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

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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 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.