Abstract
A tetrahydroquinoline oxocarbazate (PubChem CID 23631927) was tested as an inhibitor of human cathepsin L (EC 3.4.22.15) and as an entry blocker of SARS coronavirus and Ebola pseudotype virus. In the cathepsin L inhibition assay, the oxocarbazate caused a time-dependent 17-fold drop in IC50 from 6.9 nM (no pre-incubation) to 0.4 nM (4 hr pre-incubation). Slowly reversible inhibition was demonstrated in a dilution assay. A transient kinetic analysis using a single step, competitive inhibition model provided rate constants of kon = 153,000 M-1s-1 and koff = 4.40 x 10-5 s-1 (Ki = 0.29 nM). The compound also displayed cathepsin L/B selectivity of > 700-fold and was nontoxic to human aortic endothelial cells at 100 μM. The oxocarbazate and a related thiocarbazate (CID 16725315) were tested in a SARS-CoV and Ebola virus-pseudotype infection assay with the oxocarbozate, but not the thiocarbazate, demonstrating activity in blocking both SARS-CoV (IC50 = 273±49 nM) and Ebola virus (IC50 = 193±39 nM) entry into HEK 293T cells. In order to trace the intracellular action of the inhibitors with intracellular cathepsin L, the activity based probe DCG-04 was used to label the active site of cysteine proteases in 293T lysates. The reduction in active cathepsin L in inhibitor treated cells correlated well with the observed potency of inhibitors observed in the virus pseudotype infection assay. Overall, the oxocarbazate CID 23631927 was a sub-nanomolar slow-binding, reversible inhibitor of human cathepsin L that blocked SARS-CoV and Ebola pseudotype virus entry in human cells.
Footnotes
- Received February 20, 2010.
- Revision received May 13, 2010.
- Accepted May 13, 2010.
- The American Society for Pharmacology and Experimental Therapeutics