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A Multifaceted Approach to Combating Leishmaniasis, a Neglected Tropical Disease

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A Multifaceted Approach to Combating Leishmaniasis, a Neglected Tropical Disease OLD TARGETS AND NEW BEGINNINGS: A MULTIFACETED APPROACH TO COMBATING LEISHMANIASIS, A NEGLECTED TROPICAL DISEASE DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy from the Graduate School of The Ohio State University By Adam Joseph Yakovich, B.S. ***** The Ohio State University 2007 Dissertation Committee: Karl A Werbovetz, Ph.D., Advisor Approved by Pui-Kai Li, Ph.D. Werner Tjarks, Ph.D. ___________________ Ching-Shih Chen, Ph.D Advisor Graduate Program In Pharmacy ABSTRACT 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 ii 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 α- and 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 ds for GB-II-5 for L. amazonensis and L. tarentolae were determined to be 1.7 µM and 2.4 µM, iii respectively. These data taken together indicate that tubulin from L. tarentolae is a suitable alternative to tubulin from pathogenic species for drug evaluation. The K ds and IC 50 values determined for numerous GB-II-5 analogues synthesized in our lab against Leishmania tubulin has allowed us to utilize molecular modeling and molecular dynamics to better characterize the dinitroaniline binding site. Although we have learned a great deal about this site using the aforementioned methods, a crystal structure of tubulin with a dinitroaniline bound would yield the most definitive information about the binding site. Traditionally, tubulin has been very difficult to crystallize, due in part to its lack of stability and it’s propensity to polymerize at higher conditions. Recently the crystal structure of vinblastine bound to mammalian tubulin has been elucidated. The authors utilized stathmin, a 17 kDa protein which acts to regulate microtubule dynamics, to sequester tubulin into tetramers and were able to crystallize a tubulin-stathmin complex. It was thought that this approach may be useful for crystallizing Leishmania tubulin. Reports documenting the purification of mammalian stathmin indicate stathmin present in the soluble cell lysate of fetal bovine thymus will effectively inhibit tubulin polymerization by sequestering dimers. Although successful in inhibiting mammalian tubulin polymerization, preliminary attempts to sequester Leishmania tubulin with active stathmin containing fractions from fetal bovine thymus failed to show any activity. The lack of activity was not particularly surprising and it was hypothesized that variations in tubulin between Leishmania and mammals were great enough to prohibit mammalian stathmin from interacting with Leishmania iv tubulin. As such, the next step was an attempt to identify an active stathmin like protein present in Leishmania or the closely related parasite Crithidia fasciculata . Efforts to isolate an active fraction by looking for activity in terms of inhibiting tubulin polymerization, per the protocol for isolating mammalian stathmin, were unsuccessful. It was discovered that upon warming, a dense precipitate formed in the Leishmania soluble cell lysates. Since tubulin requires heat to polymerize, and that polymerization is assessed by monitoring turbidity at 350 nm, the precipitate was preventing us from monitoring tubulin polymerization or the lack thereof. Attempts to remove the precipitate by repeated warming and centrifugation steps failed to decrease baseline levels to those at which tubulin polymerization could be observed. Being as the protein of interest was likely a low molecular weight protein (LMW), size exclusion chromatography was used to fraction the soluble cell lysate by molecular weight. Although this method was useful in separating the proteins present in the cell lysate, and removing the precipitate, the LMW fractions failed to demonstrate any activity against Leishmania tubulin. Concerns that column fractionation may have decreased the stathmin like protein concentration below its active threshold were addressed by concentrating LMW containing fractions. An iCON protein concentrator was used and effectively concentrated LMW containing fractions from 0.53 mg/mL to 2.7 mg/mL. Unfortunately, these concentrated LMW fractions still failed to show activity against tubulin. At this point the search for a Leishmania stathmin like protein was terminated. This work does not prove that there is no stathmin like protein present in Leishmania , however, it indicatess that if there is such a protein present, it likely does not as large of a role in regulating microtubule dynamics as its mammalian counterpart. Perhaps future studies utilizing v coimmunoprecipitation coupled to mass spectrometry will be useful in identifying proteins that associate with and influence Leishmania microtubule dynamics. GB-II-5 is a promising antileishmanial compound, however, it is desirable to have multiple leads due to later stage attrition often seen in the drug development process. As such, our lab has recently purchased the ChemBridge CNS set of 10,000 druglike molecules. A multifaceted approach is being employed with the library whereby compounds were screened both against Leishmania donovani axenic amastigotes and also against purified Leishmania tubulin. Compounds in this library conform to Lipinski’s rule of 5, thus, it is likely that any potential lead compound identified in this screen will possess oral availability. The first stage of the screen against L. donovani axenic amastigotes identified 75 compounds which inhibited parasite growth by 50 % or more at a concentration of 10 µM. The specific activity, in terms of IC 50 , of each hit compound was then determined. Of the 75 identified active compounds, 47 were found to have IC 50 values ≤ 5 µM. Surprisingly, 11 of the originally identified active compounds failed to show activity when they were reevaluated and had IC 50 values > 25 µM. The 47 compounds with IC 50 values ≤ 5 µM were assayed against mammalian Vero cells to determine if they were selectively cytotoxic to Leishmaia cells. Of the 47 compounds, 17 were found to be at least 25-fold selective for inhibiting Leishmania proliferation. These compounds are currently being screened in our infected macrophage assay. The infected macrophage assay allows us to assess the compounds activity in clearing infection from mouse peritoneal macrophages; this vi system more closely mimics in vivo conditions. Furthermore, efforts are currently underway to examine the mechanism of action of some of the active compounds. Hopefully this endeavor will bring us closer to our ultimate goal of producing an orally available, inexpensive antileishmanial chemotherapeutic agent. vii Dedicated to my Parents: Jeffrey and Kathleen Yakovich, For everything I am and all that I have. viii ACKNOWLEDGMENTS First and foremost, I would like to thank
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