Identification of Small Molecule Lead Compounds for Visceral Leishmaniasis Using a Novel Ex Vivo Splenic Explant Model System

Melby PC (2011) Identification of Small Molecule Lead Compounds for Visceral Leishmaniasis Using a Novel Ex Vivo Splenic Explant Model System. PLoS Negl Trop Dis 5(2): e962. doi:10.1371/journal.pntd.0000962 Identification of Small Molecule Lead Compounds for Visceral Leishmaniasis Using a Novel Ex Vivo Splenic Explant Model System Yaneth Osorio 0 Bruno L. Travi 0 Adam R. Renslo 0 Alex G. Peniche 0 Peter C. Melby 0 Timothy G. Geary, McGill University, Canada 0 1 Department of Veterans Affairs Medical Center, Research Service, South Texas Veterans Health Care System , San Antonio, Texas , United States of America, 2 Department of Medicine, The University of Texas Health Science Center , San Antonio, Texas , United States of America, 3 Small Molecule Discovery Center, Sandler Center for Basic Research in Parasitic Diseases, and Department of Pharmaceutical Chemistry, University of California San Francisco , San Francisco , California, United States of America, 4 Department of Microbiology and Immunology, The University of Texas Health Science Center , San Antonio, Texas , United States of America Background: New drugs are needed to treat visceral leishmaniasis (VL) because the current therapies are toxic, expensive, and parasite resistance may weaken drug efficacy. We established a novel ex vivo splenic explant culture system from hamsters infected with luciferase-transfected Leishmania donovani to screen chemical compounds for anti-leishmanial activity. Methodology/Principal Findings: This model has advantages over in vitro systems in that it: 1) includes the whole cellular population involved in the host-parasite interaction; 2) is initiated at a stage of infection when the immunosuppressive mechanisms that lead to progressive VL are evident; 3) involves the intracellular form of Leishmania; 4) supports parasite replication that can be easily quantified by detection of parasite-expressed luciferase; 5) is adaptable to a high-throughput screening format; and 6) can be used to identify compounds that have both direct and indirect anti-parasitic activity. The assay showed excellent discrimination between positive (amphotericin B) and negative (vehicle) controls with a Z' Factor .0.8. A duplicate screen of 4 chemical libraries containing 4,035 compounds identified 202 hits (5.0%) with a Z score of , -1.96 (p,0.05). Eighty-four (2.1%) of the hits were classified as lead compounds based on the in vitro therapeutic index (ratio of the compound concentration causing 50% cytotoxicity in the HepG2 cell line to the concentration that caused 50% reduction in the parasite load). Sixty-nine (82%) of the lead compounds were previously unknown to have anti-leishmanial activity. The most frequently identified lead compounds were classified as quinoline-containing compounds (14%), alkaloids (10%), aromatics (11%), terpenes (8%), phenothiazines (7%) and furans (5%). Conclusions/Significance: The ex vivo splenic explant model provides a powerful approach to identify new compounds active against L. donovani within the pathophysiologic environment of the infected spleen. Further in vivo evaluation and chemical optimization of these lead compounds may generate new candidates for preclinical studies of treatment for VL. - Funding: This work was funded by Department of Defense, Air Force Contract No. FA7014-07-C-0034 (P.C. Melby P.I.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. New drugs are desperately needed to treat visceral leishmaniasis (VL), and this in turn requires new approaches to discover novel lead compounds that might populate a pipeline of new therapeutics for patients with VL. Current therapies for the leishmaniases are toxic, difficult to deliver, expensive, and their efficacy is hindered by parasite resistance (reviewed in [1]). The pentavalent antimony compounds, sodium stibogluconate and meglumine antimoniate, have been the mainstay of antileishmanial chemotherapy for more than 40 years. The recommended regimen involves prolonged and often repeated courses of drug administered by the intravenous or intramuscular routes. Cure rates of 80100% were common in the 1990s, but have dropped off considerably because of parasite resistance [2]. Adverse effects of antimony therapy are multiple and often doselimiting. Amphotericin B desoxycholate and the amphotericin lipid formulations are also used in the treatment of VL, and in many regions have replaced antimony as first-line therapy. The use of these drugs, however, is limited by their difficulty of administration, well-known risk of toxicity, and high cost. Parenteral treatment of VL with the aminoglycoside paromomycin (aminosidine) is used in India but not licensed in the U.S. Miltefosine, a membrane targeting alkylphospholipid, was recently licensed in India as the first oral treatment for VL, but after only a few years of use, drug resistance has emerged. The discovery of Visceral leishmaniasis is a life threatening parasitic disease present in several countries of the world. New drugs are needed to treat this disease because treatments are becoming increasingly ineffective. We established a novel system to screen for new anti-leishmanial compounds that utilizes spleen cells from hamsters infected with the parasite Leishmania donovani. The parasite strain we used was genetically engineered to emit light by the incorporation of the firefly luciferase gen. This laboratory test system has the advantage of reproducing the cellular environment where the drug has to combat the infection. The efficacy of the compounds is easily determined by measuring the light emitted by the surviving parasites in a luminometer after exposing the infected cells to the test compounds. The screening of more than 4,000 molecules showed that 84 (2.1%) of them showed anti-leishmanial activity and had an acceptable toxicity evaluation. Eighty two percent of these molecules, which had varied chemical structures, were previously unknown to have anti-leishmanial activity. Further studies in animals of these new chemical entities may identify drug candidates for the treatment of visceral leishmaniasis. new drugs for VL would have huge impact on individual patients and on populations in the endemic area as a whole. Pre-clinical in vitro studies to identify candidate drugs for treatment of leishmaniasis have employed several different approaches, each of which has significant limitations. The testing of drugs using axenically cultured parasites, usually promastigotes, has been most commonly used, but this approach is limited by 1) the discordance of anti-leishmanial activity of compounds tested in axenically cultured promastigotes (vector stage) and amastigotes (mammalian stage) [3,4], and 2) the testing of antiparasitic activity in the absence of host immune cells, which are known to pro (...truncated)