Old Targets and New Beginnings

Leishmaniasis, a broad spectrum of disease which is caused by the protozoan parasite Leishmania, currently affects 12 million people worldwide, with over 2 million of new cases occurring annually. There are several clinical manifestations, including visceral leishmaniasis, which attacks the reticuloendothelial system and has a fatality rate near 100% if untreated. All currently available therapies all suffer from drawbacks including expense, route of administration and developing resistance. Tubulin, a heterodimeric protein, comprises microtubules which are critical for eukaryotic cell mitosis. We have previously identified a series of dinitroaniline compounds which inhibit Leishmania growth in vitro by inhibiting tubulin polymerization. These compounds selectively inhibit Leishmania tubulin, indicating it may be a useful chemotherapeutic target. Evaluation of future generations of analogues will require purified Leishmania tubulin. Previously, the pathogenic species L. amazonensis has been used as a tubulin source, however, we have modified the protocol to utilize the non-pathogenic species L. tarentolae. Genetic sequencing and drug activity/affinity assays indicates that L. tarentolae tubulin is a suitable alternative for drug screening. Modeling studies with our compounds bound to Leishmania tubulin suggest the the bind to a previously uncharacterized site on á-tubulin. To confirm this, an attempt was made to acquire a crystal structure of tubulin with our compounds bound. Previously, mammalian tubulin has been crystallized by using the protein stathmin to sequester it in tetramers. Efforts were made to identify and purify a stathmin like protein in Leishmania capable of sequestering tubulin in tetramers. Unfortunately attempts to identify a low molecular weight protein present in Leishmania capable of sequestering tubulin were unsuccessful. In an ever expanding search for novel lead compounds, our lab has recently purchased the ChemBridge CNS set of 10,000 druglike molecules. These compounds were screened against Leishmania donovani axenic amastigotes. The screen of 10,000 compounds revealed 75 compounds which inhibited Leishmania growth by 50% or greater at 10 µM. Of these 75 compounds 17 were found to exhibit at least 25-fold selectivity for inhibiting Leishmania cell proliferation when compared to mammalian Vero cells.