are protozoan parasites (order Trypanosomatida, class Kinetoplastida) that cause a range of diseases and present a serious health risk to millions of people worldwide (Desjeux, 2004
; Reithinger et al.
). The incidence of infection is primarily in tropical and subtropical regions of the world (Herwaldt, 1999
). The different species of Leishmania
are responsible for distinctive conditions (Reithinger et al.
). For example, L. donovani
causes visceral leishmaniasis, a potentially fatal disease, while infection with L. major
leads to cutaneous leishmaniasis. Several compounds are available to treat these infections, but increasing levels of drug resistance combined with the high cost and toxicity of antileishmanial drugs compromises the control of the diseases (Croft et al.
; Maltezou, 2010
). These observations explain in part why the World Health Organization has identified the leishmaniases as neglected diseases and is urgently seeking novel therapeutic approaches (World Health Organization, 2007
Our aim is to identify and characterize drug targets in these parasites and to apply structure-based approaches to develop potent inhibitors that possess the right chemical properties to underpin early-stage drug discovery (Hunter, 2009
). A promising target with respect to infection with Leishmania
sp. is the NADPH-dependent short-chain dehydrogenase/reductase pteridine reductase (PTR1; EC 184.108.40.206). This enzyme is unique to trypanosomatid parasites, where it supports the provision of reduced biopterins that are necessary for metacyclogenesis (Cunningham et al.
) and which are implicated in resistance to reactive oxygen and nitrogen species in Leishmania
(Moreira et al.
). PTR1 catalyzes the reduction of biopterin to 7,8-dihydrobiopterin as well as its subsequent reduction to 5,6,7,8-tetrahydrobiopterin. Additionally, the enzyme catalyzes similar reactions in the salvage of unconjugated folates in Leishmania
(Nare, Hardy et al.
; Nare, Luba et al., 1997
). As Leishmania
are auxotrophic for pteridines (folates and pterins) and are required to obtain these nutrients from their environment to maintain growth, disrupting this salvage process represents a potential therapeutic strategy.
We have previously studied the structure–mechanism–activity relationships for the enzymes from L. major
PTR1) and Trypanosoma brucei
PTR1) and determined the structures of a series of inhibitor complexes (Gourley et al.
; Dawson et al.
; Cavazzuti et al.
; Mpamhanga et al.
), whilst the structure of T. cruzi
PTR2 has been reported by others (Schormann et al.
). We identified sequence and structural differences between Lm
PTR1 and Tb
PTR1 that explain why some inhibitors display a significant level of selectivity for one orthologue over the other (Gibson et al.
; Tulloch et al.
). Although we were able to routinely generate crystals of Tb
PTR1 that diffracted to between 2.0 and 1.0 Å resolution (Dawson et al.
), studies with Lm
PTR1 have been hampered by poor crystal quality and a lack of reproducibility. One crystal form of Lm
PTR1 diffracted to beyond 2.0 Å resolution but could only be obtained in the presence of NADPH and methotrexate (Gourley et al.
); when other ligands were present different crystal forms were obtained. The size of the asymmetric unit is increased from two to either four or eight subunits and the crystals are often mechanically twinned and diffract to lower resolution, with the diffraction pattern being anisotropic and highly mosaic (McLuskey et al.
; Schüttelkopf et al.
). An alternative source of Leishmania
PTR1 was therefore sought for our investigations. Studies with L. tarentolae
PTR1 resulted in a 2.8 Å resolution structure of the complex with NADPH, but despite its presence in the crystallization mixture the tight-binding ligand methotrexate was not observed in the electron-density maps (Zhao et al.
). This was not considered to be an improvement on the results that we had previously obtained, so we elected to initiate crystallographic studies of L. donovani
PTR1), this also being the enzyme from the pathogen that causes the most serious form of leishmaniasis. There is a high level of conservation (91% sequence identity) between the L. major
and L. donovani
enzymes and homology modelling of the latter has suggested a close structural relationship in and around the active site (Kaur et al.