Received for publication February 27, 2008.
Revised April 2, 2008.
Accepted for publication April 10, 2008.

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

Abstract

A novel small molecule thiocarbazate (PubChem SID 26681509), a potent inhibitor of human cathepsin L (EC 3.4.22.15) with an IC₅₀ of 56 nM, was developed following a 57,821 compound screen of the NIH Molecular Libraries Small Molecule Repository. After a 4 hr 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. Additionally, 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.

Key words: Thermodynamic and kinetic processes and modeling, Fluorescence techniques, Protein-binding, Enzymology, Protein targets, Neuropeptides, peptidases