Drug safety is a major concern for people that taking medicines as well as pharmaceutical companies, regulatory bodies, and basic researchers. Drug induced phospholipidosis (DIPL) is an adverse reaction to cationic amphiphilic drugs (CADs) resulting in excessive phospholipid accumulation, which in serious cases, leads to organ failure and death. DIPL is prevalent;for example, about 50% of patients prescribed the common anti-arrythmic amiodarone will develop DIPL and up to 17% will require withdrawal from the medicine. CADs are a significant fraction of both approved drugs and chemical compounds currently in testing, but their DIPL- inducing potential is currently evaluated by laborious electron microscopy or mass spectrometry methods. These methods have been judged unsatisfactory by the FDA. Human lysosomal phospholipase A2 (LPLA2, group XV PLA2) is a phospholipid processing enzyme central to DIPL. Inhibition of LPLA2 activity induces phospholipidosis in cells, and an LPLA2 enzymatic assay would enable high-throughput testing of candidate compounds. We propose work to develop a simple fluorogenic biochemical screening assay for LPLA2 activity and its application for testing the phospholipidosis-inducing potential of CADs. Our specific aims are: i) prepare quenched fluorogenic LPLA2-specific substrates, ii) characterize these "smart probe" substrates, specific for phospholipases, with recombinant LPLA2, iii) develop a high throughput screening (HTS) assay, and iv) validate the assay using control and phospholipidosis- inducing CADs. With approximately one-third of all drugs entering clinical trials failing because of toxicity and safety concerns, early detection of DIPL would decrease health care costs by identifying drugs that will induce DIPL prior to expensive clinical trials. Further, we envision this project leading eventually to patient screening for LPLA2 activity. This specific application of personalized medicine would identify individuals who are susceptible to DIPL for a given drug and guide treatment;thus preventing patient harm and improving the efficiency of healthcare in the U.S. PUBLIC HEALTH RELEVANCE: Drug safety is a major concern for people taking medicines as well as pharmaceutical groups, regulatory bodies, and basic researchers. We plan to develop and commercialize a biochemical test assessing one common type of drug toxicity called phospholipidosis. Detection of chemical compounds early in the drug- discovery process that cause phospholipidosis would prevent patient harm, decrease costs, and improve the efficiency of healthcare in the U.S.