J. Appl. Cryst.の発刊に際して

Short-chain dehydrogenases/reductases (SDR): the 2002 update.

Kinetoplastids are a group of flagellated protozoans that include the species Trypanosoma and Leishmania, which are human pathogens with devastating health and economic effects. The sequencing of the genomes of some of these species has highlighted their genetic relatedness and underlined differences in the diseases that they cause. As we discuss in this Review, steady progress using a combination of molecular, genetic, immunologic, and clinical approaches has substantially increased understanding of these pathogens and important aspects of the diseases that they cause. Consequently, the paths for developing additional measures to control these "neglected diseases" are becoming increasingly clear, and we believe that the opportunities for developing the drugs, diagnostics, vaccines, and other tools necessary to expand the armamentarium to combat these diseases have never been better.

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Kinetoplastids: related protozoan pathogens, different diseases

Background 
Recent circumstantial evidence suggests that an increasing number of Iranian patients with cutaneous leishmaniasis are unresponsive to meglumine antimoniate (Glucantime), the first line of treatment in Iran. This study was designed to determine whether the clinical responses (healing, or non-healing) were correlated with the susceptibility of Leishmania parasites to Glucantime. 
Methods and Findings 
In vitro susceptibility testing was first performed on 185 isolated parasites in the intracellular mouse peritoneal macrophage model. A strong correlation between the clinical outcome and the in vitro effective concentration 50% (EC50) values was observed. Parasites derived from patients with non-healing lesions had EC50 values at least 4-fold higher than parasites derived from lesions of healing patients. A selection of these strains was typed at the molecular level by pulsed-field gels and by sequencing the pteridine reductase 1 (PTR1) gene. These techniques indicated that 28 out of 31 selected strains were Leishmania tropica and that three were Leishmania major. The L. major isolates were part of a distinct pulsed-field group, and the L. tropica isolates could be classified in three related additional pulsed-field groups. For each pulsed-field karyotype, we selected sensitive and resistant parasites in which we transfected the firefly luciferase marker to assess further the in vitro susceptibility of field isolates in the monocyte cell line THP1. These determinations confirmed unequivocally that patients with non-healing lesions were infected with L. tropica parasites resistant to Glucantime. Additional characterization of the resistant isolates showed that resistance is stable and can be reversed by buthionine sulfoximine, an inhibitor of glutathione biosynthesis. 
Conclusions 
To the authors' knowledge, this is the first report of proven resistant parasites contributing to treatment failure for cutaneous leishmaniasis and shows that primary Glucantime-resistant L. tropica field isolates are now frequent in Iran.

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Unresponsiveness to Glucantime treatment in Iranian cutaneous leishmaniasis due to drug-resistant Leishmania tropica parasites.

Leishmaniasis is a parasitic disease caused by hemoflagellate, Leishmania spp. The parasite is transmitted by the bite of an infected female phlebotomine sandfly. The disease is prevalent throughout the world and in at least 88 countries. Nearly 25 compounds are reported to have anti-leishmanial effects but not all are in use. The pentavalent antimony compounds have remained mainstay for nearly 75 years. Pentavalent antimony is a prodrug that is reduced by glutathione to active trivalent species catalyzed by thiol-dependent-reductase. However, emergence of resistance led to the use of other compounds -amphotericin B, pentamidine, paromomycin, allopurinol etc. Amphotericin B, an antifungal macrolide polyene is characterized by the hydrophilic polyhydroxyl and hydrophobic polyene faces on it long axis. Presently, it is the only drug with highest cure rate. It acts on membrane sterols resulting in parasite cell lysis. Its lipid formulations have been developed to minimize side effects. Other anti-fungals like ketoconazole, fluconazole and terbinafine are found less effective. Recently, anticancer alkylphosphocholines have been found most effective oral compounds. These act as membrane synthetic ether-lipid analogues, and consist of alkyl chains in the lipid portions. Most promising of these are miltefosine (hexadecylphosphocholine), edelfosine (ET-18-OCH3) and ilmofosine (BM 41.440). However, the recent focus has been on identifying newer therapeutic targets in the parasite such as DNA topoisomerases. The present review describes the current understanding of different drugs against leishmaniasis, their chemistry, mode of action and the mechanism of resistance in the parasite. Future perspectives in the area of new anti-leishmanial drug targets are also enumerated. However, due to the vastness of the topic main emphasis is given on visceral leishmaniasis.

Chemotherapy of leishmaniasis: past, present and future.

Pteridine reductase (PTR1) is a short-chain reductase (SDR) responsible for the salvage of pterins in parasitic trypanosomatids. PTR1 catalyzes the NADPH-dependent two-step reduction of oxidized pterins to the active tetrahydro-forms and reduces susceptibility to antifolates by alleviating dihydrofolate reductase (DHFR) inhibition. Crystal structures of PTR1 complexed with cofactor and 7,8-dihydrobiopterin (DHB) or methotrexate (MTX) delineate the enzyme mechanism, broad spectrum of activity and inhibition by substrate or an antifolate. PTR1 applies two distinct reductive mechanisms to substrates bound in one orientation. The first reduction uses the generic SDR mechanism, whereas the second shares similarities with the mechanism proposed for DHFR. Both DHB and MTX form extensive hydrogen bonding networks with NADP(H) but differ in the orientation of the pteridine.

Pteridine reductase mechanism correlates pterin metabolism with drug resistance in trypanosomatid parasites

summary Leishmania and other trypanosomatid protozoa require reduced pteridines (pterins and folates) for growth, suggesting that inhibition of these pathways could be targeted for eective chemotherapy. This goal has not yet been realized, indicating that pteridine metabolism may be unusual in this lower eukaryote. We have investigated this possibility using both wild type and laboratory-selected antifolate-resistant strains, and with defined genetic knockouts of several pteridine metabolic genes. In Leishmania, resistance to the antifolate methotrexate is mediated through several mechanisms singly or in combination, including alterations in transport leading to reduced drug influx, overproduction (R-region amplification) or point mutation of dihydrofolate reductase-thymidylate synthase (DHFR-TS), and amplification of a novel pteridine reductase (PTR 1, encoded by the H-region). All of the proteins involved are potential targets for antifolate chemotherapy. Notably, parasites in which the gene encoding dihydrofolate reductase (DHFR) has been deleted (dhfr-ts’ knockouts) do not survive in animal models, validating this enzyme as a target for eective chemotherapy. However, the properties of pteridine reductase 1 (PTR1) suggest a reason why antifolate chemotherapy has so far not been successful in trypanosomatids. PTR1, by its ability to provide reduced pterins and folates, has the potential to act as a by-pass and}or modulator of DHFR inhibition under physiological conditions. Moreover, PTR1 is less sensitive to many antifolates targeted primarily against DHFR. These findings suggest that successful antifolate chemotherapy in Leishmania will have to target simultaneously both DHFR and PTR1.

New approaches to Leishmania chemotherapy: pteridine reductase 1 (PTR1) as a target and modulator of antifolate sensitivity

The enzyme pteridine reductase (PTR1) has recently been discovered in the protozoan parasite Leishmania and validated as a target for therapeutic intervention. PTR1 is responsible for the salvage of pteridines and also contributes to antifolate drug resistance. Structural analysis, in combination with ongoing biochemical characterization will assist the elucidation of the structure–activity relationships of this important enzyme and support a structure-based approach to discover novel inhibitors. Recombinant L. major PTR1 has been purified from an Escherichia coli expression system and used in crystallization experiments. Orthorhombic crystals have been obtained and data to 2.8 A has been measured. The space group is P21212 or P212121 with unit-cell dimensions of a = 103.9, b = 134.7, c = 96.2 A. One homotetramer, of molecular mass approximately 120 kDa, probably constitutes the asymmetric unit and gives a Matthews coefficient, Vm, of 2.8 A3 Da−1 and 56% solvent volume. Self-rotation function calculations show a single well defined non-crystallographic twofold axis with features that might represent additional elements of non-crystallographic symmetry. The detail of exactly what constitutes the asymmetric unit will be resolved by structure determination.

James Luba

Papers

Pteridine reductase mechanism correlates pterin metabolism with drug resistance in trypanosomatid parasites

New approaches to Leishmania chemotherapy: pteridine reductase 1 (PTR1) as a target and modulator of antifolate sensitivity

Crystallization of recombinant leishmania major pteridine reductase 1 (ptr1)