Wang Pilot Project Summary
Human leishmaniasis is a devastating infectious disease caused by protozoan parasites belonging to the genus of Leishmania. The disease is found in more than 90 countries and responsible for an estimated 1-2 million new infections each year worldwide. Leishmaniasis is also common in returning U.S. military personnel from Iraq and Afghanistan, with more than two thousand cases reported since 2001. Leishmania parasites cause disfiguring skin sores (cutaneous leishmaniasis or CL) and life-threatening infection of vital internal organs (visceral leishmaniasis or VL). VL (~20% of all leishmaniasis cases) is the most dangerous and fatal form of the disease, with a mortality rate close to 100% if left untreated. It is the second most deadly parasitic disease in the world (after malaria). Despite being a serious public health problem in many endemic regions, there are no vaccines or preventative chemotherapies available for leishmaniasis control. Current antileishmanial drugs have limited efficacy, serious side effects and high cost. Hence, there is a major unmet medical need for safe and effective drugs against leishmaniasis. Leishmanial CYP5122A1 is a novel cytochrome P450 (CYP) enzyme that is essential for the survival of L. donovani, a major causative agent for VL. Independent and our own studies have led us to hypothesize that a two-tier screening cascade, consisting of primary high-throughput screening (HTS) assays and secondary function and activity assays will identify novel CYP5122A1 inhibitors as therapeutic agents for leishmaniasis control. To test the hypothesis, two specific aims are proposed to answer two main questions: 1) Can the fluorescence-based inhibition assay be translated into a robust HTS assay? 2) Will the proposed two-tier screening cascade lead to discovery of novel antileishmanial compounds? Innovative experimental approaches will be employed in the proposed project, e.g., a new HPLC-MS/MS-based sterol assay, fluorescence-based CYP5122A1 and CYP51 inhibition assays, and cutting-edge high content imaging-based intracellular antileishmanial assays. If successfully completed, the proposed project will establish robust primary HTS assays and validate a two-tier screening cascade. Both are critically important for a future NIH R01 application that aims to identify novel CYP5122A1 inhibitors as therapeutic agents for leishmaniasis control.