(515u) High-Throughput Screening of the NIH Molecular Libraries Small Molecule Repository to Determine Novel, Potent, and Selective Inhibitors of Human Cathepsin L

Authors: 
Shah, P. P., University of Pennsylvania
Napper, A. D., University of Pennsylvania
Diamond, S. L., University of Pennsylvania


Cathepsin L, an endopeptidase found in lysosomes, is a papain-like cysteine protease of interest due to its involvement in many biological processes including epidermal homeostasis, hair follicle morphogenesis and cycling, extracellular matrix degradation, Severe Acute Respiratory Syndrome (SARS) coronavirus entry, and proteolytic processing of Ebola virus glycoprotein. A high-throughput screen of 57,821 compounds from the NIH Molecular Libraries Small Molecule Repository was performed to discover novel, potent, and selective inhibitors of human liver cathepsin L. The primary screen comprised small molecules (10 µM), cathepsin L (300 pM), and z-Phe-Arg-7-amido-4-methylcoumarin fluorogenic substrate (1 µM) in a 10 µL final reaction volume. Compounds were transferred to assay plates using a 100 nL hydrophobic slot pintool. Dose-response studies were performed on hits from the primary screen to determine relative potencies of compounds against cathepsin L. Our most potent hit has an IC50 equal to 100 nM and displays 322-fold greater selectivity against cathepsin L than cathepsin S and over 500-fold greater selectivity against cathepsin L than cathepsin B. This compound was determined to be a very slowly reversible inhibitor of cathepsin L. Structure-activity relationships and crystal structure docking studies are underway to predict how the compound interacts with the active site of the enzyme.