Clinical response and resistance to antimonial drugs in Leishmania
Wellcome Trust Project

Pentavalent antimonial drugs (SbV) continue to be the mainstay of antileishmanial therapy despite their toxicity, high cost, and parenteral administration during > 20 days. We remain uncertain of the structure, mechanism of action or even the biologically active component of the most commonly utilized formulations, meglumine antimonate (Glucantime¨°) and sodium stibogluconate (Pentostam¨°). Effective alternative therapies, such as pentamidine and amphotericin B, though useful, even lifesaving in certain circumstances, are potentially more toxic, more expensive and also require parenteral administration. Although certain alternative drugs and immunotherapies may have merit for treating some cases of leishmaniasis, an equally effective, less toxic therapy that is easily administrable (in the endemic setting) remains to be identified. Thus, antimonials are likely to remain the mainstay of antileishmanial therapy for the foreseeable future. Understanding how antimonial drugs work in the treatment of leishmaniasis and why they sometimes fail is fundamental to the optimal use of the existing formulations and is also likely to be instructive in the development of new therapies. Reduction of antimonials The mode of antileishmanial action of SbV is not understood although there is general agreement that SbV is converted into SbIII, which is thought to be active form of the drug. The greater susceptibility to SbV of intracellular amastigotes compared with promastigotes suggested that the reductive activation of the drug is catalysed either by the host macrophage or, the intracellular amastigote itself. Non-enzymatic reduction of metalloids by thiols has been shown to occur, but is relatively slow and not biologically relevant. The key reductase enzyme of Leishmania has yet to be identified. Bacteria and yeast possess a specific arsenate reductase, which mediates resistance to to arsenate, although it is not known whether these enzymes can also reduce antimonate to antimonite. One hypothesis is that Leishmania possess an antimonate reductase which is related to bacterial and yeast arsenate reductases, has greater activity in amastigotes than promastigotes and so mediates antimonial drug efficacy. Depletion of this enzyme activity could result in resistance. This hypothesis will be tested using two complementary avenues. 1. The key gene will be searched for using a number of approaches. A putative distant homologue of an arsenate reductase is present on chromosome 1 of L. major. The role of this putative ORF in reduction of antimonate will be investigated using standard methods. In addition, yeast expression libraries will be constructed from cDNAs of parasites and used to complement a metal-hypersensitive yeast mutant obtained by disruption of the ACR2 reductase. This should lead to identification of a leishmanial gene that can mediate antimoniate reduction. Other approaches will be to attempt PCR amplification of putative reductase genes using sequence information from the bacterial and yeast metallo-reductase genes and to continue gene mining of the Leishmania and trypanosome genome data bases. Genes of interest will be heterologously expressed and their antimoniate reductase activity characterised. Genetic manipulation experiments involving gene deletions and re- or over-expression will be carried out to determine the role of the genes in SbV sensitivity. If reduction of SbV to SbIII is rate-limiting then overexpression of this gene should reverse resistance or render wild type isolates hypersensitive to SbV. Provided the enzyme does not have another essential role, then null mutants should be refractory to SbV. 2. The reductase enzyme itself will be investigated with biochemical assays for an antimony reductase which will complement and benefit from the arsenate reductase enzymatic assays conducted at Wayne. Extracts of promastigotes and amastigotes of will be incubated with Sbv (plus NADPH or NADH as electron donor) and the resulting SbIII separated by selective extraction into organic solvents or by HPLC or TLC methods. Depending on the sensitivity required for the assay, spectrophotometric, atomic absorption spectrometry or radioisotopic methods will be developed. Similar experiments will be carried out with whole cells incubated with Sbv. If reduction of Sbv can be established, then attempts will be made to assay the reduction spectrophotometrically coupled via either thioredoxin reductase and thioredoxin, glutathione reductase and glutaredoxin or trypanothione reductase and tryparedoxin systems. All the reagents necessary for their NADPH-dependent assays are available as pure recombinant proteins in Dundee. The possibility that resistance is mediated by a decreased ability to reductively activate Sbv will be studied biochemically by comparing sensitive and resistant lines of L. panamensis. Should this resistance mechanism be established, attempts will be made to purify the enzyme activity, clone the gene and confirm its role in resistance using forward and reverse genetics. Should these investigations prove negative, then the possibility that the host macrophage is the site of reduction of Sb will be examined using similar approaches. The possibility that reduction occurs both in the host and in the parasite cannot be excluded, but drug activation within macrophages will only be investigated in this project if reduction by the parasite is ruled out.