Research Article Antitubercular Activity Analysis of...

Research ArticleIn Silico Antitubercular Activity Analysis of Benzofuran andNaphthofuran Derivatives

Prashantha Karunakar1 Chamarahalli Ramakrishnaiyer Girija2

Venkatappa Krishnamurthy1 Venkatarangaiah Krishna3

and Kunigal Venugopal Shivakumar1

1 Department of Biotechnology PES Institute of Technology BSK III Stage Bangalore 560085 India2Department of Chemistry SSMRV College Jayanagar 4th T Block Bangalore 560 041 India3 Department of Biotechnology and Bioinformatics Kuvempu University Shankaraghatta 577451 India

Correspondence should be addressed to Prashantha Karunakar prashanthakarunakarpesedu

Received 7 May 2014 Accepted 10 July 2014 Published 11 September 2014

Academic Editor W N Rom

Copyright copy 2014 Prashantha Karunakar et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

For the human health Mycobacterium tuberculosis (MTB) is the deadliest enemy since decades due to its multidrug resistantstrains During latent stage of tuberculosis infection MTB consumes nitrate as the alternate mechanism of respiration in theabsence of oxygen thus increasing its survival and virulence NarL is a nitratenitrite response transcriptional regulatory protein oftwo-component signal transduction system which regulates nitrate reductase and formate dehydrogenase for MTB adaptation toanaerobic condition Phosphorylation by sensor kinase (NarX) is the primary mechanism behind the activation of NarL althoughmany response regulators get activated by small molecule phospho-donors in the absence of sensor kinase Using in silico approachthemolecular docking of benzofuran and naphthofuran derivatives and dynamic study of benzofuran derivative were performed Itwas observed that compound Ethyl 5-bromo-3-ethoxycarbonylamino-1-benzofuran-2-carboxylate could be stabilized at the activesite for over 10 ns of simulation Here we suggest that derivatives of benzofuranmoiety can lead to developing novel antituberculosisdrugs

1 Introduction

Furan having various pharmacological and biological activ-ities such as antituberculosis [1] anti-inflammatory [2] andantibacterial [3] activities attracted the attention of syntheticchemists Antitubercular activity has been observed [4] inin vitro analysis using resazurin assay by microtiter-platemethod (REMA) of benzofuran derivatives Substituted ben-zofurans find their applications in different fields such asfluorescent sensors [5] antioxidants brightening agents anda variety of drugs and agriculture

In the major global infectious diseases Mycobacteriumtuberculosis (MTB) is second after HIVAIDS 14 millionpeople died and 87million people fell ill fromTBduring 2011In low-income and middle-income countries over 95 of TBdeaths occur and it is among top three causes of death forwomen aged from 15 to 44 [6] The availability of nutrients

in the host and their metabolism by MTB plays a majorrole in mycobacterial pathogenesis and persistence duringanaerobic condition Due to multidrug-resistant tuberculosis(MDR-TB) bacteria could not respond to either isoniazid orrifampicin the twomost powerful first-line or standard anti-TB drugs Mycobacteria can adapt to the host environmentby two-component signal transduction system (TCS) wherethey sense respond and adapt to changes in the environmentby modulating the expression of subsets of genes in responseto specific extracellular signals

There are 11 complete TCSs in MTB [7] A prototypicaltwo-component system consists of a histidine protein kin-ase (HK) and a response regulator (RR) RRs have a modulararchitecture consisting of a conserved receiver domain and avariable effector domain consisting of DNA binding elements[8] NarX-NarL and NarQ-NarP are two-component systemsthat are involved in the regulation of anaerobic respiratory

Hindawi Publishing CorporationTuberculosis Research and TreatmentVolume 2014 Article ID 697532 10 pageshttpdxdoiorg1011552014697532

2 Tuberculosis Research and Treatment

gene expression in response to nitrate and nitrite [9] NarXis a membrane-bound nitrate sensor [10] and NarL is a cyto-plasmic response regulator consisting of N-terminal receiverdomain and C-Terminal effector domain NarL is involvedin the synthesis of nitrate reductase (encoded by narGHJI)and formate dehydrogenase-N (encoded by fdnGHI operon)in the presence of nitrate and these enzymes are involvedin nitrate respiration [11] NarQ is a sensor kinase whichcan independently sense the presence of nitrate and transferthe signal to NarL Furthermore small molecule phosphor-donors such as acetyl phosphate can also phosphorylate NarLin vitro [12] The phosphorylation-dependent activation ofNarL exposes the C-Terminal domain for binding to theDNA to activate or repress the transcription [13] Duringanaerobic growth nitrate acts through NarL to activate andrepress the anaerobic respiratory gene expression [9] Thegene narL coding for NarL protein is upregulated four-fold inthe late stationary phase cultures of MTB [14] There is 35overall amino-acid identity between E coli and MTB NarLand their active sites superimpose exactly and the positions ofthe active site residues are nearly identical [30] Based on thissequence homology NarL has been predicted to be involvedin the regulation of anaerobic nitrogen metabolism in MTB(Tuberculist website httpgenolistpasteurfrTubercuList)and is identified as potential drug target (httpwwwuniprotorguniprotO53856) Blocking the active site will inhibit thephosphorylation activity of NarL and negatively affects theMTB adaptation

2 Materials and Methods

Protein-ligand interaction of ethyl naphtho[21-b]furan-2-carbothioate (C1) 2-naphtho[21-b]furan-2-yl-5-phenyl-134-oxadiazole (C2) ethyl 8-nitronaphtho[21-b]furan-2-car-boxylate (C3) 1-(2-chlorophenyl)-1-(8-nitronaphtho[21-b]furan-2-yl)urea (C4) (4-nitrophenyl)-1-(8-nitronaphtho[21-b]furan-2-yl)urea (C5) ethyl 3-amino-5-bromo-1-benzofu-ran-2-carboxylate (C6) ethyl 5-bromo-3-ethoxycarbonylamino-1-benzofuran-2-carboxylate (C7) and ethyl naph-thofuran-2-carboxylate (C8) with protein target NarL ofMycobacterium tuberculosis was carried out to understand itsantitubercular activity

21 Antitubercular Activity of Benzofuran and NaphthofuranDerivatives For the molecular docking analysis newly syn-thesized furan derivatives were considered to analyze theantitubercular activity In these synthesized furan derivativestwo compound structures were solved crystallographicallymainly C6 [15] and C7 [16] The three-dimensional atomiccoordinates of C6 and C7 were prepared using WinGX [17]Since the conformation of the benzofuran and naphthofuranderivatives have been determined from single crystal X-raydiffraction it is worthy to look at their binding pattern toobtain meaningful insights for these experimentally deter-mined conformations These studies also provide inputs fordrug design which reveal conformational variant at the activesite of protein [18]The coordinates of these compounds alongwith other derivatives were further extrapolated for dockingstudies

22 NarL Structure The NarL protein (in the asymmetricunit) is a homotetramer containing 4 identical subunitshaving their independent active sites each for subunits AB C and D respectively This structure represents theN-terminal signal receiver domain containing 4 identicalchains The coordinates of crystal structure of 19 A of thesignal receiver domain of the putative response regulatorNarL fromMycobacterium tuberculosis (PDB ID 3EUL) wasobtained from the Protein Data Bank Though protein ishomotetramer the crystallographer determined biologicallyactive monomeric form was considered for the docking anddynamics simulation study Figure 1 shows the active siteresidues Asp15 Asp16 Arg63 Ser89 and Lys111 and site ofphosphorylation Asp-61 [30] Schnell et al have indicatedthat from native PAGE analysis and the elution profile in sizeexclusion chromatography and gel-filtration chromatogra-phy NarL-N is a monomer in solution and it migrated in thenative gel as a single well-defined band Hence biologicallyactive unit has been considered for docking and moleculardynamics

23 Molecular Docking Processing Topology file and otherforce field parameters were generated for ligand using thePRODRG program [19] The Kollman charges were addedto the protein atoms and the Gasteiger partial charges wereassigned to the ligand atoms using the program AutoDockAll the ligands were converted to pdbqt format using Open-Babel tool built inside PyRx 08 docking tool Flexible torsionsfor all ligands were defined using AUTOTORS The dockingsite for the ligands on 3EULwas defined at the active site withgrid box size 48 times 56 times 48 spacing 0375 and grid centreminus9584 3945 and 1545 The autogrid module calculatedthe electrostatic map and atomic interaction maps for allatom types of the ligand molecules The Lamarckian GeneticAlgorithm (LGA) was selected with the population size of150 individuals and with a maximum number of generationsand energy evaluations of 27000 and 25million respectivelyDefault parameters like elitism (1) mutation (002) andcrossover rate (08) were considered From the estimated freeenergy of ligand binding (Δ119866) the inhibition constant (119870

119894)

for the ligandswas calculated Results from theAutoDock runwere analyzed using molecular graphics laboratory (MGL)tools [20] and the best conformation with least bindingenergy interaction was analyzed using Ligplot+ [21] PyMOLwas used for docking conformation representation [22]

To overcome any serendipity positive controls such asacetyl phosphate carbamoyl phosphate dihydroxyacetone-P phosphoramidate and monophosphoimidazole were usedthroughout the docking procedure Interaction profile fornatural phospho-donors is included in Table 1

24 Molecular Dynamics Molecular dynamics simulationwas carried out to study the real time interaction and stabilityof the top scoring compounds in the biological environmentThe study also gives the detailed description of the molecularinteractions in the solvent environment which is very closeto reality Molecular dynamics simulation was carried out for10 ns using Gromacs version 455

Tuberculosis Research and Treatment 3

ARG-63ASP-61

ASP-16

ASP-15

LYS-111SER-89

Figure 1 Active site residues of NarL are represented in ball and stick

Table 1 Energy profile of small molecule phospho-donors

SI number Compound AutoDock 42 binding energy (kcalmol) Inhibition constant1 Acetyl phosphate minus393 131mM2 Carbamoyl phosphate minus382 158mM3 Dihydroxyacetone-P minus332 367mM4 Phosphoramidate minus384 154mM5 Monophosphoimidazole minus382 158mM

25 Premolecular Dynamics Processing The system con-tained protein water molecules and appropriate number ofsodium ions all enclosed in a periodic box Hydrogens andcharges were added and protein was defined using Amber-99SB force field parameters

Using UCSF chimera visualization tool [23] protein wasprepared for dry run by explicitly adding hydrogens andAM1-BCC partial charges Protein was defined using Amber-99SB force field parameters [24] Ligand was defined usinggeneralized amber force field (GAFF) parameters [25] andAM1-BCC partial charges were added using ANTECHAM-BER [26] followed by conversion to GROMACS compatibletopology using ACPYPE [27] Complex was prepared forproduction run using chimera

26 Molecular Dynamics Simulation All MD simulationswere performed using GROMACS version 455 compiled insingle-precision mode [28 29] A simulation cell was createdin a cubic periodic box with a minimum distance of 10 Abetween the protein and the box walls The protein wasbathed with TIP3P water molecules along with four sodiumions to neutralize the system (Figure 2)

Energy minimization was performed by using 5000 stepsof steepest descent coupled with conjugate gradient method

Figure 2 Neutralization of system by addition of four sodiumions Stick model represents the water molecules and ball modelrepresents NA ions (Blue) and protein is shown as ribbon enclosedinside the box with secondary structure coloring

at every 100 steps or until the maximum force was smallerthan 100 kJmolminus1 nmminus1 The electrostatic interactions par-ticularly of long range were calculated using Particle-MeshEwald method (PME) The cutoff for distance was 1 nmThe dispersion interactions both short-range repulsive andattractive as described by Lennard-Jones had a cutoff of


Table 2 Molecular docking interaction details of benzofuran and naphthofuran derivatives against NarL protein

Compounds BElowast LElowast IC+ IElowast VElowast EElowast TIlowast TElowast

C1 minus699 minus039 754 minus788 minus782 minus006 02 089C2 minus526 minus022 14042 minus585 minus563 minus022 minus039 06C3 minus376 minus018 1750 minus525 minus537 012 minus032 149C4 minus514 minus019 17181 minus603 minus593 minus01 minus08 089C5 minus431 minus015 69653 minus55 minus555 005 minus043 119C6 minus521 minus025 15205 minus76 minus76 0 0 239C7 minus413 minus026 94059 minus562 minus535 minus027 minus018 149C8 minus441 minus025 58262 minus531 minus55 02 minus011 089BE = binding energy LE = ligand efficiency IC = inhibition constant IE = intermolecular energy VE = Van der Waals + hydrogen bonding + desolvationenergy EE = electrostatic energy TI = total internal energy TE = torsional energy lowast = kcalmol and + = micro molar

1 nm The LINCS algorithm was used to constrain bondsallowing a time step of 1 fs At every 10 steps neighbor search-ing was carried out A Parrinello-Rahman barostat pressureof 1 bar was used with a coupling constant of Tau P = 05 psand compressibility of 45e-5 (barminus1) Water protein ionsand ligand molecules were coupled separately to the thermalbath at 300K using a v-rescale coupling constant Tau T= 01 ps An initial preparatory run for 500 ps was carriedout to allow the randomization of water molecules aroundthe protein The position restrained molecular dynamics wascarried out using mdrun program The isobaric-isothermalensemble simulation was performed for 10 ns The resultswere analyzed in VMD tool and Gromacs

3 Results and Discussions

31 Molecular Docking Studies of Benzofuran and Naphthofu-ran Derivatives Chemical structures of the benzofuran andnaphthofuran derivatives considered for docking are listed inFigure 3

32 Antitubercular Activity Study of Benzofuran and Naph-thofuran Derivatives The in silico interaction of phospho-donors to NarL was carried out and it was observed thatall compounds were binding to active site though blinddocking was performed All the natural phospho-donors(acetyl phosphate carbamoyl phosphate phosphoramidateandmonophosphoimidazole) bind to active site with bindingenergy ranging from minus332 to minus393 kcalmol and inhibitionconstant from 131mM to 367mM (Table 1)

The conformation with least binding energy and moststability based on cluster analysis was taken for dockinganalysis The docking interaction details are represented inTable 2 Out of 8 compounds docked C1 shows least bindingenergy of minus699 kcalmol and C3 with highest binding energyof minus376 kcalmol From the estimated inhibition constant itis observed that micromolar concentration of all compoundsis sufficient to inhibit the protein activity by 50 (IC

50) The

less quantity of 754mM is required to have desired IC50effect

when compared to C7 which requires 940mM From theLigplot+ diagrams (Figure 4) it is observed that compoundsC1 C3 C5 and C8 show a hydrophobic interaction OneN-Hsdot sdot sdotO hydrogen bond was observed in C2 C4 C6and C7 at the active site of NarL Either the nitrogen of

ligand or nitrogen of active site residue is involved in bondformation Compounds C6 and C7 formed hydrogen bondwith site of phosphorylation residue Asp16 bromine atomand carboxylate group showing hydrophobic interaction atthe active site C2 and C4 hydrogen bonded with Lys111and Arg63 respectively Similar N-Hsdot sdot sdotO intermolecularhydrogen bonding was observed in crystal packing of C6 andC7 [15 16]

In C7 intermolecular N1-H1Asdot sdot sdotO2 (symmetry code119909 + 1 minus119910 + 12 119911 + 12) was observed with bond distanceof 2977 (4) A and angle of 171 (3)∘ However the resultof docking correlates more with intramolecular hydrogenbonding N1-H1Bsdot sdot sdotO3 with bond distance of 2835 (4) A andangle of 123 (3)∘ The hydrogen bonding length and anglesof C6 and C7 are represented in Figure 5 The hydroxylgroup appears to hold the strong intramolecular hydrogenbonded feature as observed in the crystal structure of ligandThough binding energy inhibition constant are used asimportant indicators in molecular docking studies theycannot offer complete characteristics of the interaction inall the cases thus the use of dynamic simulation becomesinevitable

Figure 6 shows the molecular docking interaction ofcompound 6 alone as it was further extrapolated to dynamicssimulation Figure 7 depicts the interaction of all ligands atthe active site of NarL This suggests that these groups areresponsible for stabilizing the interactions in the active siteand blocking the site of phosphorylation of NarL

33 Molecular Dynamics Simulation Analysis The crystallo-graphically solved (C7) structure was further extrapolatedto dynamics studies The molecular dynamics simulationshowed hydrogen bonding for most parts of 10 ns run(Figure 8) Though the compound was fixed at the active site(Pocket-1) it moved to another (pocket-2) and interactedwith Leu47 Met67 and Gln71 Ala75 and Ser78 and Tyr79(Figure 9) There is a local structural difference at the loopregion and 120572-helix region from 64 to 77 in both NarL of Mtuberculosis and E coli The residue Met67 has the maximaldisplacement of 77 A in C120572 position as compared to NarL ofM tuberculosis and E coli Even from the crystallography it isunclear whether this loop region difference is due to crystallattice or it reflects any biological relevant loop conformation[30] This movement could be due to less steric hindrance


C5

C6

O

Br

NO

O

H

O

O

C7

C1

O

S

O

carbothioate

OO2N ONH

HN

NO2

(4-Nitrophenyl)-1-(8-nitronaphtho[21-b]

C2 O

N

O

N

phenyl-134-oxadiazole

C3O O

O

O2NO

Br O

O

NH2

OC4

O OCl

O2N

NH

HN

C8

O

O

Ethyl naphtho[21-b]furan-2-carboxylate

2-carboxylate

carboxylateethoxycarbonylamino-1-benzofuran-2-carboxylate

1-(2-Chlorophenyl)-1-(8-nitronaphtho[21-b]furan-2-yl)urea

furan-2-yl)ureaEthyl naphtho[21-b]furan-2-

Ethyl 3-amino-5-bromo-1-benzofuran-

Ethyl 8-nitronaphtho[21-b]furan-2-Ethyl 5-bromo-3-

2-Naphtho[21-b]furan-2-yl-5-

Figure 3 Benzofuran and naphthofuran derivatives structures used in docking studies

with Ala 44 Met 67 and Ala 74-75 and aiding the ligandto be in the hydrophobic pocket and the remaining part ofthe ligand to be in the polar region of the protein Figure 10represents the interaction of C7 in the active site of NarL aftermolecular dynamic simulation

4 ConclusionFrom the docking study it can be noticed that the crys-tallographically determined structures C6 and C7 possess abromine atom along with the benzofuran ring It showed thatthe hydrophobic interaction at the active site and the nitro


C4

322C1

C2

C3C4

C5 C6

C7

C8

C9 C10

C11

C12

O1

N1 O2

O3

N2

C13

O4

N3

C14C15

C16

Cl

C17

C18

C19

N

CACO

CBCG

CD

NECZ

NH1

NH2

Arg63

Pro65

Asp16

Asp61

His91

Ala90

Ser89

C1

C1

C2

C3C4

C5C6

C7C8

C9

C10

C11

C12

O1

C13

SO2

C14C15

Phe20

Lys111

Ala90

Asp61

N

C O

C2

257

C1 C2C3 C4

C5 C6

C7 C8

C9

C10

C11

C12

O1

C13N1

N2C14

O2

C15C16

C17C18C19

C20

CA

CB

CG

CD

CE

NZLys111

His17

Arg63

Phe20

Asp61

Asp112

Asp15

Asp16

His91

C5

C1

C2

C3

C4

C5C6

C7

C8

C9 C10 C11C12O1

N1

O2

O3

N2C13

O4N3C14C15

C16C17C18

C19

N4O5O6

Lys111

His17

Asp16

Pro18

Asp61

Ala90

Arg63

Ser89His91

C3

C1C2

C3C4

C5C6

C7C8

C9

C10

C11

C12

O1

NO2

O3

C13O4 O5

C14C15

Lys111

Phe20

Ala90

Asp61

His17

Ser89

Leu19

Arg63

C6

259

C1

C2

C3

C4

C5

C6

C7C8

C9

C12

C13C14

N1

O1

O2O3

O4

Br

O5

C10

C11

N

CACO

CB

CG OD1OD2

Asp16

His17

Asp61

Arg63

Lys111

Ser89Pro65

His91

C7

281

C1C2C3

C4C5C6

C7

C8

O1

Br

N

C9O2

O3

C10

C11

Arg63

NCA

CO

CB

CGOD1

OD2

Asp16

His91

Met64

Pro65

Lys111

Asp61

C8

C1C2

C3C4

C5C6

C7C8

C9

C10

C11

C12

O1

C13O2 O3

C14C15

Arg63

Lys111

Asp61

Asp16

Met64

Pro65

Ala90

His17

Ser89

His91

Figure 4 2D representation using Ligplot+ of all compounds interacting at the active site of NarL Hydrogen bonds are represented by dashedlines


H1

AH1

1AA1

4BH1

C14

4CH1

3BH1

3AH1

C13C12

O3

O2

N1

H1

1200

C7C8

C9

C5

C6H6

26

O5O4

Br

C1 C2

C3C4

O1

H3

H2

0BH1C10 1BH1

0AH1

OD2 OD1

ASP-16

CBN

C

CG11

(a)

H

H H

H

HHHH

HH

C

CC

C

C

C

C C

C

C

C

O

O

O

N

N

OD2 OD1

ASP-16CG

CB

Br

28

1299

(b)

Figure 5 N-Hsdot sdot sdotO intermolecular hydrogen bonding of C6 (a) and C7 (b) compared with crystal packing

ARG63 (B)

ASP61 (B)HIS91 (B)

SER89 (B)

LYS111 (B)

HIS17 (B)

ASP16 (B)

PRO65 (B)

Figure 6 Interaction of C6 at the active site of NarL

group forms hydrogen bonding with aspartic acid 16 therebystabilizing the complex From the molecular dynamics sim-ulation study on the compounds it can be suggested thatall benzofuran and naphthofuran derivatives have affinity tointeract at the active site of NarL and have the potential to

act as lead molecules From the molecular dynamics studyit was observed that local structural difference at the loopand 120572-helix regions from 64 to 77 residues is flexible for themovement of ligand towards Met67 This movement couldbe due to less steric hindrance with Ala 44 Met 67 and Ala


ARG63 (B)

HIS91 (B)

ASP61 (B)

SER89 (B)

LYS111 (B)

PRO65 (B)

ASP16 (B)

HIS17 (B)

Figure 7 Interaction of all ligands at the active site of NarL

Distance

Dist

ance

(nm

)

Time (ps)0 2000 4000 6000 8000 10000

6

5

4

3

2

1

Ldl

2000 4000 6000 8000 10000

2

15

1

05

0

0

Num

ber

Time (ps)

Hydrogen bonds

Hydrogen bonds

Figure 8 Distance plot of C7 from the active site residue Asp-61 through the 10 ns

74-75 and aiding the ligand to be in the hydrophobic pocketand the remaining part of the ligand to be in the polar regionof the protein Also there is no hydrogen bonding betweenthe ligand and theNarL protein Another crystallographicallydetermined structure C8 shows only hydrophobic interactionin the docking studies

From the docking analysis of naphthofuran andbenzofuran derivatives it can be concluded that in most of

the interactions nitro group formed hydrogen bond withAsp16 bromine atom and carboxylate group showinghydrophobic interaction at the active site Bindingenergy and inhibition constant are also minimal showingthat these compounds have high affinity towards NarLprotein This suggests that these groups are responsiblefor stabilizing the interactions in the active site therebyblocking the site of phosphorylation of NarL Hence these


HIS-91 ARG-63

GLN-71

SER-89

LYS-111

HIS-17 ASP-16

ASP-61PRO-65

MET-67

ALA-75

LEU-47

SER-78

TYR-79

Figure 9 Movement of ligand C7 interaction from pocket-1 (orange) to pocket-2 (blue)

C1

C2

C3C4

C5

C6

C7C8

C9

C12

C13C14

N1

O1

O2O3

O4

O5

C10

C11

Tyr79

Ala75

Ser78

Leu47

Met67

Gln71

Figure 10 Interaction of C7 in the active site of NarL after molecular dynamic simulation

compounds may act as a potential antitubercular leadmolecule

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Authors thank Professor V P Vaidya Department ofChemistry Kuvempu University Shankaraghatta India for

providing the furan derivatives formolecular docking studiesand Dr Roshan Makam Department of Biotechnology PESInstitute of Technology Bangalore India for his usefulinteraction on dynamics studies

References

[1] N R Tawari R Bairwa M K Ray M G R Rajan and M SDegani ldquoDesign synthesis and biological evaluation of 4-(5-nitrofuran-2-yl)prop- 2-en-1-one derivatives as potent antitu-bercular agentsrdquo Bioorganic ampMedicinal Chemistry Letters vol20 no 21 pp 6175ndash6178 2010


[2] J-S Shin S-J Park S Ryu et al ldquoPotent anti-inflammatoryeffect of a novel furan-2 5-dione derivative BPD mediated bydual suppression of COX-2 activity and LPS-induced inflam-matory gene expression via NF-120581B inactivationrdquo British Journalof Pharmacology vol 165 no 6 pp 1926ndash1940 2011

[3] C Kirilmis M Ahmedzade S Servi M Koca A Kizirgiland C Kazaz ldquoSynthesis and antimicrobial activity of somenovel derivatives of benzofuran part 2 The synthesis andantimicrobial activity of some novel 1-(1-benzofuran-2-yl)-2-mesitylethanone derivativesrdquo European Journal of MedicinalChemistry vol 43 no 2 pp 300ndash308 2008

[4] V N Telvekar A Belubbi V K Bairwa and K SatardekarldquoNovel N1015840-benzylidene benzofuran-3-carbohydrazide deriva-tives as antitubercular and antifungal agentsrdquo Bioorganic ampMedicinal Chemistry Letters vol 22 no 6 pp 2343ndash2346 2012

[5] O Oter K Ertekin C Kirilmis M Koca and M AhmedzadeldquoCharacterization of a newly synthesized fluorescent benzofu-ran derivative and usage as a selective fiber optic sensor forFe(III)rdquo Sensors and Actuators B Chemical vol 122 no 2 pp450ndash456 2007

[6] WHO ldquoWorld Health Organization Global tuberculosis con-trol WHO report 2012rdquo 2012 httpappswhointirisbitst-ream106657593819789241564502 engpdf

[7] S T Cole R Brosch J Parkhill et al ldquoDeciphering the biologyof mycobacterium tuberculosis from the complete genomesequencerdquo Nature vol 393 no 6685 pp 537ndash544 1998

[8] R Gao T R Mack and A M Stock ldquoBacterial response reg-ulators versatile regulatory strategies from common domainsrdquoTrends in Biochemical Sciences vol 32 no 5 pp 225ndash234 2007

[9] R S Rabin and V Stewart ldquoEither of two functionally redun-dant sensor proteins NarX and NarQ is sufficient for nitrateregulation in Escherichia coli K-12rdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 89 no18 pp 8419ndash8423 1992

[10] R Cavicchioli I Schroder M Constanti and R P Gun-salus ldquoThe NarX and NarQ sensor-transmitter proteins ofEscherichia coli each require two conserved histidines fornitrate-dependent signal transduction to NarLrdquo Journal ofBacteriology vol 177 no 9 pp 2416ndash2424 1995

[11] H Wang and R P Gunsalus ldquoCoordinate regulation of theEscherichia coli formate dehydrogenase fdnGHI and fdhF genesin response to nitrate nitrite and formate roles for NarL andNarPrdquo Journal of Bacteriology vol 185 no 17 pp 5076ndash50852003

[12] I Schroder C D Wolin R Cavicchioli and R P GunsalusldquoPhosphorylation and dephosphorylation of the NarQ NarXand NarL proteins of the nitrate-dependent two-componentregulatory system of Escherichia colirdquo Journal of Bacteriologyvol 176 no 16 pp 4985ndash4992 1994

[13] J H Zhang G Xiao R P Gunsalus and W L Hubbell ldquoPhos-phorylation triggers domain separation in the DNA bindingresponse regulator NarLrdquo Biochemistry vol 42 no 9 pp 2552ndash2559 2003

[14] Y Hu and A R Coates ldquoIncreased levels of sigJ mRNA inlate stationary phase cultures of Mycobacterium tuberculosisdetected by DNA array hybridisationrdquo FEMS MicrobiologyLetters vol 202 no 1 pp 59ndash65 2001

[15] A J Yamuna P Karunakar C R Girija V P Vaidya andV Krishnamurthy ldquoEthyl 3-amino-5-bromo-1-benzofuran-2-carboxylaterdquoActa Crystallographica E Structure Reports Onlinevol 69 no 5 p o775 2013

[16] P Karunakar V Krishnamurthy C R Girija V Krishna VP Vaidya and A J Yamuna ldquoEthyl 5-bromo-3-ethoxycarbo-nylamino-1-benzofuran-2-carboxylaterdquo Acta CrystallographicaE vol 69 no 3 article o342 2013

[17] L J Farrugia ldquoWinGX suite for small-molecule single-crystalcrystallographyrdquo Journal of Applied Crystallography vol 32 no4 pp 837ndash838 1999

[18] S K Nayak S B Mallik S P Kanaujia et al ldquoCrystal structuresand binding studies of atovaquone and its derivatives withcytochrome bc1 a molecular basis for drug designrdquo CrystEng-Comm vol 15 no 24 pp 4871ndash4884 2013

[19] D M F van Aalten ldquoPRODRG a program for generatingmolecular topologies and unique molecular descriptors fromcoordinates of small moleculesrdquo Journal of Computer-AidedMolecular Design vol 10 no 3 pp 255ndash262 1996

[20] M F Sanner ldquoPython a programming language for softwareintegration and developmentrdquo Journal of Molecular Graphicsand Modelling vol 17 no 1 pp 57ndash61 1999

[21] A CWallace R A Laskowski and J MThornton ldquoLIGPLOTa program to generate schematic diagrams of protein-ligandinteractionsrdquoProtein Engineering vol 8 no 2 pp 127ndash134 1995

[22] W L DeLanoThe PyMOLMolecular Graphics System DeLanoScientific San Carlos Calif USA 2002

[23] E F Pettersen T D Goddard C C Huang et al ldquoUCSFChimeramdasha visualization system for exploratory research andanalysisrdquo Journal of Computational Chemistry vol 25 no 13pp 1605ndash1612 2004

[24] V Hornak R Abel A Okur B Strockbine A Roitberg andC Simmerling ldquoComparison of multiple amber force fieldsand development of improved protein backbone parametersrdquoProteins Structure Function and Bioinformatics vol 65 no 3pp 712ndash725 2006

[25] J Wang R M Wolf J W Caldwell P A Kollman and D ACase ldquoDevelopment and testing of a general amber force fieldrdquoJournal of Computational Chemistry vol 25 no 9 pp 1157ndash11742004

[26] J Wang W Wang P A Kollman and D A Case ldquoAutomaticatom type and bond type perception in molecular mechanicalcalculationsrdquo Journal of Molecular Graphics and Modelling vol25 no 2 pp 247ndash260 2006

[27] A W S da Silva and W F Vranken ldquoACPYPEmdashantechamberpython parser interfacerdquo BMC Research Notes vol 5 no 1article 367 2012

[28] B Hess C Kutzner D van der Spoel and E LindahlldquoGRGMACS 4 algorithms for highly efficient load-balancedand scalable molecular simulationrdquo Journal of Chemical Theoryand Computation vol 4 no 3 pp 435ndash447 2008

[29] D van der Spoel E Lindahl B Hess G Groenhof A E Markand H J C Berendsen ldquoGROMACS fast flexible and freerdquoJournal of Computational Chemistry vol 26 no 16 pp 1701ndash1718 2005

[30] R Schnell D Agren and G Schneider ldquo19 A structure of thesignal receiver domain of the putative response regulator NarLfromMycobacterium tuberculosisrdquoActaCrystallographica F vol64 no 12 pp 1096ndash1100 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


gene expression in response to nitrate and nitrite [9] NarXis a membrane-bound nitrate sensor [10] and NarL is a cyto-plasmic response regulator consisting of N-terminal receiverdomain and C-Terminal effector domain NarL is involvedin the synthesis of nitrate reductase (encoded by narGHJI)and formate dehydrogenase-N (encoded by fdnGHI operon)in the presence of nitrate and these enzymes are involvedin nitrate respiration [11] NarQ is a sensor kinase whichcan independently sense the presence of nitrate and transferthe signal to NarL Furthermore small molecule phosphor-donors such as acetyl phosphate can also phosphorylate NarLin vitro [12] The phosphorylation-dependent activation ofNarL exposes the C-Terminal domain for binding to theDNA to activate or repress the transcription [13] Duringanaerobic growth nitrate acts through NarL to activate andrepress the anaerobic respiratory gene expression [9] Thegene narL coding for NarL protein is upregulated four-fold inthe late stationary phase cultures of MTB [14] There is 35overall amino-acid identity between E coli and MTB NarLand their active sites superimpose exactly and the positions ofthe active site residues are nearly identical [30] Based on thissequence homology NarL has been predicted to be involvedin the regulation of anaerobic nitrogen metabolism in MTB(Tuberculist website httpgenolistpasteurfrTubercuList)and is identified as potential drug target (httpwwwuniprotorguniprotO53856) Blocking the active site will inhibit thephosphorylation activity of NarL and negatively affects theMTB adaptation

2 Materials and Methods

Protein-ligand interaction of ethyl naphtho[21-b]furan-2-carbothioate (C1) 2-naphtho[21-b]furan-2-yl-5-phenyl-134-oxadiazole (C2) ethyl 8-nitronaphtho[21-b]furan-2-car-boxylate (C3) 1-(2-chlorophenyl)-1-(8-nitronaphtho[21-b]furan-2-yl)urea (C4) (4-nitrophenyl)-1-(8-nitronaphtho[21-b]furan-2-yl)urea (C5) ethyl 3-amino-5-bromo-1-benzofu-ran-2-carboxylate (C6) ethyl 5-bromo-3-ethoxycarbonylamino-1-benzofuran-2-carboxylate (C7) and ethyl naph-thofuran-2-carboxylate (C8) with protein target NarL ofMycobacterium tuberculosis was carried out to understand itsantitubercular activity

21 Antitubercular Activity of Benzofuran and NaphthofuranDerivatives For the molecular docking analysis newly syn-thesized furan derivatives were considered to analyze theantitubercular activity In these synthesized furan derivativestwo compound structures were solved crystallographicallymainly C6 [15] and C7 [16] The three-dimensional atomiccoordinates of C6 and C7 were prepared using WinGX [17]Since the conformation of the benzofuran and naphthofuranderivatives have been determined from single crystal X-raydiffraction it is worthy to look at their binding pattern toobtain meaningful insights for these experimentally deter-mined conformations These studies also provide inputs fordrug design which reveal conformational variant at the activesite of protein [18]The coordinates of these compounds alongwith other derivatives were further extrapolated for dockingstudies

22 NarL Structure The NarL protein (in the asymmetricunit) is a homotetramer containing 4 identical subunitshaving their independent active sites each for subunits AB C and D respectively This structure represents theN-terminal signal receiver domain containing 4 identicalchains The coordinates of crystal structure of 19 A of thesignal receiver domain of the putative response regulatorNarL fromMycobacterium tuberculosis (PDB ID 3EUL) wasobtained from the Protein Data Bank Though protein ishomotetramer the crystallographer determined biologicallyactive monomeric form was considered for the docking anddynamics simulation study Figure 1 shows the active siteresidues Asp15 Asp16 Arg63 Ser89 and Lys111 and site ofphosphorylation Asp-61 [30] Schnell et al have indicatedthat from native PAGE analysis and the elution profile in sizeexclusion chromatography and gel-filtration chromatogra-phy NarL-N is a monomer in solution and it migrated in thenative gel as a single well-defined band Hence biologicallyactive unit has been considered for docking and moleculardynamics

23 Molecular Docking Processing Topology file and otherforce field parameters were generated for ligand using thePRODRG program [19] The Kollman charges were addedto the protein atoms and the Gasteiger partial charges wereassigned to the ligand atoms using the program AutoDockAll the ligands were converted to pdbqt format using Open-Babel tool built inside PyRx 08 docking tool Flexible torsionsfor all ligands were defined using AUTOTORS The dockingsite for the ligands on 3EULwas defined at the active site withgrid box size 48 times 56 times 48 spacing 0375 and grid centreminus9584 3945 and 1545 The autogrid module calculatedthe electrostatic map and atomic interaction maps for allatom types of the ligand molecules The Lamarckian GeneticAlgorithm (LGA) was selected with the population size of150 individuals and with a maximum number of generationsand energy evaluations of 27000 and 25million respectivelyDefault parameters like elitism (1) mutation (002) andcrossover rate (08) were considered From the estimated freeenergy of ligand binding (Δ119866) the inhibition constant (119870

119894)

for the ligandswas calculated Results from theAutoDock runwere analyzed using molecular graphics laboratory (MGL)tools [20] and the best conformation with least bindingenergy interaction was analyzed using Ligplot+ [21] PyMOLwas used for docking conformation representation [22]

To overcome any serendipity positive controls such asacetyl phosphate carbamoyl phosphate dihydroxyacetone-P phosphoramidate and monophosphoimidazole were usedthroughout the docking procedure Interaction profile fornatural phospho-donors is included in Table 1

24 Molecular Dynamics Molecular dynamics simulationwas carried out to study the real time interaction and stabilityof the top scoring compounds in the biological environmentThe study also gives the detailed description of the molecularinteractions in the solvent environment which is very closeto reality Molecular dynamics simulation was carried out for10 ns using Gromacs version 455


ARG-63ASP-61

ASP-16

ASP-15

LYS-111SER-89



















50) The








C5

C6

O

Br

NO

O

H

O

O

C7

C1

O

S

O

carbothioate

OO2N ONH

HN

NO2


C2 O

N

O

N


C3O O

O

O2NO

Br O

O

NH2

OC4

O OCl

O2N

NH

HN

C8

O

O


2-carboxylate











C4

322C1

C2

C3C4

C5 C6

C7

C8

C9 C10

C11

C12

O1

N1 O2

O3

N2

C13

O4

N3

C14C15

C16

Cl

C17

C18

C19

N

CACO

CBCG

CD

NECZ

NH1

NH2

Arg63

Pro65

Asp16

Asp61

His91

Ala90

Ser89

C1

C1

C2

C3C4

C5C6

C7C8

C9

C10

C11

C12

O1

C13

SO2

C14C15

Phe20

Lys111

Ala90

Asp61

N

C O

C2

257

C1 C2C3 C4

C5 C6

C7 C8

C9

C10

C11

C12

O1

C13N1

N2C14

O2

C15C16

C17C18C19

C20

CA

CB

CG

CD

CE

NZLys111

His17

Arg63

Phe20

Asp61

Asp112

Asp15

Asp16

His91

C5

C1

C2

C3

C4

C5C6

C7

C8

C9 C10 C11C12O1

N1

O2

O3

N2C13

O4N3C14C15

C16C17C18

C19

N4O5O6

Lys111

His17

Asp16

Pro18

Asp61

Ala90

Arg63

Ser89His91

C3

C1C2

C3C4

C5C6

C7C8

C9

C10

C11

C12

O1

NO2

O3

C13O4 O5

C14C15

Lys111

Phe20

Ala90

Asp61

His17

Ser89

Leu19

Arg63

C6

259

C1

C2

C3

C4

C5

C6

C7C8

C9

C12

C13C14

N1

O1

O2O3

O4

Br

O5

C10

C11

N

CACO

CB

CG OD1OD2

Asp16

His17

Asp61

Arg63

Lys111

Ser89Pro65

His91

C7

281

C1C2C3

C4C5C6

C7

C8

O1

Br

N

C9O2

O3

C10

C11

Arg63

NCA

CO

CB

CGOD1

OD2

Asp16

His91

Met64

Pro65

Lys111

Asp61

C8

C1C2

C3C4

C5C6

C7C8

C9

C10

C11

C12

O1

C13O2 O3

C14C15

Arg63

Lys111

Asp61

Asp16

Met64

Pro65

Ala90

His17

Ser89

His91



H1

AH1

1AA1

4BH1

C14

4CH1

3BH1

3AH1

C13C12

O3

O2

N1

H1

1200

C7C8

C9

C5

C6H6

26

O5O4

Br

C1 C2

C3C4

O1

H3

H2

0BH1C10 1BH1

0AH1

OD2 OD1

ASP-16

CBN

C

CG11

(a)

H

H H

H

HHHH

HH

C

CC

C

C

C

C C

C

C

C

O

O

O

N

N

OD2 OD1

ASP-16CG

CB

Br

28

1299

(b)


ARG63 (B)

ASP61 (B)HIS91 (B)

SER89 (B)

LYS111 (B)

HIS17 (B)

ASP16 (B)

PRO65 (B)





ARG63 (B)

HIS91 (B)

ASP61 (B)

SER89 (B)

LYS111 (B)

PRO65 (B)

ASP16 (B)

HIS17 (B)


Distance

Dist

ance

(nm

)

Time (ps)0 2000 4000 6000 8000 10000

6

5

4

3

2

1

Ldl

2000 4000 6000 8000 10000

2

15

1

05

0

0

Num

ber

Time (ps)

Hydrogen bonds

Hydrogen bonds






HIS-91 ARG-63

GLN-71

SER-89

LYS-111

HIS-17 ASP-16

ASP-61PRO-65

MET-67

ALA-75

LEU-47

SER-78

TYR-79


C1

C2

C3C4

C5

C6

C7C8

C9

C12

C13C14

N1

O1

O2O3

O4

O5

C10

C11

Tyr79

Ala75

Ser78

Leu47

Met67

Gln71





Acknowledgments



References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















ARG-63ASP-61

ASP-16

ASP-15

LYS-111SER-89



















50) The








C5

C6

O

Br

NO

O

H

O

O

C7

C1

O

S

O

carbothioate

OO2N ONH

HN

NO2


C2 O

N

O

N


C3O O

O

O2NO

Br O

O

NH2

OC4

O OCl

O2N

NH

HN

C8

O

O


2-carboxylate











C4

322C1

C2

C3C4

C5 C6

C7

C8

C9 C10

C11

C12

O1

N1 O2

O3

N2

C13

O4

N3

C14C15

C16

Cl

C17

C18

C19

N

CACO

CBCG

CD

NECZ

NH1

NH2

Arg63

Pro65

Asp16

Asp61

His91

Ala90

Ser89

C1

C1

C2

C3C4

C5C6

C7C8

C9

C10

C11

C12

O1

C13

SO2

C14C15

Phe20

Lys111

Ala90

Asp61

N

C O

C2

257

C1 C2C3 C4

C5 C6

C7 C8

C9

C10

C11

C12

O1

C13N1

N2C14

O2

C15C16

C17C18C19

C20

CA

CB

CG

CD

CE

NZLys111

His17

Arg63

Phe20

Asp61

Asp112

Asp15

Asp16

His91

C5

C1

C2

C3

C4

C5C6

C7

C8

C9 C10 C11C12O1

N1

O2

O3

N2C13

O4N3C14C15

C16C17C18

C19

N4O5O6

Lys111

His17

Asp16

Pro18

Asp61

Ala90

Arg63

Ser89His91

C3

C1C2

C3C4

C5C6

C7C8

C9

C10

C11

C12

O1

NO2

O3

C13O4 O5

C14C15

Lys111

Phe20

Ala90

Asp61

His17

Ser89

Leu19

Arg63

C6

259

C1

C2

C3

C4

C5

C6

C7C8

C9

C12

C13C14

N1

O1

O2O3

O4

Br

O5

C10

C11

N

CACO

CB

CG OD1OD2

Asp16

His17

Asp61

Arg63

Lys111

Ser89Pro65

His91

C7

281

C1C2C3

C4C5C6

C7

C8

O1

Br

N

C9O2

O3

C10

C11

Arg63

NCA

CO

CB

CGOD1

OD2

Asp16

His91

Met64

Pro65

Lys111

Asp61

C8

C1C2

C3C4

C5C6

C7C8

C9

C10

C11

C12

O1

C13O2 O3

C14C15

Arg63

Lys111

Asp61

Asp16

Met64

Pro65

Ala90

His17

Ser89

His91



H1

AH1

1AA1

4BH1

C14

4CH1

3BH1

3AH1

C13C12

O3

O2

N1

H1

1200

C7C8

C9

C5

C6H6

26

O5O4

Br

C1 C2

C3C4

O1

H3

H2

0BH1C10 1BH1

0AH1

OD2 OD1

ASP-16

CBN

C

CG11

(a)

H

H H

H

HHHH

HH

C

CC

C

C

C

C C

C

C

C

O

O

O

N

N

OD2 OD1

ASP-16CG

CB

Br

28

1299

(b)


ARG63 (B)

ASP61 (B)HIS91 (B)

SER89 (B)

LYS111 (B)

HIS17 (B)

ASP16 (B)

PRO65 (B)





ARG63 (B)

HIS91 (B)

ASP61 (B)

SER89 (B)

LYS111 (B)

PRO65 (B)

ASP16 (B)

HIS17 (B)


Distance

Dist

ance

(nm

)

Time (ps)0 2000 4000 6000 8000 10000

6

5

4

3

2

1

Ldl

2000 4000 6000 8000 10000

2

15

1

05

0

0

Num

ber

Time (ps)

Hydrogen bonds

Hydrogen bonds






HIS-91 ARG-63

GLN-71

SER-89

LYS-111

HIS-17 ASP-16

ASP-61PRO-65

MET-67

ALA-75

LEU-47

SER-78

TYR-79


C1

C2

C3C4

C5

C6

C7C8

C9

C12

C13C14

N1

O1

O2O3

O4

O5

C10

C11

Tyr79

Ala75

Ser78

Leu47

Met67

Gln71





Acknowledgments



References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























50) The








C5

C6

O

Br

NO

O

H

O

O

C7

C1

O

S

O

carbothioate

OO2N ONH

HN

NO2


C2 O

N

O

N


C3O O

O

O2NO

Br O

O

NH2

OC4

O OCl

O2N

NH

HN

C8

O

O


2-carboxylate











C4

322C1

C2

C3C4

C5 C6

C7

C8

C9 C10

C11

C12

O1

N1 O2

O3

N2

C13

O4

N3

C14C15

C16

Cl

C17

C18

C19

N

CACO

CBCG

CD

NECZ

NH1

NH2

Arg63

Pro65

Asp16

Asp61

His91

Ala90

Ser89

C1

C1

C2

C3C4

C5C6

C7C8

C9

C10

C11

C12

O1

C13

SO2

C14C15

Phe20

Lys111

Ala90

Asp61

N

C O

C2

257

C1 C2C3 C4

C5 C6

C7 C8

C9

C10

C11

C12

O1

C13N1

N2C14

O2

C15C16

C17C18C19

C20

CA

CB

CG

CD

CE

NZLys111

His17

Arg63

Phe20

Asp61

Asp112

Asp15

Asp16

His91

C5

C1

C2

C3

C4

C5C6

C7

C8

C9 C10 C11C12O1

N1

O2

O3

N2C13

O4N3C14C15

C16C17C18

C19

N4O5O6

Lys111

His17

Asp16

Pro18

Asp61

Ala90

Arg63

Ser89His91

C3

C1C2

C3C4

C5C6

C7C8

C9

C10

C11

C12

O1

NO2

O3

C13O4 O5

C14C15

Lys111

Phe20

Ala90

Asp61

His17

Ser89

Leu19

Arg63

C6

259

C1

C2

C3

C4

C5

C6

C7C8

C9

C12

C13C14

N1

O1

O2O3

O4

Br

O5

C10

C11

N

CACO

CB

CG OD1OD2

Asp16

His17

Asp61

Arg63

Lys111

Ser89Pro65

His91

C7

281

C1C2C3

C4C5C6

C7

C8

O1

Br

N

C9O2

O3

C10

C11

Arg63

NCA

CO

CB

CGOD1

OD2

Asp16

His91

Met64

Pro65

Lys111

Asp61

C8

C1C2

C3C4

C5C6

C7C8

C9

C10

C11

C12

O1

C13O2 O3

C14C15

Arg63

Lys111

Asp61

Asp16

Met64

Pro65

Ala90

His17

Ser89

His91



H1

AH1

1AA1

4BH1

C14

4CH1

3BH1

3AH1

C13C12

O3

O2

N1

H1

1200

C7C8

C9

C5

C6H6

26

O5O4

Br

C1 C2

C3C4

O1

H3

H2

0BH1C10 1BH1

0AH1

OD2 OD1

ASP-16

CBN

C

CG11

(a)

H

H H

H

HHHH

HH

C

CC

C

C

C

C C

C

C

C

O

O

O

N

N

OD2 OD1

ASP-16CG

CB

Br

28

1299

(b)


ARG63 (B)

ASP61 (B)HIS91 (B)

SER89 (B)

LYS111 (B)

HIS17 (B)

ASP16 (B)

PRO65 (B)





ARG63 (B)

HIS91 (B)

ASP61 (B)

SER89 (B)

LYS111 (B)

PRO65 (B)

ASP16 (B)

HIS17 (B)


Distance

Dist

ance

(nm

)

Time (ps)0 2000 4000 6000 8000 10000

6

5

4

3

2

1

Ldl

2000 4000 6000 8000 10000

2

15

1

05

0

0

Num

ber

Time (ps)

Hydrogen bonds

Hydrogen bonds






HIS-91 ARG-63

GLN-71

SER-89

LYS-111

HIS-17 ASP-16

ASP-61PRO-65

MET-67

ALA-75

LEU-47

SER-78

TYR-79


C1

C2

C3C4

C5

C6

C7C8

C9

C12

C13C14

N1

O1

O2O3

O4

O5

C10

C11

Tyr79

Ala75

Ser78

Leu47

Met67

Gln71





Acknowledgments



References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















C5

C6

O

Br

NO

O

H

O

O

C7

C1

O

S

O

carbothioate

OO2N ONH

HN

NO2


C2 O

N

O

N


C3O O

O

O2NO

Br O

O

NH2

OC4

O OCl

O2N

NH

HN

C8

O

O


2-carboxylate











C4

322C1

C2

C3C4

C5 C6

C7

C8

C9 C10

C11

C12

O1

N1 O2

O3

N2

C13

O4

N3

C14C15

C16

Cl

C17

C18

C19

N

CACO

CBCG

CD

NECZ

NH1

NH2

Arg63

Pro65

Asp16

Asp61

His91

Ala90

Ser89

C1

C1

C2

C3C4

C5C6

C7C8

C9

C10

C11

C12

O1

C13

SO2

C14C15

Phe20

Lys111

Ala90

Asp61

N

C O

C2

257

C1 C2C3 C4

C5 C6

C7 C8

C9

C10

C11

C12

O1

C13N1

N2C14

O2

C15C16

C17C18C19

C20

CA

CB

CG

CD

CE

NZLys111

His17

Arg63

Phe20

Asp61

Asp112

Asp15

Asp16

His91

C5

C1

C2

C3

C4

C5C6

C7

C8

C9 C10 C11C12O1

N1

O2

O3

N2C13

O4N3C14C15

C16C17C18

C19

N4O5O6

Lys111

His17

Asp16

Pro18

Asp61

Ala90

Arg63

Ser89His91

C3

C1C2

C3C4

C5C6

C7C8

C9

C10

C11

C12

O1

NO2

O3

C13O4 O5

C14C15

Lys111

Phe20

Ala90

Asp61

His17

Ser89

Leu19

Arg63

C6

259

C1

C2

C3

C4

C5

C6

C7C8

C9

C12

C13C14

N1

O1

O2O3

O4

Br

O5

C10

C11

N

CACO

CB

CG OD1OD2

Asp16

His17

Asp61

Arg63

Lys111

Ser89Pro65

His91

C7

281

C1C2C3

C4C5C6

C7

C8

O1

Br

N

C9O2

O3

C10

C11

Arg63

NCA

CO

CB

CGOD1

OD2

Asp16

His91

Met64

Pro65

Lys111

Asp61

C8

C1C2

C3C4

C5C6

C7C8

C9

C10

C11

C12

O1

C13O2 O3

C14C15

Arg63

Lys111

Asp61

Asp16

Met64

Pro65

Ala90

His17

Ser89

His91



H1

AH1

1AA1

4BH1

C14

4CH1

3BH1

3AH1

C13C12

O3

O2

N1

H1

1200

C7C8

C9

C5

C6H6

26

O5O4

Br

C1 C2

C3C4

O1

H3

H2

0BH1C10 1BH1

0AH1

OD2 OD1

ASP-16

CBN

C

CG11

(a)

H

H H

H

HHHH

HH

C

CC

C

C

C

C C

C

C

C

O

O

O

N

N

OD2 OD1

ASP-16CG

CB

Br

28

1299

(b)


ARG63 (B)

ASP61 (B)HIS91 (B)

SER89 (B)

LYS111 (B)

HIS17 (B)

ASP16 (B)

PRO65 (B)





ARG63 (B)

HIS91 (B)

ASP61 (B)

SER89 (B)

LYS111 (B)

PRO65 (B)

ASP16 (B)

HIS17 (B)


Distance

Dist

ance

(nm

)

Time (ps)0 2000 4000 6000 8000 10000

6

5

4

3

2

1

Ldl

2000 4000 6000 8000 10000

2

15

1

05

0

0

Num

ber

Time (ps)

Hydrogen bonds

Hydrogen bonds






HIS-91 ARG-63

GLN-71

SER-89

LYS-111

HIS-17 ASP-16

ASP-61PRO-65

MET-67

ALA-75

LEU-47

SER-78

TYR-79


C1

C2

C3C4

C5

C6

C7C8

C9

C12

C13C14

N1

O1

O2O3

O4

O5

C10

C11

Tyr79

Ala75

Ser78

Leu47

Met67

Gln71





Acknowledgments



References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















C4

322C1

C2

C3C4

C5 C6

C7

C8

C9 C10

C11

C12

O1

N1 O2

O3

N2

C13

O4

N3

C14C15

C16

Cl

C17

C18

C19

N

CACO

CBCG

CD

NECZ

NH1

NH2

Arg63

Pro65

Asp16

Asp61

His91

Ala90

Ser89

C1

C1

C2

C3C4

C5C6

C7C8

C9

C10

C11

C12

O1

C13

SO2

C14C15

Phe20

Lys111

Ala90

Asp61

N

C O

C2

257

C1 C2C3 C4

C5 C6

C7 C8

C9

C10

C11

C12

O1

C13N1

N2C14

O2

C15C16

C17C18C19

C20

CA

CB

CG

CD

CE

NZLys111

His17

Arg63

Phe20

Asp61

Asp112

Asp15

Asp16

His91

C5

C1

C2

C3

C4

C5C6

C7

C8

C9 C10 C11C12O1

N1

O2

O3

N2C13

O4N3C14C15

C16C17C18

C19

N4O5O6

Lys111

His17

Asp16

Pro18

Asp61

Ala90

Arg63

Ser89His91

C3

C1C2

C3C4

C5C6

C7C8

C9

C10

C11

C12

O1

NO2

O3

C13O4 O5

C14C15

Lys111

Phe20

Ala90

Asp61

His17

Ser89

Leu19

Arg63

C6

259

C1

C2

C3

C4

C5

C6

C7C8

C9

C12

C13C14

N1

O1

O2O3

O4

Br

O5

C10

C11

N

CACO

CB

CG OD1OD2

Asp16

His17

Asp61

Arg63

Lys111

Ser89Pro65

His91

C7

281

C1C2C3

C4C5C6

C7

C8

O1

Br

N

C9O2

O3

C10

C11

Arg63

NCA

CO

CB

CGOD1

OD2

Asp16

His91

Met64

Pro65

Lys111

Asp61

C8

C1C2

C3C4

C5C6

C7C8

C9

C10

C11

C12

O1

C13O2 O3

C14C15

Arg63

Lys111

Asp61

Asp16

Met64

Pro65

Ala90

His17

Ser89

His91



H1

AH1

1AA1

4BH1

C14

4CH1

3BH1

3AH1

C13C12

O3

O2

N1

H1

1200

C7C8

C9

C5

C6H6

26

O5O4

Br

C1 C2

C3C4

O1

H3

H2

0BH1C10 1BH1

0AH1

OD2 OD1

ASP-16

CBN

C

CG11

(a)

H

H H

H

HHHH

HH

C

CC

C

C

C

C C

C

C

C

O

O

O

N

N

OD2 OD1

ASP-16CG

CB

Br

28

1299

(b)


ARG63 (B)

ASP61 (B)HIS91 (B)

SER89 (B)

LYS111 (B)

HIS17 (B)

ASP16 (B)

PRO65 (B)





ARG63 (B)

HIS91 (B)

ASP61 (B)

SER89 (B)

LYS111 (B)

PRO65 (B)

ASP16 (B)

HIS17 (B)


Distance

Dist

ance

(nm

)

Time (ps)0 2000 4000 6000 8000 10000

6

5

4

3

2

1

Ldl

2000 4000 6000 8000 10000

2

15

1

05

0

0

Num

ber

Time (ps)

Hydrogen bonds

Hydrogen bonds






HIS-91 ARG-63

GLN-71

SER-89

LYS-111

HIS-17 ASP-16

ASP-61PRO-65

MET-67

ALA-75

LEU-47

SER-78

TYR-79


C1

C2

C3C4

C5

C6

C7C8

C9

C12

C13C14

N1

O1

O2O3

O4

O5

C10

C11

Tyr79

Ala75

Ser78

Leu47

Met67

Gln71





Acknowledgments



References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















H1

AH1

1AA1

4BH1

C14

4CH1

3BH1

3AH1

C13C12

O3

O2

N1

H1

1200

C7C8

C9

C5

C6H6

26

O5O4

Br

C1 C2

C3C4

O1

H3

H2

0BH1C10 1BH1

0AH1

OD2 OD1

ASP-16

CBN

C

CG11

(a)

H

H H

H

HHHH

HH

C

CC

C

C

C

C C

C

C

C

O

O

O

N

N

OD2 OD1

ASP-16CG

CB

Br

28

1299

(b)


ARG63 (B)

ASP61 (B)HIS91 (B)

SER89 (B)

LYS111 (B)

HIS17 (B)

ASP16 (B)

PRO65 (B)





ARG63 (B)

HIS91 (B)

ASP61 (B)

SER89 (B)

LYS111 (B)

PRO65 (B)

ASP16 (B)

HIS17 (B)


Distance

Dist

ance

(nm

)

Time (ps)0 2000 4000 6000 8000 10000

6

5

4

3

2

1

Ldl

2000 4000 6000 8000 10000

2

15

1

05

0

0

Num

ber

Time (ps)

Hydrogen bonds

Hydrogen bonds






HIS-91 ARG-63

GLN-71

SER-89

LYS-111

HIS-17 ASP-16

ASP-61PRO-65

MET-67

ALA-75

LEU-47

SER-78

TYR-79


C1

C2

C3C4

C5

C6

C7C8

C9

C12

C13C14

N1

O1

O2O3

O4

O5

C10

C11

Tyr79

Ala75

Ser78

Leu47

Met67

Gln71





Acknowledgments



References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















ARG63 (B)

HIS91 (B)

ASP61 (B)

SER89 (B)

LYS111 (B)

PRO65 (B)

ASP16 (B)

HIS17 (B)


Distance

Dist

ance

(nm

)

Time (ps)0 2000 4000 6000 8000 10000

6

5

4

3

2

1

Ldl

2000 4000 6000 8000 10000

2

15

1

05

0

0

Num

ber

Time (ps)

Hydrogen bonds

Hydrogen bonds






HIS-91 ARG-63

GLN-71

SER-89

LYS-111

HIS-17 ASP-16

ASP-61PRO-65

MET-67

ALA-75

LEU-47

SER-78

TYR-79


C1

C2

C3C4

C5

C6

C7C8

C9

C12

C13C14

N1

O1

O2O3

O4

O5

C10

C11

Tyr79

Ala75

Ser78

Leu47

Met67

Gln71





Acknowledgments



References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















HIS-91 ARG-63

GLN-71

SER-89

LYS-111

HIS-17 ASP-16

ASP-61PRO-65

MET-67

ALA-75

LEU-47

SER-78

TYR-79


C1

C2

C3C4

C5

C6

C7C8

C9

C12

C13C14

N1

O1

O2O3

O4

O5

C10

C11

Tyr79

Ala75

Ser78

Leu47

Met67

Gln71





Acknowledgments



References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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