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Structures of the Pteridine Reductase Structures of the Pteridine Reductase (PTR1) from (PTR1) from Trypanosoma bruceiTrypanosoma brucei

Complexed with Folate Analog Complexed with Folate Analog InhibitorsInhibitors..

Viviane Paula MartiniViviane Paula Martiniaa, Jorge Iulek, Jorge Iulekaa, William Nigel , William Nigel HunterHunterbb, Lindsay Brian Tulloch, Lindsay Brian Tullochbb

aaDepartment of Chemistry, Biotechnology Center, State University Department of Chemistry, Biotechnology Center, State University of Ponta Grossa - PR, Brazil.of Ponta Grossa - PR, Brazil.

bbDivision of Biological Chemistry and Molecular Microbiology, Division of Biological Chemistry and Molecular Microbiology, School of Life Sciences, University of Dundee, UK.School of Life Sciences, University of Dundee, UK.

e-mail: e-mail: w.n.hunter@dundee.ac.ukw.n.hunter@dundee.ac.uk, , iulek@uepg.briulek@uepg.brIntroductionIntroduction

Human African Trypanosomiasis (HAT, or African Sleeping Sickness). The disease

is caused through infection by the flagellated protozoan trypanosomatid parasites

Trypanosoma brucei rhodesiense (Eastern and Southern Africa) and Trypanosoma

brucei gambiense (Western and Central Africa), which is transmitted through the bite of

the tsetse fly (Glossina spp) [1]. The infections by T. b. rhodesiense are more severe

than the ones by T. b. gambienses. The treatment is always difficult, specially when the

disease reaches an advanced phase with the involvement of the central nervous

system, because few drugs are available and have rather low efficiency. Pentamidine®,

Suramin®, Melarsoprol® and Eflornithine® (DFMO) are some of the used drugs,

however, parasite resistant forms are being each time more frequently found, besides

the undesirable side effects [2]. There is the urgent need for new, more efficient

treatments [3]. The enzymes dihydrofolate reductase-thymidylate synthase and

pteridine reductase (PTR) are involved in pterin/folate dependent metabolism and

together represent an important target for chemotherapy of parasitic leishmanias and

trypanosomes [4,5]. X-ray crystallography was used to elucidate the structure of PTR1

from Trypanosoma brucei in complex with folate analogues. Such complex structures

are models for studies in computational chemistry and molecular modeling and were

used for initial virtual screening procedures with the program eHiTS [6].

ObjectivesObjectives Experimental Methods and ResultsExperimental Methods and Results

Some Complexed Some Complexed TbTbPTR1PTR1 CrystalsCrystals

a c dbFigure 2 Some crystals of the TbPTR1 complexed with a) WSG3066, b) WSG3067, c) Melamine and d) Folate, shown in mother liquid, grown with 1 mmol/L NADP+, 1 mmol/L of the ligand (inhibitor or folate), 20 mmol/L of dithiothreitol and 1% (m/V) of dimethyl sulphoxide, buffer tris-HCl (20 mmol/L and pH 7,5). The method used was hanging drop vapour diffusion. The data collection was carried out at the Daresbury Synchrotron; images were processed with either mosflm/scala [8] or xds/xscale [9]. Electron Density of Inhibitors at the Electron Density of Inhibitors at the

TbTbPTR1PTR1 Active Site Active Site Table I: Crystallographic Summary (values in parenthesis Table I: Crystallographic Summary (values in parenthesis correspond to the highest resolution shell)correspond to the highest resolution shell)

a

dc

b

Superposition of the Crystallized Superposition of the Crystallized LigandsLigands at the Active Siteat the Active Site

Table II: Active Site ContentsTable II: Active Site Contents

Conclusions and Conclusions and Further PerspectivesFurther Perspectives

ReferencesReferences[1] Ollivier, G. & Legros, D. 2001. African human

trypanosomiasis: History of treatment successes

and failures. Tropical Medicine & International

Health 6: 855-863.

[2] WHO (2000). WHO Report on Global

Survaeillance of Epidemic-Prone Infectious Diseases.

World Health Organisation:

http://www.who.int/csr/resources/publications/surveillance/en/WHO_Report_Infectious_Diseases.pdf

[3] L.T. Webster In "The Pharmacological Basis of

Therapeutics" Section X. Chemotherapy of Parasitic

Infections, p.954 - 959, 978 - 998 e 1008 - 1017.

Eigth Edition, Pergamon Press. New York. (1990).

[4] B. Nare, J. Luba, L. Hardy & S.M.Beverley, New

approaches to Leishmania chemotherapy: pteridine

reductase 1 (PTR1) as a target and modulator of

antifolate sensitivity. Parasitology, 114, S101-110,

(1997).

[5] J. E. Hyde, The dihydrofolate reductase-

thymidylate synthase gene in the drug resistence of

malaria parasites. Pharmacology and Therapeutics,

48, 45-59, (1990).

[6] Zsolt Zsoldos, Darryl Reid, Aniko Simon, Sayyed

Bashir Sadjad, A. Peter Johnson, 2006. eHiTS: A new

fast, exhaustive flexible ligand docking system.

Journal of Molecular Graphics and Modelling, in

press.

[7] John J. Irwin and Brian K. Shoichet, 2005. ZINC -

A Free Database of Commercially Available

Compounds for Virtual Screening. J. Chem. Inf.

Model, 45, 177-182.

[8] Leslie, A. G. I, Mosflm: Integration of

macromolecular diffraction data. (1999) Acta Cryst.

D55, 1696-1702

[9] Kabsch, W., XDS: Automatic processing of

rotation diffraction data from crystals of initially

unknown symmetry and cell constants. (1993) J.

Appl. Cryst. 26, 795-800.

[10] DeLano, W.L. The PyMOL Molecular Graphics

System (2002) DeLano Scientific, San Carlos, CA,

USA. http://www.pymol.org[11] Refinement of Macromolecular Structures by the Maximum-Likelihood Method" G.N. Murshudov, A.A.Vagin and E.J.Dodson, (1997) in Acta Cryst. D53, 240-255.[12] Emsley, P. and Cowtan, K., Coot: Model-Building Tools for Molecular Graphics. (2004) Acta Cryst. D60, 2126-2132.

V.P.M. thanks CAPES fellowship, W.N.H and University of Dundee. J.I. thanks CAPES, fellowship number BEX V.P.M. thanks CAPES fellowship, W.N.H and University of Dundee. J.I. thanks CAPES, fellowship number BEX 2000/04-0 and SimBioSys Inc. for the eHiTS academic license. W.N.H. thanks The Wellcome Trust, 2000/04-0 and SimBioSys Inc. for the eHiTS academic license. W.N.H. thanks The Wellcome Trust,

BBSRC(UK) and synchrotron beam time from the ESRF and SLS.BBSRC(UK) and synchrotron beam time from the ESRF and SLS.

• To crystallize complexes of inhibitors bound to TbPTR1 and solve their structures; • To map the active site of the enzyme and to characterize the enzyme-ligand interactions visually and through computational methods [6];• To understand the enzymatic mechanism and its specificities, particularly, to guide the planning and discovery of new compounds that are powerful, but selective, inhibitors for the parasitic enzyme.

Eight complexes have been

crystallized; five structures were refined,

of which four inhibitors. The structures

provided important information about

the interactions each inhibitor makes

with the TbPTR1, supplying data to plan

more powerful inhibitors and, in a wider

way, to assist in the rational

development of therapeutical drugs

against HAT. Further analyses are

being/will be carried out with

computational methods, including

docking (virtual screening) procedures

trained with the structural data (eHiTS,

[6]) using the Zinc [7] and other

molecular databases.

Figure 3 Active site of the TbPTR1 with the corresponding ligands: a) trianterene (TTR), b) Cyromazin (CYR), c) WSG3066 (synthesis code) and d) WSG3067 (synthesis code). Electron Density maps are contoured at 1.5 σ. Figure produced by Pymol [10].

Figure 4 TbPTR1 active site with the ligands TTR, CYR, WSG3066 and WSG3067 superposed at their corresponding experimentally determined positions. Several key interactions are present in most of (if not all) the structures. Special detail to the stacking formed by a ligand aromatic ring, the Phe97 side chain and the cofactor nicotinamide (NAP) ring. Figure produced by Pymol [10].

Crystallographic Crystallographic Asymmetric UnitAsymmetric Unit

Figure 5 Illustration of the 4 monomers in the asymmetric unit which represents the functional unit; each monomer contains 268 residues. Modelling and refinement was made with the programs refmac [11] and coot [12]. Figure produced by Pymol [10].

Table III: Some Partial Docking ResultsTable III: Some Partial Docking ResultsThe ranking and the score are shown with and without the weight training using the crystallographic structures. A subset of the Zinc database was

used/created based upon the ligand stacking rings. Analyses of the docked poses is currently underway and partial observations point that the most of

best scored molecules conserve the key active site interactions.

* GOL: glycerol; EDO: ethylene glycol; DTT: dithiothreitol; DTO: oxidized dithiothreitol

Figure 1 (a) Human African Trypanosomiasis (HAT), (b) Distribution and abundance of HAT, (c) Flagellated protozoan trypanosomatid parasites Trypanosoma brucei, (d) tsetse fly (Glossina ssp). Figure adapted form www.sgpp.org/african_sleeping_sickness.shtml

a bd

c