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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
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