TY - JOUR T1 - A Small-Molecule Oxocarbazate Inhibitor of Human Cathepsin L Blocks Severe Acute Respiratory Syndrome and Ebola Pseudotype Virus Infection into Human Embryonic Kidney 293T cells JF - Molecular Pharmacology JO - Mol Pharmacol SP - 319 LP - 324 DO - 10.1124/mol.110.064261 VL - 78 IS - 2 AU - Parag P. Shah AU - Tianhua Wang AU - Rachel L. Kaletsky AU - Michael C. Myers AU - Jeremy E. Purvis AU - Huiyan Jing AU - Donna M. Huryn AU - Doron C. Greenbaum AU - Amos B. Smith III AU - Paul Bates AU - Scott L. Diamond Y1 - 2010/08/01 UR - http://molpharm.aspetjournals.org/content/78/2/319.abstract N2 - 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 severe acute respiratory syndrome (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 preincubation) to 0.4 nM (4-h preincubation). 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 × 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 (PubChem CID 16725315) were tested in a SARS coronavirus (CoV) and Ebola virus-pseudotype infection assay with the oxocarbazate but not the thiocarbazate, demonstrating activity in blocking both SARS-CoV (IC50 = 273 ± 49 nM) and Ebola virus (IC50 = 193 ± 39 nM) entry into human embryonic kidney 293T cells. To trace the intracellular action of the inhibitors with intracellular cathepsin L, the activity-based probe biotin-Lys-C5 alkyl linker-Tyr-Leu-epoxide (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 subnanomolar, slow-binding, reversible inhibitor of human cathepsin L that blocked SARS-CoV and Ebola pseudotype virus entry in human cells. ER -