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Kinetic Characterization and Molecular Docking of a Novel, Potent, and Selective Slow-Binding Inhibitor of Human Cathepsin L

Institute for Medicine and Engineering (P.P.S., M.P.B., J.E.P., H.J., A.D.N., S.L.D.) and the Department of Chemistry (M.C.M., D.M.H., B.S.C., A.B.S.), Penn Center for Molecular Discovery, University of Pennsylvania, Philadelphia, Pennsylvania; and University of Pittsburgh Drug Discovery Institute, Pittsburgh, Pennsylvania (H.J.G., E.R.S.)

A novel small molecule thiocarbazate (PubChem SID 26681509), a potent inhibitor of human cathepsin L (EC 3.4.22.1 [EC] 5) with an IC₅₀ of 56 nM, was developed after a 57,821-compound screen of the National Institutes of Health Molecular Libraries Small Molecule Repository. After a 4-h preincubation with cathepsin L, this compound became even more potent, demonstrating an IC₅₀ of 1.0 nM. The thiocarbazate was determined to be a slow-binding and slowly reversible competitive inhibitor. Through a transient kinetic analysis for single-step reversibility, inhibition rate constants were k_on = 24,000 M^-1s^-1 and k_off = 2.2 x 10^-5 s^-1 (K_i = 0.89 nM). Molecular docking studies were undertaken using the experimentally derived X-ray crystal structure of papain/CLIK-148 (1cvz. pdb). These studies revealed critical hydrogen bonding patterns of the thiocarbazate with key active site residues in papain. The thiocarbazate displayed 7- to 151-fold greater selectivity toward cathepsin L than papain and cathepsins B, K, V, and S with no activity against cathepsin G. The inhibitor demonstrated a lack of toxicity in human aortic endothelial cells and zebrafish. In addition, the thiocarbazate inhibited in vitro propagation of malaria parasite Plasmodium falciparum with an IC₅₀ of 15.4 µM and inhibited Leishmania major with an IC₅₀ of 12.5 µM.

Address correspondence to: Scott L. Diamond, Penn Center for Molecular Discovery, University of Pennsylvania, 1024 Vagelos Laboratories, Philadelphia, PA 19104-6383. E-mail: sld{at}seas.upenn.edu