Severe Acute Respiratory Syndrome 3CL Research Articles

Stable Benzotriazole Esters as Mechanism-Based Inactivators of the Severe Acute Respiratory Syndrome 3CL Protease

SummarySevere acute respiratory syndrome (SARS) is caused by a newly emerged coronavirus that infected more than 8000 individuals and resulted in more than 800 fatalities in 2003. Currently, there is no effective treatment for this epidemic. SARS-3CLpro has been shown to be essential for replication and is thus a target for drug discovery. Here, a class of stable benzotriazole esters was reported as mechanism-based inactivators of 3CLpro, and the most potent inactivator exhibited a kinact of 0.0011 s−1 and a Ki of 7.5 nM. Mechanistic investigation with kinetic and mass spectrometry analyses indicates that the active site Cys145 is acylated, and that no irreversible inactivation was observed with the use of the C145A mutant. In addition, a noncovalent, competitive inhibition became apparent by using benzotriazole ester surrogates in which the bridged ester-oxygen group is replaced with carbon.

Inhibition of the severe acute respiratory syndrome 3CL protease by peptidomimetic α,β-unsaturated esters

The proteolytic processing of polyproteins by the 3CL protease of severe acute respiratory syndrome coronavirus is essential for the viral propagation. A series of tripeptide α,β-unsaturated esters and ketomethylene isosteres, including AG7088, are synthesized and assayed to target the 3CL protease. Though AG7088 is inactive (IC 50 > 100 μM), the ketomethylene isosteres and tripeptide α,β-unsaturated esters containing both P1 and P2 phenylalanine residues show modest inhibitory activity (IC 50 = 11–39 μM). The Phe-Phe dipeptide inhibitors 18a– e are designed on the basis of computer modeling of the enzyme–inhibitor complex. The most potent inhibitor 18c with an inhibition constant of 0.52 μM is obtained by condensation of the Phe-Phe dipeptide α,β-unsaturated ester with 4-(dimethylamino)cinnamic acid. The cell-based assays also indicate that 18c is a nontoxic anti-SARS agent with an EC 50 value of 0.18 μM.

Discovery of Potent Anilide Inhibitors against the Severe Acute Respiratory Syndrome 3CL Protease

A diversified library of peptide anilides was prepared, and their inhibition activities against the SARS-CoV 3CL protease were examined by a fluorogenic tetradecapeptide substrate. The most potent inhibitor is an anilide derived from 2-chloro-4-nitroaniline, l-phenylalanine and 4-(dimethylamino)benzoic acid. This anilide is a competitive inhibitor of the SARS-CoV 3CL protease with K(i) = 0.03 muM. The molecular docking experiment indicates that the P1 residue of this anilide inhibitor is distant from the nucleophilic SH of Cys145 in the active site.

Synthesis and Evaluation of Keto-Glutamine Analogues as Potent Inhibitors of Severe Acute Respiratory Syndrome 3CLpro

The 3C-like proteinase (3CL(pro)) of severe acute respiratory syndrome (SARS) coronavirus is a key target for structure-based drug design against this viral infection. The enzyme recognizes peptide substrates with a glutamine residue at the P1 site. A series of keto-glutamine analogues with a phthalhydrazido group at the alpha-position were synthesized and tested as reversible inhibitiors against SARS 3CL(pro). Attachment of tripeptide (Ac-Val-Thr-Leu) to these glutamine-based "warheads" generated significantly better inhibitors (4a-c, 8a-d) with IC(50) values ranging from 0.60 to 70 microM.