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 IC 50 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 IC 50 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 ϭ 24,000 M Ϫ1 s Ϫ1 and k off ϭ 2.2 ϫ 10 Ϫ5 s Ϫ1 (K i ϭ 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 IC 50 of 15.4 M and inhibited Leishmania major with an IC 50 of 12.5 M.

Discovery of Potent Small-Molecule Inhibitors of Multidrug-Resistant Plasmodium falciparum Using a Novel Miniaturized High-Throughput Luciferase-Based Assay

“…A cellular cytotoxicity assay was conducted with human aortic endothelial cells (HAEC) and human hepatocellular liver carcinoma cells (HepG2) as previously described (27) to measure viability after 24 h of incubation.…”

Molecular Docking of Cathepsin L Inhibitors in the Binding Site of Papain

“…15,24 Compound 1 demonstrated selectivity towards cathepsin L versus other cysteine proteases, including cathepsins V, S, B, and K, with the greatest selectivity index observed for cathepsin K (150). 23 The details of a structure activity relationship study for cathepsin L inhibition in the carbazate series are being published separately 25 , but reveal that removal of the NHBoc group causes a dramatic loss of cathepsin L inhibitory activity. This single synthetic change to the carbazate scaffold of 1 removes both the potential for hydrogen bonding with Asp 148 and the hydrophobic contact of the tert-butoxy group in the S3 subsite, resulting in a 400-fold reduction in cathepsin L inhibition.…”

“…Most notably, the rate of inhibitor dissociation (k off ) from cathepsin L was extremely slow, leading to a K i = k off / k on = 0.890 nM. 23 Alternative reaction schemes for steady-state and irreversible inhibitor binding were also tested, but did not fit the data as well as the 5-parameter model. Taken together, the results from the docking and kinetic analyses suggest a covalent but slowly reversible mechanism of inhibition that is aided by strong non-covalent interactions.…”

“…23 It was also necessary to estimate an additional parameter, representing the time-delay until the first experimental reading was taken (ca. 150 sec).…”

The Identification, Characterization and Optimization of Small Molecule Probes of Cysteine Proteases: Experiences of the Penn Center for Molecular Discovery with Cathepsin B and Cathepsin L

“…Second, docking studies of 15a (R 1 =H, R 2 = o -ethylphenyl) placed the C2 carbonyl in close proximity to the active site cysteine xxxix. The kinetic characterization of 15a revealed a slowly reversible inhibition xl. Additional studies provided further insight into the mode of action of these inhibitors.…”