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Partial Characterization of PF13_0027: A PutativePhosphatase of Plasmodium falciparumChristopher CampbellUniversity of South Florida, cocampbell@gmail.com

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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

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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:

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Permission: PD-USGov-HHS-CDC

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http://www.dpd.cdc.gov/dpdx/images/ParasiteImages/M-R/Malaria/malaria_LifeCycle.gif

Maps

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