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.
Footnotes
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This work was supported by the National Institutes of Health National Human Genome Research Institute [Grant U54-HG003915] (to S.L.D.) and the National Institutes of Health National Institute of Allergy and Infectious Diseases [Grants T32-AI55400 (to R.L.K.), U01-AI070369 (to P.B.), U54-AI057168 (to P.B.)].
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.110.064261.
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ABBREVIATIONS:
- SARS
- severe acute respiratory syndrome
- CoV
- coronavirus
- CID
- PubMed compound identification
- CID 16725315
- ([(S)-2-{N′-[(2-ethyl-phenylcarbamoyl)-methylsulfanylcarbonyl]-hydrazino}-1-(1H-indol-3-ylmethyl)-2-oxo-ethyl]-carbamic acid tert-butyl ester
- DCG-04
- biotin-Lys-C5 alkyl linker-Tyr-Leu-epoxide
- AMC
- 7-amido-4-methylcoumarin
- DMSO
- dimethyl sulfoxide
- VSV-G
- vesicular stomatitis virus glycoprotein
- HEK
- human embryonic kidney
- Cat-L
- cathepsin L
- SID
- PubChem substance identification
- SID 26681509
- tert-butyl N-[(2S)-1-[2-[2-(2-ethylanilino)-2-oxoethyl]sulfanylcarbonylhydrazinyl]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]carbamate
- SID 46493575
- tert-butyl N-[(2S)-1-[2-[2-(3,4-dihydro-2H-quinolin-1-yl)-2-oxoethoxy]carbonylhydrazinyl]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]carbamate.
- Received February 20, 2010.
- Accepted May 13, 2010.
- Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics
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