Chiang Mai J. Sci. 2016; 43(5) 931

Chiang Mai J. Sci. 2016; 43(5) : 931-945http://epg.science.cmu.ac.th/ejournal/Contributed Paper

Molecular Docking Study of Mycobacteriumtuberculosis Dihydrofolate Reductase in Complexwith 2,4-diaminopyrimidines AnaloguesPimonluck Sittikornpaiboon [a], Pisanu Toochinda [a], Chawanee Thongpanchang [b],

Ubolsree Leartsakulpanich [b] and Luckhana Lawtrakul* [a]

[a] School of Bio-Chemical Engineering and Technology, Sirindhorn International Institute of Technology,

Thammasat University, Thailand.

[b] National Center for Genetic Engineering and Biotechnology, National Science and Technology

Development Agency, Thailand.

*Author for correspondence; e-mail: luckhana@siit.tu.ac.th

Received: 8 July 2015

Accepted: 23 September 2015

ABSTRACT

The molecular docking approach was used to determine the binding affinities andthe interactions of Mycobacterium tuberculosis dihydrofolate reductase (mtbDHFR) in complexwith 2, 4-diaminopyrimidines analogues (PYR analogous). This approach can classify compoundsinto low and high affinity agents which can be further developed as a possible dihydrofolatereductase inhibitor for tuberculosis treatment. Our study provides insight into the importantinteractions of mtbDHFR with PYR analogues which lead to the design of effective agentsagainst mtbDHFR.

Keywords: molecular modeling, stability constant, binding energy, protein, ligands

1. INTRODUCTION

Tuberculosis (TB) is a bacterial infectiousdisease caused by Mycobacterium tuberculosis.In Thailand, TB is a notorious lethalinfectious disease, and the major cause ofdeath among AIDs patients [1]. The WorldHealth Organization estimates that newmultidrug-resistant TB (MDR-TB) incidentsare up to 450,000 cases globally, and 170,000MDR-TB patients died in 2012 [2].Tuberculosis treatment can take 4 to 9 monthsto be complete by combination of first-linedrugs such as isoniazid, rifampin, rifapentine,ethambutol, and pyrazinamide. Due to a long

treatment process, drugs resistant bacteriacan emerge if the drugs are not takenproperly. The treatment for MDR-TB requiresa combination of second-line drugs, whichcan cause unwanted side effects, longertreatment time, as well as a higher treatmentcost relative to first line drugs (up to 100 times,2012). According to the rapid growth ofMDR-TB and the fatality of the disease,a new effective drug/treatment for TB isurgently needed.

Dihydrofolate reductase (DHFR), anenzyme in the folate biosynthesis pathway,

932 Chiang Mai J. Sci. 2016; 43(5)

has long been recognized as a target foranticancer, antimalarial, and antibacterialagents. Folates are required for synthesis ofnucleotides and many amino acids; therefore,inhibition of this enzyme leads to cell death[3]. Due to the essential function and therelatively conserve property of the DHFRenzymes across various organisms, it isreasonably to hypothesize that DHFRs ofpathogenic organisms could be good drugtargets.

Crystal structures of Mycobacteriumtuberculosis dihydrofolate reductase(mtbDHFR) and Plasmodium falciparumdihydrofolate reductase (pfDHFR) revealedthat the enzymes exhibit similar overallstructure containing central β-sheet flankedby four α-helices [4]. Structure based drugdesign has been successfully used to designan effective antimalarial against the pfDHFRdomain of the bifunctional P. falciparumdihydrofolate reductase - thymidylate synthase(pfDHFR-TS) [5]. It is likely that somedeveloped antimalarial pfDHFR compoundswould possess cross activity againstmtbDHFR.

Molecular modeling is an efficientcomputational tool widely used in structuralbiology to aid the design and evaluate theefficacy of compounds prior the synthesis,which is a costly and time consuming process.From the previous studies, molecularsuperposition was used to study the structuralsimilarity between mtbDHFR and pfDHFR[6-7]. The root-mean-square deviation(RMSD) of 147 alpha carbon amino acidbetween pfDHFR and mtbDHFR are lowwith a value of 1.05 . Moreover, theinhibitors bind in the same binding pocketof mtbDHFR and pfDHFR. Moleculardocking and quantum chemistry calculationswere used to study the interaction betweenmtbDHFR and its inhibitors. The resultsprovide an explanation of the DHFR and

inhibitor interactions at the molecular level,such as the importance characteristics ofinhibitors that are required to promote tightbinding with the target, and to identify thekey binding residue of mtbDHFR that assistsin binding of the inhibitors to mtbDHFR.This molecular modeling approach showspromise and potential in the evaluation ofnovel inhibitors.

In this work, molecular modeling wasemployed to investigate the interactionsbetween mtbDHFR target and 2, 4-diaminopyrimidine analogues (pyrimethamineanalogous; PYR analogous) to determine thebinding affinities and the mode of interaction.Fifty pyrimethamine analogues were screenedfor binding in the active site of mtbDHFRusing the molecular docking approach.Results from this study provide generalcharacteristics of effective mtbDHFRinhibitors, which can lead to furtherdevelopment of novel drugs for tuberculosistreatment. This classification technique canprovide reliable screening of drug candidateswhich can save a lot of money and time fordrug development.

2. MATERIALS AND METHODS

Antifolates, pyrimethamine analogous[8-9] previously shown to inhibit pfDHFRand parasite growth were chosen. Theirbinding affinity toward mtbDHFR wasdetermined. Their initial conformation weregenerated by the means of GaussView andtheir geometry optimizations were performedusing density functional calculations atB3LYP/6-31G (d,p) level as included inthe GAUSSIAN03 program package [10].Crystal structures of mtbDHFR binary andternary complexes were taken from the RCSBProtein Data Bank, which are PDB: 1DF7,1DG5, 1DG7, 1DG8 [11]; 4KL9, 4KM2,4KM0, and 4KNE [12]. All inhibitors, ligands,and water molecules were removed from the

Chiang Mai J. Sci. 2016; 43(5) 933

crystal structure by using Discovery StudioVisualizer 4.0 program [13] and the resultingstructure was then used for the molecularmodeling study.

Molecular docking of mtbDHFR withinhibitors was carried out using the AutoDock4.2 software package [14]. All rotatable bondswithin the inhibitor molecules were allowedto be freely flexible during the dockingsimulation, whereas the protein is set as arigid molecule. Polar hydrogen atoms wereadded to the protein by using the hydrogenmodule in AutoDock Tools (ADT). Gasteigercharges were assigned for the molecules.The Lamarckian Genetic Algorithm was usedat 100 dockings for each inhibitor. The gridsize was set at specified grid points of64 points in all three dimensions, centered atan inhibitor binding pocket with a grid pointspacing of 0.375 . All other parameterswere run at the program’s default settings.The best-fit conformations of the similarbinding mode with their parent x-raystructures with the lowest binding energyand high percentage frequency concurrentlywere selected for predicting the preferredorientation of the inhibitor to mtbDHFRwhen bound to each other in order to forma stable complex.

3. RESULTS AND DISCUSSION

3.1 X-ray Crystallographic Structures ofmtbDHFR

The information regarding nine crystalstructures of mtbDHFR in this study are listedin Table 1. Three structures are mtbDHFRin binary complexes with the cofactorNADPH, the other six structures are ternarycomplexes of mtbDHFR-NADPH-inhibitor.The inhibitors are methotrexate (MTX),trimethoprim (TMP), 4-bromo WR99210(WRB), pyrimethamine (PYR), and cycloguanil(CYC). To compare the sequences ofmtbDHFR of nine structures, the sequencealignment of all PDB structures wereperformed using Discovery studio visualizer4.0. The sequence alignment showed that allmtbDHFR structures contain 159 identicalamino acid sequences, with no polymorphismfound in these sequences. The mtbDHFRstructures were superimposed by alphacarbon (α-carbon) of 159 amino acid residues,and the RMSD between all mtbDHFRstructures are shown in Table 2. All ninemtbDHFR structures derived from tworesearch groups show high similarity, with anaverage RMSD of 159 α-carbon atomsbetween 0.61 and 0.82 .

Table 1. Deposited mtbDHFR crystal structures from RCSB protein databank.

a 2′-monophosphoadenosine-5′-diphosphate (ATR) is a NADPH that lacks a nicotinamide ribose moiety.b MTX is methotrexate; TMP is trimethoprim; WRB is 4-bromo WR99210; PYR is pyrimethamine; CYC is cycloguanil.

PDB1DG81DF7

1DG51DG74KL94KLX4KM04KM24KNE

Ligand a

Glycerol, NADPHGlycerol, NADPH,SO

42-

Glycerol, NADPHGlycerol, NADPHNADPHATRATRATRATR

Inhibitor b

MTX

TMPWRB

PYRTMPCYC

Ki (nM)

11

88,000187

9101,4301,260

Resolution ( )2.001.70

2.001.801.391.231.301.402.00

Reference[11][11]

[11][11][12][12][12][12][12]

934 Chiang Mai J. Sci. 2016; 43(5)

Table 2. The RMSD in of α-carbon atoms superposition of all x-ray mtbDHFR structures.

PDB1DG81DF71DG51DG74KL94KLX4KM04KM24KNEAverage

1DG80.000.310.270.240.631.141.140.981.110.65

1DF70.310.000.350.230.731.111.110.961.060.65

1DG50.270.350.000.270.621.041.040.921.010.61

1DG70.240.230.270.000.671.091.080.951.050.62

4KL90.630.730.620.670.001.221.201.071.200.82

4KLX1.141.111.041.091.220.000.160.530.320.73

4KM01.141.111.041.081.200.160.000.520.310.73

4KM20.980.960.920.951.070.530.520.000.470.71

4KNE1.111.061.011.051.200.320.310.470.000.73

3.2 The Reliability of Molecular DockingCalculations

In order to verify the reliability ofcalculation methods, the available crystalstructures of mtbDHFR-inhibitor complex(1DF7, 1DG7, 1DG5, 4KM0, 4KNE, and4KM2) were selected to perform moleculardocking calculations. The inhibitors wereremoved from the binding site and dockedagain by our calculations. Four crystalstructures with PDB codes 4KLX, 4KM0,4KM2, and 4KNE [12] are mtbDHFRcomplexed with 2′-monophosphoadenosine-5′-diphosphate (ATR) which is a NADPHthat lacks the electron density for the

nicotinamide ribose moiety. However, themain part of x-ray structures of ATR andNADPH are identical. Therefore, the ATRmolecule can be replaced with NADPHmolecule by superimpose their identicaladenosine diphosphate parts together.Moreover the binding pocket of NADPH isspecific, therefore the relaxation of NADPHmolecule after replacement is not needed.

AutoDock calculations utilize a bindingenergy evaluation to identify the optimalbinding modes. The inhibition constant (K

i)

is the reciprocal of the equilibrium constant(K) as shown in Figure 1.

Figure 1. The model of molecular docking study of antifolate drug into the binding site ofmtbDHFR and the formulas to calculate equilibrium constant (K) and inhibition constant (K

i).

Chiang Mai J. Sci. 2016; 43(5) 935

The binding energy (B.E.) of mtbDHFRinhibitor in molecular docking is related toboth of K and K

i values by equation:

B.E. = -RT ln K = RT ln Ki.

The inhibition constant (Ki) and the

binding energy (B.E.) at T = 298.15 K ofmolecular docking models and fromexperiments are presented in Table 3.

Table 3. RMSD of molecular superposition between docked inhibitors and their x-raystructures. Binding energy (B.E.) and inhibition constant (K

i) from the experimental data and

from docking calculations.

Inhibitors

MethotrexateBr-WR99210TrimethoprimPyrimethamine

CycloguanilTrimethoprim

PDB code

1DF71DG71DG54KM04KNE4KM2

RMSD ( )

3.391.670.640.610.440.75

Ki (nM)

Exp.11187

88,000910

1,2601,430

Docking3.45

223.464,7501,520352.174,520

B.E. (kcal/mol)Exp.

-10.86-9.18-5.53-8.24-8.05-7.97

Docking-11.55-9.07-7.26-7.94-8.80-7.29

Figure 2. The molecular superposition models of all docked inhibitors (gray stick model)into mtbDHFR (gray ribbon) where x-ray inhibitors are shown as black stick model. (a)Methotrexate, (b) Br-WR99210, (c) Trimethoprim (PDB code: 1DG5), (d) Pyrimethamine,(e) Cycloguanil and (f) Trimethoprim (PDB code: 4KM2).

936 Chiang Mai J. Sci. 2016; 43(5)

After the molecular docking process,the docked inhibitors and those present in thex-ray structure reference were superimposedinto the binding site of mtbDHFR as shownin Figure 2. The interaction mode of dockedinhibitors with mtbDHFR is very similar tothat of the original mode from the x-raystructures with RMSD values lower than1.00 for PYR, CYC, and TMP (Table 3).MTX and WRB molecules consist of a flexiblelinker that is likely to contribute to higherRMSD values (3.39 and 1.67 for MTXand WRB, respectively).

Molecular docking simulation wasutilized to predict the B.E. and K

i of the

inhibitors to mtbDHFR. The validation ofmolecular docking approach was verifiedby computing the correlation coefficientbetween the B.E. and K

i from docking with

the values from the experiment. A lower valueof K

i suggests a high possibility in binding

complex of mtbDHFR with inhibitor, andalso indicates the effectiveness of the inhibitorattached to the target. Based on the availableexperimental K

i values of mtbDHFR with

TMP from the literature, the results providetwo different values where one is 88,000 nM(Li et al, 2000) while the other is 1,430 nM(Dias et al., 2014), as shown in Table 3. Theexperimental K

i value of mtbDHFR with

PYR is 910 nM from Li et al. (2000), but theK

i value determined by our group is 6,075

nM (unpublished data). This indicates that Ki

varies considerably with different groupsand different techniques. However, the K

i

and B.E. obtained from docking calculationsfor mtbDHFR with TMP are similar(Table 3). The correlation coefficientbetween experimental data and our dockingcalculations of all inhibitors K

i is 0.65

while the correlation coefficient of their B.E.is 0.86. Therefore, we used B.E. fromdocking calculations to analyze the propertiesof compounds binding to mtbDHFR for

further study.These results suggest that docking

calculations in this study provide reliableresults with high correlation coefficient ofB.E. values with the experimental data and itis able to be used for the molecular dockingstudy of PYR analogues into the binding siteof mtbDHFR as a possible inhibitor used fortuberculosis treatment. In general, a morenegative number in B.E. results in a strongerinteraction between mtbDHFR and acompound, indicates a higher potential of thecompound to be optimized for tuberculosistreatment.

3.3 Molecular Docking Calculations ofmtbDHFR with PYR Analogues

We have the experimental anti-mtbDHFR biological activity values ofonly two compounds, P1 (PYR) and P157,from the BIOTEC Laboratory (personalcommunication). P157 is highly effectiveagainst mtbDHFR whereas P1 is not effective,the values of their K

i and B.E. are shown in

Table 4. In order to classify the binding affinityof PYR analogues with mtbDHFR, the B.E.from docking calculations of P1 and P157were compared with the availableexperimental values. The B.E. values fromdocking calculations agreed with theexperimental data and showed highclassification performance in order to separatenon-active compound (P1) and activecompound (P157), Table 4.

Table 4. Binding energy (B.E.) and inhibitionconstant (Ki

) of mtbDHFR with P1 and P157from the experimental data and from dockingcalculations.

Inhibitors

P1P157

Ki (nM)

Exp.6,07520.35

Docking1,520458.46

B.E. (kcal/mol)Exp.-7.12-10.49

Docking-7.94-8.65

Chiang Mai J. Sci. 2016; 43(5) 937

In order to gain more information abouttheir anti-mtbDHFR biological activity,the molecular structures of mtbDHFR incomplexes with these compounds areneeded. Nevertheless, only crystal structure ofmtbDHFR with PYR is existing, other PYR

analogues are unavailable. Therefore wehave to use the molecular docking calculationsto predict the interaction between aminoacid residues in mtbDHFR binding pocketwith PYR analogues.

Figure 3. The chemical structures and the model of molecular docking study of P1 andP 157 in mtbDHFR. P1 and P157 are shown in pink and yellow, respectively. NADPH moleculeis shown as stick model. The hydrophobic binding residues are highlighted in cyan color.

The molecular docking calculations showthe interaction between pyrimidine ring ofPYR analogues with Phe31, and the H-bondsbetween the primary amine groups (NH

2) of

2, 4-diaminopyrimidine ring with O atom ofAsp27 side-chain (OD1), and O atom frombackbones of Ile5, as indicated by dash linesin Figure3. For high affinities P157 molecule,it has one more H-bond with Ile 94 andhydrophobic interactions between itshydrophobic part (substituent at C-5 ofpyrimidine ring) with Ile20, and nicotinamidering of the cofactor NADPH, which wereabsent in case of P1 and therefore resultedin poor binding with mtbDHFR.

Fifty PYR analogues [8-9] screened in thisstudy can be classified into two series (series I

and series II), which are distinguished bydifference substituent groups at C(5) and C(6)of the 2, 4-diaminopyrimidine ring. Chemicalstructures of PYR series I and II are presentedin Table 5 and Table 6, respectively. None ofbinding affinities experimental data ofthese PYR analogues with mtbDHFR.Therefore molecular docking techniquewas used to predict the anti-DHFR biologicalactivity and the mode of binding of theseanalogues in the active site pocket.

All PYR analogues were docked into thebinding pocket of mtbDHFR crystal structure(PDB code 4KM0). The lowest bindingenergy (B.E.) and inhibition constant (K

i)

from molecular docking calculations arealso indicated in Table 5 and Table 6.

938 Chiang Mai J. Sci. 2016; 43(5)

Table 5. Chemical structures of PYR series I analogues, binding energy (B.E.) and inhibitionconstant (K

i) from docking calculations.

a compound that could be an effective mtbDHFR inhibitor (B.E. ≤ -8.65 kcal/mol)

CompoundsP1P17P13P15P20P30P16a

P26P29a

P12a

P33a

P31a

P45P41P46P42a

P47a

P43a

P39P44a

P38P32a

P40a

R1

HHCl

HClHHClHHClHClHClHClHClClClCl

R2

ClMeCl

HHClHHClHHHHHHHHHHHHH

“OCH2O”

R3

EtEtEtEtEtEt

(CH2)

3COOMe

(CH2)

3COOMe

(CH2)

3COOMe

(CH2)3Ph

(CH2)3Ph

(CH2)3Ph

(CH2)

3OH

(CH2)

3OH

(CH2)

3OCOCH

3

(CH2)

3OCOCH

3

(CH2)

3OCOC

6H

5

(CH2)

3OCOC

6H

5

nC6H

13

(CH2)

3OCOOCH

2C

6H

5

Me(CH

2)

3C

6H

4-(p-OMe)

(CH2)

2O(CH

2)

3OPh

B.E. (kcal/mol)-7.94-7.94-8.43-8.19-7.64-8.09-8.77-8.39-8.74-10.11-9.80-10.15-7.73-8.19-8.49-8.86-9.79-10.45-8.46-10.14-7.85-10.55-9.91

Ki (nM)

1,5201,520661.83991.352,5301,180375.78712.50389.4939.1065.3836.382,150999.69602.50320.2966.4821.81632.9036.891,77018.3654.62

Chiang Mai J. Sci. 2016; 43(5) 939

Table 6. Chemical structures of PYR series II, binding energy (B.E.) and inhibition constant(K

i) from docking calculations.

CompoundsP91

P85

P82

P115

P102

P110

P103

P135

P131

P130

B.E. (kcal/mol)-8.46

-8.18

-7.80

-7.89

-7.37

-7.43

-7.59

-8.44

-7.14

-8.50

Ki (nM)

626.30

1,010

1,920

1,650

3,960

3,580

2,740

655.87

5,800

584.30

R

940 Chiang Mai J. Sci. 2016; 43(5)

Table 6. Continued.

CompoundsP98a

P105

P108

P107

P112

P113

P114

P134

P140a

P169a

P121

B.E. (kcal/mol)-8.81

-8.52

-7.88

-8.36

-8.16

-8.25

-8.16

-8.41

-8.84

-9.07

-8.40

Ki (nM)

347.87

565.06

1,680

740.36

1,040

902.60

1,050

685.71

331.03

226.01

701.92

R

Chiang Mai J. Sci. 2016; 43(5) 941

Table 6. Continued.

CompoundsP96

P125

P90

P89

P99

P157

B.E. (kcal/mol)-8.13

-7.78

-8.18

-8.01

-8.18

-8.65

Ki (nM)

1,100

2,000

1,010

1,350

1,010

458.46

R

a compound that could be an effective mtbDHFR inhibitor (B.E. ≤ -8.65 kcal/mol)

Figure 4. Superposition of 50 PYR analoguesafter molecular docking calculations. PYR isshown as stick model. PYR series I and seriesII analogues are shown in blue and magentalines, respectively.

As Figure 4 shows, from dockingcalculations 50 PYR analogous are bound inthe same active site of mtbDHFR with itspyrimidine ring taking place the interior ofthe binding cavity by H-bonds with Ile5 andAsp27 residues.

Fourteen PYR analogues (P16, P29, P12,P33, P31, P42, P47, P43, P44, P32, P40, P98,P140, and P169) are considered as a possiblecompound structure for further developmentto be an effective mtbDHFR inhibitor usedfor anti-tuberculosis chemotherapy becausetheir B.E. are lower than -8.65 kcal/mol,which is the B.E. of a potent mtbDHFRinhibitor, P157.

942 Chiang Mai J. Sci. 2016; 43(5)

It is noted that PYR analogues in series Iwith the m-chlorophenyl or p-chlorophenylsubstituent (Cl at R1 or R2 position) showedhigher binding affinity than their correspondingunsubstituted. As Cl atom makes otherhydrophobic interactions with nicotiamidemoiety of NADPH and with a hydrophobicamino acid Ile 20 or Ile50 for m-chlorophenylor p-chlorophenyl substitution, respectively.

Additionally, the bulky group at R3

position is favorable which causes the stericinteraction with the surrounding amino acidresidues. This can explain why PYR analoguesseries II with ethyl substituent at R3 showedlower affinities with mtbDHFR than PYRanalogues series I.

In order to understand the interactionsthat promotes the potent inhibitors to bebound with mtbDHFR, the interactions offourteen potent inhibitors were compared

with the interaction of PYR (P1) in mtbDHFRbinding pocket. The non-potent (P1) andfourteen potent PYR analogues from series Iand II, based on docking simulation, weresuperimposed in the mtbDHFR binding site,Figure 5. All PYR analogues from bothseries I and series II are bound in the samebinding pocket of mtbDHFR. The 2, 4-diaminopyrimidine ring of all analogues isoriented in a similar configuration as a resultof specific hydrogen bonding of 2,4-diaminopyrimidine ring with amino acidresidues within the mtbDHFR bingingpocket. Based on H-bonds analysis, aminogroups (-NH

2) at N(2) and N(4) of 2,

4-diaminopyrimidine ring in all PYRanalogues are specifically bound to O ofAsp27 side-chain (OD1), and O frombackbones of Ile5 and Ile94, respectively.

Figure 5. Structural superposition of P1 and 14 potent PYR analogues from docking simulationin mtbDHFR. Analogues from series I and II are shown in pink and yellow, respectively.NADPH molecule is shown as stick model. The hydrophobic binding residues are highlightedin cyan color.

Chiang Mai J. Sci. 2016; 43(5) 943

Figure 6. Interaction of non-potent P1 and high potent PYR analogues series I in mtbDHFRbinding pocket. Hydrogen bonds are represented as a green dash line with the distance inAngstroms. The hydrophobic binding residues are highlighted in cyan color.

Figure 7. Interaction of P157 and potent PYR analogues series II in mtbDHFR bindingpocket. Hydrogen bonds are represented as a green dash line with the distance in Angstroms.The hydrophobic binding residues are highlighted in cyan color.

944 Chiang Mai J. Sci. 2016; 43(5)

The interactions of high affinities PYRanalogues series I and II with mtbDHFRamino acid residues are shown in Figure 6and Figure 7, respectively. The 2, 4-diaminopyrimidine ring of all ligandsinteract with Phe31 (pi-pi stacked), Ile5, andAla7 via hydrophobic interaction.

All 14 potent analogues have onecommon characteristic, the hydrophobicinteractions with Ile20 amino acid residue ofmtbDHFR and nicotinamide ring of thecofactor NADPH. However, the parts ofmolecules in PYR series I and II analogues,which have these specific hydrophobicinteraction are different. The inhibitionpotencies of PYR analogues series I and IIare ascribed to their hydrophobic part of R3

substituent (C-6 of pyrimidine ring) and Rgroup substituent at the long side chain(C-5 of pyrimidine ring), respectively, thatinteract with Ile20 and NADPH.

4. CONCLUSIONS

PYR series I and II analogues bound tothe mtbDHFR enzyme at the same bindingsite due to the hydrogen bond of aminogroups of pyrimidine ring with Asp27 (OD1),Ile5 (O), and Ile94 (O). The binding affinityof PYR series I analogues are higher thanPYR series II analogues as a result of thehydrophobic bulky group substituent (R3) atC-6 of the pyrimidine ring of PYR series I,which involves a hydrophobic interactionwith Ile20. In addition, the phenyl group atC-5 of the pyrimidine ring of potent PYRseries I hydrophobically interacts with Phe31(pi-pi T shaped), Ile94, and the nicotinamidering of the cofactor. Four potent PYR seriesII analogues are able to have a hydrophobicinteraction with Ile20 amino acid residue andNADPH by using their hydrophobic part atthe long side chain (C-5 of pyrimidine ring).Our study predicts 14 PYR analogues withhigh binding affinities as potent inhibitors

for further development of anti-tuberculosiscompounds.

ACKNOWLEDGEMENTS

This research was supported by a grantfrom the Thammasat University ResearchFund. The authors gratefully acknowledgethe Center of Nanotechnology, KasetsartUniversity for the GAUSSIAN03 programpackage. We also thank Dr. WichaiPornthanakasem and Ms. Wanwipa Ittaratfor help preparing, purified mtbDHFR andK

i measurement of PYR and P157 that was

mentioned in the paper.

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