Experimental Parasitology 120 (2008) 73–79

Contents lists available at ScienceDirect

Experimental Parasitology

journal homepage: www.elsevier.com/ locate /yexpr

Leish­mania donovani pteridine reductase 1: Biochemical properties

and structure-modeling studies

Pranav Kumar a, Ashutosh Kumar d, Shyam Sundar Verma c, Namrata Dwivedi c, Nasib Singh b, Mohammad Imran Siddiqi d, Rama Pati Tripathi c, Anuradha Dube b, Neeloo Singh a,*

a Drug Target Discovery and Devel­op­ment, Central­ Drug Research­ Institute, Ch­attat Manzil­, P.O. Box No. 173, Lucknow 226001, Indiab Parasitol­ogy, Central­ Drug Research­ Institute, Ch­attat Manzil­, P.O. Box No. 173, Lucknow 226001, Indiac Medicinal­ Ch­emistry, Central­ Drug Research­ Institute, Ch­attat Manzil­, P.O. Box No. 173, Lucknow 226001, Indiad Mol­ecul­ar and Structural­ Biol­ogy Divisions of Central­ Drug Research­ Institute, Central­ Drug Research­ Institute, Ch­attat Manzil­, P.O. Box No. 173, Lucknow 226001, India

a r t i c l e i n f o a b s t r a c t

Articl­e h­istory:

Received 24 May 2007

Received in revised form 16 May 2008

Accepted 19 May 2008

Available online 2 June 2008

Pteridine reductase 1 (PTR1, EC 1.5.1.33) is a NADPH dependent short-chain reductase (SDR) responsible

for the salvage of pterins in the protozoan parasite Leish­mania. This enzyme acts as a metabolic bypass

for drugs targeting dihydrofolate reductase, therefore, for successful antifolate chemotherapy to be devel-

oped against Leish­mania, it must target both enzyme activities. Based on homology model drawn on

recombinant pteridine reductase isolated from a clinical isolate of L. donovani, we carried out molecu-

lar modeling and docking studies with two compounds of dihydrofolate reductase specificity showing

promising antileishmanial activity in vitro. Both the inhibitors appeared to fit well in the active pocket

revealing the tight binding of the carboxylic acid ethyl ester group of pyridine moiety to pteridine reduc-

tase and identify the important interactions necessary to assist the structure based development of novel

pteridine reductase inhibitors.

© 2008 Elsevier Inc. All rights reserved.

Index Descrip­tors and Abbreviations:

Pteridine reductase 1

Clinical isolate

Recombinant protein

Antileishmanial screening

Flow cytometry

Structural modeling

Leish­mania donovani

Antileishmanial drug screening

PTR1, pteridine reductase 1

DHFR–TS, dihydrofolate reductase–thymi-

dylate synthase

DHPR, dihydropteridine reductase

MTX, methotrexate

GFP, green fluorescence protein

pABA, p­ara-aminobenzoic acid

SAG, sodium antimony gluconate

1. Intro­duc­tio­n

Infection with pathogenic Leish­mania results in a spectrum of

human diseases, with an annual incidence of 2 million cases in 88

countries (www.who.int/tdr/disease/leish). Leish­mania have a dige-

netic life cycle, first residing in the gut of phlebotomine sand flies

where they replicate as procyclic promastigotes. During a blood

meal, the parasites are transmitted and engulfed by vertebrate mono-

nuclear phagocytic system cells, where they will then transform into

the amastigote stage and divide within the acidified phagolysosomes

(Burchmore and Barrett, 2001). No effective vaccines are available

against Leish­mania infection. (Handman, 2001; Carter et al., 2007)

and treatment relies mainly on chemotherapy. In order to generate

0014-4894/$ - see front matter © 2008 Elsevier Inc. All rights reserved.

doi:10.1016/j.exppara.2008.05.005

* Corresponding author. Fax: +91 0522 2223405.

E-mail­ address: neeloo888@yahoo.com (N. Singh).

an adequate armory of drugs to treat visceral leishmaniasis, new and

effective drug targets are required to combat this disease. Enzymes

or metabolites present in the parasite but absent from their mamma-

lian host are considered as ideal targets for rational drug design. Thus

pteridine reductase 1 (PTR1, EC 1.5.1.33) of Leish­mania is an excellent

target due to the unusual salvage of pterin from the host. On the

other hand the host synthesize pterin derivatives de novo from GTP

and lack PTR11 activity (Nichol et al., 1985).

Biochemical studies indicate that this enzyme is a NADPH

dependent pterin reductase and active as a tetramer (Bello

et al., 1994; Wang et al., 1997; Kumar et al., 2004). PTR1 reduces

biopterin to H2-biopterin and H4-biopterin; it is also capable of

1 The PTR1 gene amplified from genomic DNA of L. donovani field isolate described

in this report have been submitted to GeneBank and assigned the Accession No:

AY547305.

74 P. Kumar et al­. / Exp­erimental­ Parasitol­ogy 120 (2008) 73–79

(

s

2

e

(

r

i

r

d

3

a

t

(

(

T

(

o

b

n

Y1

H

O

2

Table­ 1

Chemical structures of the screened compounds

S. No. Compound Structure/formula/molecular weight

1. (4-Fluoro-phenyl)-6-

methyl-2-thioxo-1,2,

3,4-tetrahydro-

pyrimidine-5-carboxylic

acid ethyl ester

2. 2,6-Dimethyl [3-O-benzyl-

1,2-O isopropylidene-

b-l-th­reo-pentofuronose

-4-yl]-1-phenyl-1,4-

dihydro pyridine-3,5-

dicarboxylic acid

diethyl ether

N

O

OO

H3C CH3

H3C

O

OCH2CH3H3CH2CO

O

OCH2Ph

CH3

C33H39NO8

Exact Mass: 577.27Mol. Wt.: 577.66

reducing folate to 7,8-dihydrofolate and tetrahydrofolate. PTR1

contributes about 10% of the reduction of folates in wild-type

cells while the remaining 90% is due to the activity of dihydrofo-

late reductase–thymidylate synthase (DHFR-TS, EC 1.5.1.3) (Nare

et al., 1997). Since PTR1 is significantly less inhibited by meth-

otrexate than dihydrofolate reductase–thymidylate synthase

(Nare et al., 1997), this activity allows it to act as a metabolic by-

pass of dihydrofolate reductase–thymidylate synthase. Clearly, if

antifolate chemotherapy is to be developed against Leish­mania,

it must target both dihydrofolate reductase and PTR1 activities

(Nare et al., 1997; Leblanc et al., 1998; Schüttelkopf et al., 2005).

It has been seen that low levels of tetrahydrobiopterin favour

metacyclogenesis, which involves the differentiation of the non-

infective procyclic promastigotes to highly infective metacyclic

forms within the sand fly (Sacks and Perkins, 1984). We have

found that regulation of PTR1 is growth phase dependent and

it degrades in the stationary phase of the growth, when parasite

undergoes metacyclogenesis (Kumar et al., 2007). Therefore,

when using PTR1 as a drug target for antileishmanial screening,

the parasites should be treated with inhibitor during their expo-

nential phase of growth.

Reduced pterins and folates are essential for the growth of

Leish­mania parasites but antipteridines have not shown much

promise clinically against Leish­mania in contrast to other proto-

zoal infections (Hardy et al., 1997). This establishes the need for

continued effort and research in this direction. We have focused

on a virulent clinical isolate of L. donovani when investigating

the role of pteridine metabolism for antiparasite chemotherapy

(Singh, 2002). Earlier we have overexpressed L. donovani PTR1 in

E. col­i and purified the recombinant protein (Kumar et al., 2004).

We have also overexpressed PTR1 tagged at the N-terminal with

green fluorescent protein (GFP) in Leish­mania cells (Kumar et al.,

2007). The promastigotes/amastigotes showing GFP fluorescence,

have been used in the present study for antileishmanial drug

screening using flow cytometry (Singh and Dube, 2004), to test a

collection of pteridine analogs for activity, in an effort to identify

compounds specifically targeting this enzyme. With this analysis

we have obtained good activity in vitro of two compounds. We

then combined our screening results with structural studies uti-

lizing the homology model drawn for L. donovani PTR1, in order

to confirm the chemistry behind this biochemical action.

2. Mate­rials and me­th­o­ds

2.1. Comp­ounds

Twenty compounds with structures related to thiones, known

inhibitors of dihydrofolate reductase, were synthesized and used for

screening for their antileishmanial activity in vitro in this study. Two

compounds were found to be highly active (Table 1). Compound 1 (4-flu-

oro-phenyl)-6-methyl-2-thioxo-1,2,3,4-tetrahydro-pyrimidine-5-car-

boxylic acid ethyl ester) was prepared by our earlier reported method

(Dwivedi et al., 2005) using a three component Biginelli reaction of an

4-flurorobenzaldehyde, ethylacetoacetate and ammonium acetate in

presence of tetrabutylammonium hydrogen sulphate as a phase trans-

fer catalyst. The compound was characterized on the basis of its spec-

troscopic data and microanalysis. Light yellow solid, m.p. 194–195 °C

(Shailza et al., 2004) IR (KBr) m cm¡1: 3327, 3175, 3106, 2988, 1683,

1575. FBAMS: 295 [M+H]+, 1H NMR (CDCl3, 200 MHz): d 9.60 (br s, 1H,

NH); 9.01 (br s, 1H, NH); 7.28 (dd, J = 8.5 Hz, 2.2 Hz, 2H, ArH); 6.97 (dd,

J = 8.5 Hz, 2.2 Hz, 2 H, ArH); 5.33 (d, J = 2.3 Hz, 1H, H-4), 4.08 (q, J = 7.1Hz,

2H, OCH2CH3); 2.36 (s, 3H, 6-CH3); 1.68 (t, J = 7.1 Hz, 3H, OCH2CH3); 13C

NMR (CDCl3, 50 MHz): d174.89, 165.83, 160.04, 144.79, 139.69, 128.02,

128.86, 115.80, 115.38, 102.15, 60.36, 54.92, 18.16, 14.142. C33H39NO8

Calcd: C 57.13, H 5.14, N 9.52%. Found: C 57.42, H 5.35, N 9.50%.

Compound 2 was prepared following our earlier method

Tewari et al., 2004) and characterized on the basis of its spectro-

copic data and microanalysis as shown below.

4-(3-O-Benzyl-1,2-O-isopropylidene-b-l-th­reo-furanos-4-yl)-

,6-dimethyl-1,4-dihydro-pyridine-3,5-dicarboxylic acid ethyl

ster (2):

A mixture of aniline (5.0 g, 53.76 mmol), ethyl acetoacetate

6.78 mL, 53.76 mmol) and Amberlite IR-120 resin (5.0 g) was

efluxed in anhydrous toluene (30 ml) for 3 h. The water formed

n the reaction was removed using a Dean Stark apparatus. The

esin was filtered off and the solvent was evaporated and the resi-

ue obtained was chromatographed on SiO2 gel to give the enamine,

-phenylamino-but-2-enoic acid ethyl ester (Jendralla et al., 1990)

s a light yellow colored oil. Yield (90%) and used as such. A mix-

ure of 1,2-O-isopropylidne-3-O-benzyl-a-d-xyl­o-penta-15-dialdose

1.8 g, 6.40 mmol), 3-phenylamino-but-2-enoic acid ethyl ester

1.32 mL, 6.40 mmol), ethylacetoacetate (0.736 mL, 6.40 mmol) and

BAHS (0.2 g) was magnetically stirred at 80 °C in diethylene glycol

4 mL) for 1.5 h. After cooling the reaction mixture, it was poured

nto crushed ice. The crude product, thus obtained, was purified

y column chromatography on silica gel using chloroform/metha-

ol (98:2) as eluent to afford the compound 2 as a colorless foam.

ield (89%); FABMS: m/z = 577 [M+H]+, IR (Neat) = 2983, 1691 cm¡1.

H NMR (200 MHz, CDCl3) d = 7.42–7.15(m, 10H, ArH), 5.90 (d, J = 4.0

z, 1H, H-19), 4.75 (d, J = 8.0 Hz, 1H, H-4), 4.58 (d, J = 11.9 Hz, 1H,

CHAPh), 4.51 (d, J = 13.0 Hz, 1H, OCHBPh), 4.41- 4.04 (m, 5H,H-29, £ OCH2CH3); 3.89 (d, J = 3.2, 1H, H-49), 3.78 (d, J = 3.0 Hz, 1H, H-39),

P. Kumar et al­. / Exp­erimental­ Parasitol­ogy 120 (2008) 73–79 75

Fig. 1. The sequence and three-dimensional model of L. donovani PTR1. (A) Sequence alignment of PTR1 from L. donovani and PTR1 from L. major. The secondary-structure ele-

ments of PTR1 are indicated with arrows for b-sheets and coils for a-helices. Residues involved in ligand binding are marked with “ ¤ ”. (B) A molecular model for L. donovani

PTR1 was constructed using coordinates from the L. major PTR1 (PDB code 1E7W). Helix is represented in magenta and sheet is represented in blue. Docked MTX shown in

blue ball and stick model and NADPH represented by yellow ball and stick model defines the ligand and cofactor binding sites, respectively, on the PTR1. (C) Ribbon diagram

of L. donovani PTR1 tetramer model. Each subunit is given a different color and labeled A–D. Ligand and cofactor are represented by blue and yellow ball and stick models.

(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

2.06, 2.00 (each s, 6H, CH3), 1.48, 1.28 (each s, 6H, (CH3)2C), 1.23,1.08

(each, t, J = 7.0 Hz, 6H, 2 £ OCH2CH3). 13C NMR (50 MHz, CDCl3)

d = 169.0, 168.6; 147.5, 147.3; 140.7,138.7; 130.8, 129.5, 129.2, 129.2,

128.5, 127.7, 127.1; 111.3, 109.9; 105.1; 103.8, 103.0; 83.3, 82.6, 82.1;

72.0; 61.2; 32.9; 27.2, 26.6, 18.4, 1.8.1; 14.6, 14.5. C33H39NO8 Calcd:

C 68.61, H 6.8, N 2.42. Found: C 68.81, H 6.54, N 2.62.

76 P. Kumar et al­. / Exp­erimental­ Parasitol­ogy 120 (2008) 73–79

2.2. Biol­ogical­ eval­uation of comp­ounds in vitro

Leish­mania donovani promastigotes and amastigotes of a clinical

isolate 2001, expressing GFP tagged to the N-terminal of the PTR1

gene (Kumar et al., 2007), were used for the in vitro evaluation of

compounds for antileishmanial activity. The test was performed by

flow cytometry as described (Singh and Dube, 2004). Briefly, mid

log phase promastigotes (2 £ 106 cells/ml) in medium M199 sup-

plemented with 15% heat inactivated fetal calf serum, were seeded

in 48-well tissue culture plates (Greiner Bio-One, Germany). They

were co-incubated with different drug concentrations. Untreated

cells served as control. After incubation at 26 oC for 48 h, the para-

sites were washed and suspended in PBS, followed by flow cytomet-

ric analysis. J774A.1 macrophages (105 cells/well) in 6-well culture

plates (Nunc, IL, USA) were infected with GFP-PTR1 chimera express-

ing promastigotes in a ratio of 10:1 (parasites/macrophage) and incu-

bated at 37 °C in 5% CO2 for 8–12 h. At 18 h after the amastigotes had

entered macrophages, free amastigotes were eliminated and macro-

phages were treated with various concentrations of the compounds.

At different time intervals after treatment, infected macrophages

were harvested, washed with PBS and analyzed by flow cytometry

(Becton–Dickinson, Franklin Lakes, NJ) equipped with a 15-mV,

488-nm, air-cooled argon ion laser. Cells were gated appropriately

and then macrophages-containing amastigotes were detected and

sorted according to their relative fluorescence intensities compared

to those of uninfected cells. At least 10,000 cells were acquired for

each analysis and were analyzed by Cell Quest software. The infec-

tion rate of macrophages was measured by putting markers in his-

tograms for uninfected and infected cultures. Miltefosine was used

as the reference drug. Experiments were performed in triplicate. In

vitro antileishmanial activity was expressed as the concentration

inhibiting parasite growth by 50% (IC50 ± SD) after 48 h incubation

period. MTT (Dutta et al., 2005) was used to measure cytotoxicity of

compounds against uninfected macrophages.

2.3. Mol­ecul­ar model­ing and docking

For homology modeling of the L. donovani PTR1 we used

restrained based modeling implemented in the MODELER

program (Sali and Blundell, 1993) interfaced with InsightII 2000.1

(http://www.accelrys.com.) using L. major PTR1 (sharing 91%

sequence identity with L. donovani PTR1, Fig. 1A) crystal structure

as template [PDB code 1E7W (Gourley et al., 2001)]. Discover_3

was used for further refinement of the L. donovani PTR1 model

by energy minimization with CVFF force field (1000 iterations).

PROCHECK (Laskowski et al., 1993) was used for stereochemical

analysis of models.

Molecular docking of the compounds in the active site of

this L. donovani PTR1 homology model was carried out using a

modern docking engine LigandFit available with Cerius 2_4.10

[Cerius2, Version 4.10; Accelrys, Inc. San Diego, CA, USA, 2005,

http://www.accelrys.com]. This algorithm makes use of a shape

comparison filter in combination with a Monte Carlo conforma-

tional search for generating ligand poses consistent with the

active site shape. Candidate poses are minimized in the context

of the active site using a grid-based method for evaluating pro-

tein–ligand interaction energies. The docking was carried out

with the following nondefault settings in LigandFit: site parti-

tioning 2 in order to fully access the potential docking orienta-

tion of the active site, maximum trials variable table values to

help the pseudorandom conformational analysis, and the CFF

force field option used for the grid energy calculations (Venkat-

achalam et al., 2002).

The docked conformations were scored using different scor-

ing functions available with Cerius 2. Each scoring function

may rank binding models differently, and so a consensus

scoring approach was used with six different scoring func-

tions: (i) Dock score (Cerius2, Version 4.10; Accelrys, Inc. San

Diego, CA, USA, 2005, http://www.accelrys.com); (ii) Ligscore1

& Ligscore2 (Krammer et al., 2005); (iii) Plp1 and Plp2 (Gehl-

haar et al., 1995,1999); (iv) JAIN (Jain, 1996); (v) Potential mean

force (PMF) (Muegge and Martin, 1999); and (vi) Ludi (Bohm,

1992). Docking was carried out in the presence of cofactor

NADPH, because studies in Leish­mania have shown that PTR1

has much stronger affinity (5- to 10-fold) for substrates as well

as inhibitors after binding of the cofactor NADPH (Nare et al.,

1997).

3. Re­sults and disc­ussio­n

PTR1-GFP chimera, in which the N-terminus of PTR1 gene from

a clinical isolate of L. donovani (2001), is fused to GFP was devel-

oped by us as described elsewhere (Kumar et al., 2007). Brightly

fluorescent promastigotes were obtained after electroporation and

selection. It is essential that the growth kinetics of parasite be kept

in mind for drug screening studies. Therefore, based on growth

curve analysis, 2 £ 106 promastigotes/ml was used for initial seed-

ing. At this seeding concentration it was seen that mid log phase

reached at 48 h and stationary phase reached at 96 h of growth. So

for further study we took a grown culture at 24 h as early log phase,

48 h as mid log phase, 72 h as late log phase and 96 h as station-

ary phase. The drugs were therefore incubated with promastigotes

for 48 h before flow cytometry analysis. This was done keeping in

mind that PTR1 is regulated with the growth stage of the L. dono-

vani promastigote with high activity in the logarithmic stage of the

parasite and lower activity (approximately 70% of the log phase

values) in the stationary phase (Cunningham et al., 2001; Kumar

et al., 2007). However, unlike promastigotes, it has been seen that

amastigote PTR1 level does not decline in stationary phase (Cunn-

ingham et al., 2001).

As established by us earlier (Singh and Dube, 2004), fluorescent

promastigotes and amastigotes were used in flow cytometry to

quantify the parasitocidal effect of the compounds. We tested the

effect of several concentrations of the compounds against prom-

astigotes. Since, the amastigote macrophage model is considered

as the gold standard for establishing the drug sensitivity profile

of an antileishmanial compound, promastigotes expressing the

PTR1-GFP chimera, were used to infect J774 macrophage cell line

at day 4th of seeding when the parasites had reached stationary

phase and were predominantly metacyclics (Kumar et al., 2007).

Our Leish­mania promastigote transfectants proliferated and were

infective to macrophages resulting in fluorescent amastigotes thus

maintaining the normal characteristics of the parental wild-type.

Both the compounds showed similar effect both on promastigotes

and amastigotes (Table 2). From these results, we can infer that both

compounds were able to bio transform within the macrophages to

give a more active compound. Our results also indicate that both

the compounds were five times more active on L. donovani amastig-

otes than on promastigotes. The absence of toxicity on macrophage

cell line as determined by the MTT assay (Dutta et al., 2005) indi-

cates a therapeutic index and justifying in vivo experiments on the

L. donovani hamster model which is underway in our laboratory.

The activity of 2,4-diaminopteridines, 2,4-diaminoquinazolines,

and 2,4-diaminopyrimidines against recombinant pteridine reduc-

tase 1 using promastigotes have earlier shown promising results

(Hardy et al., 1997). Since dihydropyridines and pyrimidinones

(thiones) are known inhibitors of dihydrofolate reductase and

exhibit antitubercular activity against M. tubercul­osis, a pathogen

residing in macrophages like Leish­mania, we were interested to see

whether they exhibit any pteridine reductase inhibitory activities

and therefore this study was undertaken. The high intensity and

stability of fluorescence of the PTR1-GFP-expressing chimera in

P. Kumar et al­. / Exp­erimental­ Parasitol­ogy 120 (2008) 73–79 77

Table­ 3

Docking scores of compounds

Compound/ Scores Compound 1 Compound 2

LigandFit DockScore 42.477 40.867

Ligscore1 4.090 1.210

Ligscore2 5.550 1.280

Plp1 80.630 61.370

Plp2 82.390 63.880

JAIN 2.290 2.840

PMF 85.320 161.070

Ludi 401.00 659.00

Consensus score 6 6

Table­ 2

Effi­cacy of compounds in vitro against both promastigotes and macrophage-amasti-

gote system by flow cytometry

S. No. Compound Promastigotes

IC50 (M)

Amastigotes

IC50 (M)

1. (4-Fluoro-phenyl)-6-methyl-2-thi-

oxo-1,2,3,4-tetrahydro-pyrimidine

-5-carboxylic acid ethyl ester

0.101 ± 6.1 0.0231 ± 1.7

2. 2,6-Dimethyl [3-O-benzyl-1,2-O

isopropylidene-b-l-th­reo-pentofuro-

nose-4-yl]-1-phenyl-1,4-dihydro

pyridine-3,5-dicarboxylic acid

diethyl ether

0.0908 ± 9.7 0.018 ± 3.4

3. Sodium antimony gluconate — 0.0121 ± 2.6

L. donovani promastigotes and amastigotes allowed drug testing by

flow cytometry and the inhibition of growth by two antileishma-

nial compounds was demonstrated.

A three-dimensional model of L. donovani PTR1 was drawn

(Fig. 1B), based on the crystal structure of L. major PTR1 [PDB

code 1E7W (Gourley et al., 2001)]. The functional enzyme is tet-

ramer in solution (Wang et al., 1997; Kumar et al., 2004) and rep-

resented by a ribbon diagram of the L. donovani PTR1 tetramer

(Fig. 1C), carries two active sites on each side of the assembly,

separated by only 25 Å. The PTR1 subunit is a single a/b-domain

comprising seven-stranded parallel b-sheet with three a-helices

on either side. Additionally, it has two minor helices, at the car-

boxyl side of the sixth b -sheet. The arrangement of these ele-

ments of secondary structure is similar to that of the classical

Rossmann fold. The catalytic center is mainly constructed from

a single chain using residues at the C-terminal regions of b1, b4,

b5 and b6 and the N-terminal sections of a1 and a5 (Fig. 1B). We

docked biopterin into the active site of PTR1 from L. donovani

model. After docking, we checked for all possible interactions

of biopterin with the cofactor and L. donovani PTR1. Most of the

possible interactions were as observed in the structure of PTR1

from L. major. The cofactor binds in the active site in an extended

conformation; with nicotinamide creating the floor of the cata-

lytic center and Phe113 forming an overhang under which the

pterin-binding pocket is formed (Fig. 2A). Such extensive inter-

action between substrate and nicotinamide is unique to PTR1

Fig. 2. The interaction of biopterin and MTX with PTR1 and NADPH. (A) Interaction of bi

resented by yellow. (B) MTX is represented in blue and the same color code for the NAD

to colour in this figure legend, the reader is referred to the web version of this article.)

amongst the SDR family members (Gourley et al., 2001). Asp 181

acquires a proton from the solvent and passes this over onto the

substrate in performance with Tyr194, which is the active-site

base. Lys198 helps to position the nicotinamide of the cofactor

NADPH for hydride transfer to Tyr194.

We docked MTX into the active site of PTR1 from L. donovani

model. Previous crystallographic and modeling analysis shows

that MTX interacts with L. major and L. tarentol­ae PTR1 mainly

using the pterin moiety (Gourley et al., 2001; Zhao et al., 2003).

The result of this docking attempt shows that the observed bind-

ing mode of MTX in the crystal structures is, as expected, repro-

duced in L. donovani model (Fig. 2B). The pterin moiety of MTX

interacts significantly with L. donovani PTR1 and cofactor NADPH

making five hydrogen bonds with NADPH and with residues Tyr

194, Ser 111, Arg 17 of L. donovani PTR1. On the other hand the

pABA group and glutamyl tail of MTX are relatively flexible and

only loosely associated with PTR1 making only a single hydro-

gen bond with Tyr 191. When multiple conformations are visual-

ized, different orientations of this side chain are observed. The

absence of molecular restraints and the availability of space in

that area of the active site occupied by pABA group and glutamyl

tail provide broad-spectrum activity of PTR1. MTX is a more

potent inhibitor of DHFR than PTR1 (Hardy et al., 1997) because

the pABA group and glutamyl tail makes more extensive interac-

tions, including two direct salt-bridge associations with residues

in the DHFR active site (Knighton et al., 1994). Previous studies

on the crystal structure of L. major PTR1 in complex with CB3717

opterin with PTR1 and NADPH. Biopterin is represented in brown and NADPH is rep-

PH is used. PTR1 residues are shown in magenta. (For interpretation of the references

78 P. Kumar et al­. / Exp­erimental­ Parasitol­ogy 120 (2008) 73–79

Fig. 3. Predicted binding models for (A) compound 1 (purple) and (B) compound 2 (green) in the binding site of PTR1 (magenta). Cofactor NADPH is colored in yellow. (For

interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

(Schüttelkopf et al., 2005) and MTX (Gourley et al., 2001) in com-

parison with TAQ (McLuskey et al., 2004) showed that active

site of PTR1 mainly interacts with the pterin or quinazoline ring

while the pABA group and glutamyl tail are relatively flexible

and not associated much with the active site of PTR1. Analysis

of the docked MTX in the active site of three dimensional model

structure of L. donovani PTR1 suggested that the pABA group and

glutamyl tail might not contribute that much to enzyme inhibi-

tion and it is hypothesized that smaller entity like the pterin moi-

ety may bind effectively to the active site and can be used for the

development of pharmacophores.

To understand the mechanism underlying the highly selec-

tive inhibition of L. donovani PTR1 by the selected compounds,

we analyzed the binding modes of the both the compounds in

the active site of L. donovani PTR1 model constructed by us.

LigandFit program was used for molecular docking of both the

compounds. The flexible fitting option was selected for genera-

tion of alternative conformations on the fly, as was the diverse

conformer option to ensure the solutions generated cover a

broad range of conformations with similar low-energy docking

scores, and a maximum of 30 top scoring diverse ligand poses

were returned for each of the compounds. The conformer with

best consensus score was taken as the best docked conforma-

tion. The LigandFit scores for the compounds are presented in

Table 3.

Binding mode analysis of both the inhibitors shows that they

fit well in the active site pocket and are involved in a number of

hydrogen-bonding and Van der Waals interactions with the active

site residues. The binding conformations of these two compounds

are shown in Fig. 3. The docking results indicate that the carbox-

ylic acid ethyl ester group of pyridine moiety plays important role

in the tight binding with PTR1, which is revealed from the binding

of both the compounds. An oxygen atom of the carboxylic acid

ethyl ester group in both the compound interacts extensively with

the side chain nitrogen of Arg17 and backbone nitrogen atom of

Ala 230. The presence of the ester group in the inhibitors was

found to be very important for binding as both the oxygen atoms

of this group act as a hydrogen bond acceptor and form strong

interactions with Arg 17 and Ala 230 which are very important

catalytic residues. Compound 1 displays similar binding mode

and almost similar interactions to the PTR1 active site residues

as shown by compound 2 and therefore, no significant difference

in the biological activities of both the compounds was observed.

It is interesting to note from our docking studies that, contrary

to our expectations, none of these inhibitors were found to inter-

act directly with the NADPH; however docking studies without

NADPH in the PTR1 active site were not successful. The NADPH

here may play a steric role in the binding of these inhibitors. Over-

all, our docking simulations predicted that the compound 1 PTR1

binding interaction was similar to the compound 2 interaction and

these observations were found to be in a good agreement with the

experimental data from the inhibitory assays.

The traditional chemistry based approach of finding new

candidate molecules from known active compounds or natural

products has driven antiparasitic drug discovery in the past.

Recently, drug discovery strategy has employed a search for

specific inhibitors of known biological drug targets with the

aid of protein crystal structures and/or high throughput screen-

ing assays. The GFP based high throughput screening assay for

antileishmanials along with the molecular modeling and dock-

ing details provided in this work identifies the important inter-

actions necessary to assist the structure-based development of

novel enzyme inhibitors of potential therapeutic value for this

parasitic disease.

Ac­kno­wle­dgme­nts

Support for this research was provided from CSIR India

funded network project SMM003 ‘Molecular biology of selected

pathogens for developing drug targets’ and Department of

Biotechnology, India, No. BT/PR5452/BRB/10/430/2004. P.K.,

A.K. and N.S. acknowledge Council of Scientific and Industrial

Research, India for fellowship. We are grateful to Dr Shyam Sun-

dar, BHU, Varanasi, India, for providing the clinical isolate used

in this study.

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