Old targets and new beginnings: a multifaceted approach to combating Leishmaniasis, a neglected tropical disease

Leishmaniasis, a broad spectrum of disease which is caused by the protozoan parasite Leishmania, currently affects 12 million people in 88 countries worldwide.There are over 2 million of new cases of leishmaniasis occurring annually.Clinical manifestations of leishmaniasis range from potentially disfiguring cutaneous leishmaniasis to the most severe manifestation, visceral leishmaniasis, which attacks the reticuloendothelial system and has a fatality rate near 100% if left untreated.All currently available therapies all suffer from drawbacks including expense, route of administration and developing resistance.In the laboratory of Dr. Karl Werbovetz our primary goal is the identification and development of an inexpensive, orally available antileishmanial chemotherapeutic agent.Previous efforts in the lab have identified a series of dinitroaniline compounds which have promising in vitro activity in inhibiting the growth of Leishmania parasites.It has since been discovered that these compounds exert their antileishmanial effects by binding to tubulin and inhibiting polymerization.Remarkably, although mammalian and Leishmania tubulins are ~84 % identical, the dinitroaniline compounds show no effect on mammalian tubulin at concentrations greater than 10-fold the IC 50 value determined for inhibiting Leishmania tubulin iii polymerization.These results indicate that Kinetoplastid tubulin may present a useful chemotherapeutic target.Ongoing drug development efforts with the dinitroaniline compounds will require that the next generation analogues be analyzed in vitro against not only the parasite but also purified parasite tubulin.Previously, tubulin has been purified from the pathogenic Leishmania amazonensis.There are several drawbacks with utilizing L. amazonensis as a tubulin source, including, low growth densities, expense of growth medium and risk of infection to laboratory personnel.Leishmania tarentolae utilizes the gecko lizard as a host, and does not infect humans.Furthermore, L. tarentolae can be grown to high densities and can be cultured in inexpensive medium.To assess the suitability of L. tarentolae tubulin as a viable alternative to the corresponding protein from L. amazonensis for compound screening, both the αand β-tubulin genes were sequenced for comparisons with pathogenic species.Both α-and β-tubulin protein sequences are at least 98% identical to sequences from closely related Leishmania species.Additionally, two in vitro experiments were conducted to examine the degree of dinitroaniline binding site congruency between L. amazonensis and L. tarentolae tubulin.IC 50 values, in terms of inhibiting tubulin polymerization, for our lead compound GB-II-5 were 6.7 µM and 6.8 µM for L. amazonensis and L. tarentolae tubulin, respectively.Dissociation constants (K d ), determined by fluorescence quenching, were used to compare binding affinities of GB-II-5 for tubulin from both L. amazonensis and L. tarentolae.The K d s for GB-II-5 for L. amazonensis and L. tarentolae were determined to be 1.7 µM and 2.4 µM, viii