Apweiler R, Attwood TK, Bairoch A, Bateman A, Binns D, Bork P, Das U, Daugherty L, Duquenne L et al: InterPro: the integrative protein signature database. Nucleic acids research 2009, 37(Database issue):D211-215.
284. Quevillon E, Silventoinen V, Pillai S, Harte N, Mulder N, Apweiler R, Lopez R: InterProScan: protein domains identifier. Nucleic acids research 2005, 33(Web Server issue):W116-120.
285. Sigrist CJ, Cerutti L, de Castro E, Langendijk-Genevaux PS, Bulliard V, Bairoch A, Hulo N: PROSITE, a protein domain database for functional characterization and annotation. Nucleic acids research 2010, 38(Database issue):D161-166.
286. de Castro E, Sigrist CJ, Gattiker A, Bulliard V, Langendijk-Genevaux PS, Gasteiger E, Bairoch A, Hulo N: ScanProsite: detection of PROSITE signature matches and ProRule-associated functional and structural residues in proteins. Nucleic acids research 2006, 34(Web Server issue):W362-365.
287. Gough J, Karplus K, Hughey R, Chothia C: Assignment of homology to genome sequences using a library of hidden Markov models that represent all proteins of known structure. Journal of molecular biology 2001, 313(4):903-919.
288. Schultz J, Milpetz F, Bork P, Ponting CP: SMART, a simple modular architecture research tool: identification of signaling domains. Proceedings of the National Academy of Sciences of the United States of America 1998, 95(11):5857-5864.
145
289. Letunic I, Doerks T, Bork P: SMART 6: recent updates and new developments. Nucleic acids research 2009, 37(Database issue):D229-232.
290. Zuckerkandl E, Pauling L: Molecules as documents of evolutionary history. Journal of theoretical biology 1965, 8(2):357-366.
291. Saitou N, Nei M: The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 1987, 4(4):406-425.
292. Cole C, Barber JD, Barton GJ: The Jpred 3 secondary structure prediction server. Nucleic acids research 2008, 36(Web Server issue):W197-201.
293. Bryson K, McGuffin LJ, Marsden RL, Ward JJ, Sodhi JS, Jones DT: Protein structure prediction servers at University College London. Nucleic acids research 2005, 33(Web Server issue):W36-38.
294. Jones DT: Protein secondary structure prediction based on position-specific scoring matrices. Journal of molecular biology 1999, 292(2):195-202.
295. Blom N, Gammeltoft S, Brunak S: Sequence and structure-based prediction of eukaryotic protein phosphorylation sites. Journal of molecular biology 1999, 294(5):1351-1362.
296. Blom N, Sicheritz-Ponten T, Gupta R, Gammeltoft S, Brunak S: Prediction of post-translational glycosylation and phosphorylation of proteins from the amino acid sequence. Proteomics 2004, 4(6):1633-1649.
297. Petersen TN, Brunak S, von Heijne G, Nielsen H: SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 2011, 8(10):785-786.
298. Zuegge J, Ralph S, Schmuker M, McFadden GI, Schneider G: Deciphering apicoplast targeting signals--feature extraction from nuclear-encoded precursors of Plasmodium falciparum apicoplast proteins. Gene 2001, 280(1-2):19-26.
146
299. Waller RF, Keeling PJ, Donald RG, Striepen B, Handman E, Lang-Unnasch N, Cowman AF, Besra GS, Roos DS, McFadden GI: Nuclear-encoded proteins target to the plastid in Toxoplasma gondii and Plasmodium falciparum. Proceedings of the National Academy of Sciences of the United States of America 1998, 95(21):12352-12357.
300. Waller RF, Reed MB, Cowman AF, McFadden GI: Protein trafficking to the plastid of Plasmodium falciparum is via the secretory pathway. The EMBO journal 2000, 19(8):1794-1802.
301. Bologna G, Yvon C, Duvaud S, Veuthey AL: N-Terminal myristoylation predictions by ensembles of neural networks. Proteomics 2004, 4(6):1626-1632.
302. Arnold K, Bordoli L, Kopp J, Schwede T: The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 2006, 22(2):195-201.
303. Kiefer F, Arnold K, Kunzli M, Bordoli L, Schwede T: The SWISS-MODEL Repository and associated resources. Nucleic acids research 2009, 37(Database issue):D387-392.
304. Schwede T, Kopp J, Guex N, Peitsch MC: SWISS-MODEL: An automated protein homology-modeling server. Nucleic acids research 2003, 31(13):3381-3385.
305. Stewart AE, Dowd S, Keyse SM, Mcdonald NQ: Crystal structure of the MAPK phosphatase Pyst1 catalytic domain and implications for regulated activation. America 1999, 6:174-182.
306. Bordoli L, Kiefer F, Arnold K, Benkert P, Battey J, Schwede T: Protein structure homology modeling using SWISS-MODEL workspace. Nature Protocols 2009, 4(1):1-13.
307. Laskowski RA, Rullmannn JA, MacArthur MW, Kaptein R, Thornton JM: AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J Biomol NMR 1996, 8(4):477-486.
308. Vriend G: WHAT IF: a molecular modeling and drug design program. J Mol Graph 1990, 8(1):52-56, 29.
147
309. Colovos C, Yeates TO: Verification of protein structures: patterns of nonbonded atomic interactions. Protein science : a publication of the Protein Society 1993, 2(9):1511-1519.
310. Trivedi V, Nag S: In silico characterization of atypical kinase PFD0975w from Plasmodium kinome: a suitable target for drug discovery. Chemical biology & drug design 2012, 79(4):600-609.
311. Bowie JU, Luthy R, Eisenberg D: A method to identify protein sequences that fold into a known three-dimensional structure. Science 1991, 253(5016):164-170.
312. Luthy R, Bowie JU, Eisenberg D: Assessment of protein models with three-dimensional profiles. Nature 1992, 356(6364):83-85.
313. Burgoyne NJ, Jackson RM: Predicting protein interaction sites: binding hot-spots in protein-protein and protein-ligand interfaces. Bioinformatics 2006, 22(11):1335-1342.
314. Hendlich M, Rippmann F, Barnickel G: LIGSITE: automatic and efficient detection of potential small molecule-binding sites in proteins. Journal of molecular graphics & modelling 1997, 15(6):359-363, 389.
315. Liang J, Edelsbrunner H, Woodward C: Anatomy of protein pockets and cavities: measurement of binding site geometry and implications for ligand design. Protein science : a publication of the Protein Society 1998, 7(9):1884-1897.
316. Liang J, Edelsbrunner H, Fu P, Sudhakar PV, Subramaniam S: Analytical shape computation of macromolecules: II. Inaccessible cavities in proteins. Proteins 1998, 33(1):18-29.
317. Liang J, Edelsbrunner H, Fu P, Sudhakar PV, Subramaniam S: Analytical shape computation of macromolecules: I. Molecular area and volume through alpha shape. Proteins 1998, 33(1):1-17.
318. Kaminski GA, Friesner RA, Tirado-Rives J, Jorgensen WL: Evaluation and Reparametrization of the OPLS-AA Force Field for Proteins via Comparison with Accurate Quantum Chemical Calculations on Peptides. Journal of Physical Chemistry 2001(105):6474-6487.
148
319. Friesner RA, Banks JL, Murphy RB, Halgren TA, Klicic JJ, Mainz DT, Repasky MP, Knoll EH, Shelley M, Perry JK et al: Glide: a new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. J Med Chem 2004, 47(7):1739-1749.
320. Halgren TA, Murphy RB, Friesner RA, Beard HS, Frye LL, Pollard WT, Banks JL: Glide: a new approach for rapid, accurate docking and scoring. 2. Enrichment factors in database screening. J Med Chem 2004, 47(7):1750-1759.
321. Friesner RA, Murphy RB, Repasky MP, Frye LL, Greenwood JR, Halgren TA, Sanschagrin PC, Mainz DT: Extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes. J Med Chem 2006, 49(21):6177-6196.
322. Plouffe D, Brinker A, McNamara C, Henson K, Kato N, Kuhen K, Nagle A, Adrian F, Matzen JT, Anderson P et al: In silico activity profiling reveals the mechanism of action of antimalarials discovered in a high-throughput screen. Proc Natl Acad Sci U S A 2008, 105(26):9059-9064.
323. Lipinski C: Lead- and drug-like compounds: the rule-of-five revolution. Drug Discov Today 2004, 1(4):337-341.
324. Ham S: Studies on Menadione as an Inhibitor of the cdc25 Phosphatase. Bioorganic Chemistry 1997, 25:33-36.
325. Brohm D, Philippe N, Metzger S, Bhargava A, Müller O, Lieb F, Waldmann H: Solid-phase synthesis of dysidiolide-derived protein phosphatase inhibitors. Journal of the American Chemical Society 2002, 124:13171-13178.
326. Dobson S, May T, Berriman M, Del Vecchio C, Fairlamb aH, Chakrabarti D, Barik S: Characterization of protein Ser/Thr phosphatases of the malaria parasite, Plasmodium falciparum: inhibition of the parasitic calcineurin by cyclophilin-cyclosporin complex. Molecular and biochemical parasitology 1999, 99:167-181.
327. Boutros T, Chevet E, Metrakos P: Mitogen-activated protein (MAP) kinase/MAP kinase phosphatase regulation: roles in cell growth, death, and cancer. Pharmacol Rev 2008, 60(3):261-310.
149
328. Contour-Galcera MO, Sidhu A, Prevost G, Bigg D, Ducommun B: What's new on CDC25 phosphatase inhibitors. Pharmacol Ther 2007, 115(1):1-12.
329. Ludin P, Woodcroft B, Ralph S, Maser P: In silico prediction of antimalarial drug target candidates. INternational journal for parasitology 2012, 2:191-199.
330. Low H, Chua CS, Sim T-S: Regulation of Plasmodium falciparum Pfnek3 relies on phosphorylation at its activation loop and at threonine 82. Cellular and molecular life sciences : CMLS 2009, 66:3081-3090.
331. Surachetpong W, Singh N, Cheung KW, Luckhart S: MAPK ERK signaling regulates the TGF-beta1-dependent mosquito response to Plasmodium falciparum. PLoS pathogens 2009, 5:e1000366.
332. Gagaring K, Borboa R, Francek C, Chen Z, Buenviaje J, Plouffe D, Winzeler E, Brinker A, Diagana T, Taylor J et al: Novartis-GNF Malaria Box Dataset. In. Edited by Novartis: Genomics Institute of the Novartis Research Foundation (GNF); 2010.
333. Guiguemde WA, Shelat AA, Bouck D, Duffy S, Crowther GJ, Davis PH, Smithson DC, Connelly M, Clark J, Zhu F et al: Chemical genetics of Plasmodium falciparum. Nature 2010, 465(7296):311-315.
334. McGovern SL, Shoichet BK: Information decay in molecular docking screens against holo, apo, and modeled conformations of enzymes. J Med Chem 2003, 46(14):2895-2907.
150
Appendices
151
Appendix A: Transfection Plasmids
Figure AI: The helper plasmid codes for the piggyBac transposase used in random insertional mutagenesis. This helper plasmid was used in both the initial transfection that knocked out PF13_0027 and in the complementation experiments.
152
Appendix A: continued
Figure AII: Plasmid used for the initial transfection knocking out PF13_0027. The region between the ITRs was randomly inserted into the N-terminal region of PF13_0027.
153
Appendix A: continued
Figure AIII: Plasmid used for complementation of C9. Complementation of C9 with PF13_0027 required this construct with the N-terminal intergenic promoter region (above schematic) and 3’CAM termination sequence. To differentiate transfected clones a BSD selection marker was used in this second round of transfection.
pL-BACII-HBH
PF13_0027
PF13_0027
154
Appendix B: Primers
Table BI: Primer list
Gene Primers
PCR
PF13_0027 5′-CACCCTACCCCTGTATTATTTCCTACCCTC-3 5′-GTTTATGTAATTATTTATTACTATAAATGGTC-3
BSD 5′-GCCCTCCCACACATAACCAGAGGGCA-3′ 5′-GCCTTTGTCTCAAGAAGAATCCACCCT-3′
hDHFR 5′-ATGGTTGGTTCGCTAAACTG-3′ 5′-TTAATCATTCTTCTCATATACTTCAAA-3′
Episome 5′-AGATGTCCTAAATGCACAGCGAC-3′ 5′-CAGGAAACAGCTATGAC-3′
RT-PCR
PF13_0027 5′-CACCATGGAATATAAAAGCATCGATTTTG-3′ 5′-GTTTATGTAATTATTTATTACTATAAATGGTC-3′
18S RNA 5′-AACCTGGTTGATCTTGCCA-3′ 5′-GTATTGTTATTTCTTGTCACTACCTCTC-3′
qRT-PCR
PF13_0027 5′-TCGATTTTGAGGAGCTGAA-3′ 5′-GGGTAAAACATCCTTTTTGTT-3′
Southern Blot
Probe 5′-ATAAGAAGAAGTATATAATGAATTATATATAATGCT-3′
5′-CATATGTATTTTTTTTGTAATTTCTGTGTTTAT-3′ Site-directed Mutagenesis
D345A 5′-CTTTATGTACAAACCAAGTAAAAGCTAATAATACATCATTTATAAAAC-3′ 5′-GTTTTATAAATGATGTATTATTAGCTTTTACTTGGTTTGTACATAAAG-3′
C383A 5′-CAAAATAATATCCTTATTATAGCTAATCATGGAATGAAAAATCCTAC-3′ 5′-GTAGGATTTTTCATTCCATGATTAGCTATAATAAGGATATTATTTTG-3′
K388A/ K394A
5′-GTAATCATGGAATGGCAAATCCTACATCAGAAGCAACAAATAGTATAAGTC-3′ 5′-GACTTATACTATTTGTTGCTTCTGATGTAGGATTTGCCATTCCATGATTAC-3′
155
Appendix C: Flow Cytometry Gating
Figure CI: Gating used to sort the different developmental stages of asexual P. falciparum cultures by flow cytometry. Ring stages with a lower DNA content fluoresce at a lower wavelength in both the FL1-A and FL3-A channels when stained with ethidium bromide, compared to the later stages. Throughout development, as the parasites mature the wavelength of light fluorescing off the infected cells in both channels increases. By using tightly synchronized cultures, we are able to determine the precise pattern and format the gate accordingly to sort different developmental stages. The relative abundance of each developmental stage is then measured and plotted. Overlaying these plots give us a graph that compared the relative abundances and allows us to determine the cell cycle timing and progression.
Schizonts
Trophozoites
Rings
Uninfected cells
156
Appendix D: Whole genome sequencing.
Table DI: Transposon insertions and SNPs in the genomes of NF54, C9 and the complemented parasite lines. A single SNP in the putative clathrin coat assembly protein AP180 was present in all samples. This experiment also validated that the complementation of C9 was episomal
Clone Chromosome Position pB Gene Epi SNP SNP gene
NF54 - - N N N 1 PFIT_1246000
C9 13 271811 Y PF13_0027 N 1 PFIT_1246000
E3 13 271811 Y PF13_0027 Y 1 PFIT_1246000
E8 13 271811 Y PF13_0027 Y 1 PFIT_1246000
157
Appendix E: Southern Blot of complemented parasites
Figure EI: Southern blot analysis of the transfected clones. Using a probe specific to the BSD selection cassette produced signals representative of the complementation plasmid (arrows).
158
Appendix F: PCR Validation of complemented clones
Figure FI: PCRs used to validate the complemented parasite clones. The hDHFR sequence (567 bp) is only present in the parasites that carried the original transposon used to knock out PF13_0027 and was present in all sequences except NF54. The BSD sequence (393 bp) was present in only the positive transfectants and was not present in C9 or NF54. The third reaction was used to test for the presence of the intact complementation plasmid as an indication of episomal retention (608 bp) and was present in all the clones. The fourth reaction used the Southern blot probe (711 bp) as an additional validation that the selected clones carried the complementation plasmid.
159
Appendix G: Statistical Analysis Tables
Table GI: Growth assay analysis for NF54, C9, E3 and E8
Number of families 1
Number of comparisons per family 6
Alpha 0.05
Dunn's multiple comparisons test Significant?
NF54 vs. C9 Yes
NF54 vs. E3 No
NF54 vs. E8 No
C9 vs. E3 Yes
C9 vs. E8 Yes
E3 vs. E8 No
Kruskal-Wallis test
P value 0.0038
Do the medians vary signif. (P < 0.05) Yes
Number of groups 4
Kruskal-Wallis statistic 13.44
160
Appendix G: continued
Table GII: Invasion assay analysis for NF54, C9, E3 and E8
Number of families 1
Number of comparisons per family 6
Alpha 0.05
Dunn's multiple comparisons test Significant?
C9 vs. E3 Yes
C9 vs. E8 Yes
C9 vs. NF54 Yes
E3 vs. E8 No
E3 vs. NF54 No
E8 vs. NF54 No
Kruskal-Wallis test
P value 0.0152
Do the medians vary signif. (P < 0.05) Yes
Number of groups 4
Kruskal-Wallis statistic 8.128
161
Appendix G: continued
Table GIII: Growth assay analysis for NF54, C9, D345A, C383A, and K388A/ K394A
Number of families 1
Number of comparisons per family 10
Alpha 0.05
Dunn's multiple comparisons test Significant?
NF54 vs. C9 Yes
NF54 vs. C383A Yes
NF54 vs. D345A Yes
NF54 vs. K388A K394A No
C9 vs. C383A No
C9 vs. D345A No
C9 vs. K388A K394A Yes
C383A vs. D345A No
C383A vs. K388A K394A Yes
D345A vs. K388A K394A Yes
Kruskal-Wallis test
P value 0.0031
Exact or approximate P value? Exact
Do the medians vary signif. (P < 0.05) Yes
Number of groups 5
Kruskal-Wallis statistic 11.05
162
Appendix G: continued
Table GIV: Invasion assay analysis for NF54, C9, D345A, C383A, and K388A/ K394A
Number of families 1
Number of comparisons per family 10
Alpha 0.05
Dunn's multiple comparisons test Significant?
NF54 vs. C9 Yes
NF54 vs. C383A Yes
NF54 vs. D345A Yes
NF54 vs. K388A K394A No
C9 vs. C383A No
C9 vs. D345A No
C9 vs. K388A K394A Yes
C383A vs. D345A No
C383A vs. K388A K394A Yes
D345A vs. K388A K394A Yes
Kruskal-Wallis test
P value 0.0003
Exact or approximate P value? Exact
Do the medians vary signif. (P < 0.05) Yes
Number of groups 5
Kruskal-Wallis statistic 12.23
163
Appendix H: Bioinformatics
Table HI: Bioinformatics Resources
Tool Version URL
Databases
PlasmoDB 9.2 http://plasmodb.org/plasmo/
GenBank 194 http://www.ncbi.nlm.nih.gov/genbank/
ChEMBL-NTD
https://www.ebi.ac.uk/chemblntd
Prosite 20.89 http://prosite.expasy.org/
Superfamily 1.75 http://supfam.cs.bris.ac.uk/SUPERFAMILY/
Prediction and Characterization
ProtParam
http://web.expasy.org/protparam/
CDD
http://www.ncbi.nlm.nih.gov/cdd/
CDART
http://www.ncbi.nlm.nih.gov/Structure/lexington/lexington.cgi
InterProScan 4.8 http://www.ebi.ac.uk/Tools/pfa/iprscan/
SMART 7 http://smart.embl-heidelberg.de/
NCBI BLASTP
http://blast.ncbi.nlm.nih.gov/Blast.cgi
JPRED 3 http://www.compbio.dundee.ac.uk/www-jpred/
PSIPRED 3.2 http://bioinf.cs.ucl.ac.uk/psipred/
Net Phos 2.0 2 http://www.cbs.dtu.dk/services/NetPhos/
Net PhosK 1.0 1 http://www.cbs.dtu.dk/services/NetPhosK/
Predotar 1.03 http://urgi.versailles.inra.fr/predotar/predotar.html
PATS 1.2.1 http://gecco.org.chemie.uni-frankfurt.de/pats/pats-index.php
Modeling
Swiss-Model
http://swissmodel.expasy.org/
Structure Validation
ANOLEA
http://protein.bio.puc.cl/anolea/index.html
GROMOS
http://www.gromacs.org/Documentation/Terminology/Force_Fields/GROMOS
QMEAN6
http://swissmodel.expasy.org/qmean/cgi/index.cgi
PROCHECK
http://www.ebi.ac.uk/thornton-srv/software/PROCHECK/
DSSP 2 http://swift.cmbi.ru.nl/gv/dssp/
PROMOTIF 3 http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=n/a&template=doc_promotif.html
ERRAT 2 http://nihserver.mbi.ucla.edu/ERRATv2/
Verify3D
http://nihserver.mbi.ucla.edu/Verify_3D/
RAMPAGE
http://mordred.bioc.cam.ac.uk/~rapper/rampage.php
Pocket Finder
http://www.modelling.leeds.ac.uk/pocketfinder/help.html
Q-Site Finder
http://www.modelling.leeds.ac.uk/qsitefinder/
CASTp
http://sts.bioengr.uic.edu/castp/calculation.php
WHAT-IF 8 http://swift.cmbi.ru.nl/whatif/
164
Appendix I: Content Permissions
Malaria life cycle:
Content Providers: CDC/Alexander J. da Silva, Ph.D/ Melanie Moser
Permission: PD-USGov-HHS-CDC
This image is in the public domain and thus free of any copyright restrictions. As a matter of courtesy we request that the content provider be credited and notified in any public or private usage of the image.
http://www.dpd.cdc.gov/dpdx/images/ParasiteImages/M-R/Malaria/malaria_LifeCycle.gif
Maps
Malaria Atlas Project
Creative Commons Attribution 3.0 Unported (CC by 3.0)
Free to share (copy, distribute and transmit the work); remix (adapt the work); make commercial use of the work. http://creativecommons.org/licenses/by/3.0/
http://www.map.ox.ac.uk/
Top Related