Evaluation of peptide-aldehyde inhibitors using R188I mutant of SARS 3CL protease as a proteolysis-resistant mutant

Abstract

The 3C-like (3CL) protease of the severe acute respiratory syndrome (SARS) coronavirus is a key enzyme for the virus maturation. We found for the first time that the mature SARS 3CL protease is subject to degradation at 188Arg/189Gln. Replacing Arg with Ile at position 188 rendered the protease resistant to proteolysis. The R188I mutant digested a conserved undecapeptide substrate with a K(m) of 33.8 microM and k(cat) of 4753 s(-1). Compared with the value reported for the mature protease containing a C-terminal His-tag, the relative activity of the mutant was nearly 10(6). Novel peptide-aldehyde derivatives containing a side-chain-protected C-terminal Gln efficiently inhibited the catalytic activity of the R188I mutant. The results indicated for the first time that the tetrapeptide sequence is enough for inhibitory activities of peptide-aldehyde derivatives.

Synthesis and evaluation of phenylisoserine derivatives for the SARS-CoV 3CL protease inhibitor

“…[1][2][3] The key enzyme in the processing of polyproteins pp1a and pp1ab, translated by the viral RNA genome of SARS-CoV is called a 3C-like protease (3CL protease). 10 Although our group additionally found that tetrapeptide aldehyde Ac-Thr-Val-Cha-His-H (1) showed high inhibitory activity with an IC 50 value of 98 nM against SARS 3CL R188I mutant protease in 2008, 12 there are generally enzymatic digestions of peptide chains and a-proton racemization. For this reason, a variety of 3CL protease inhibitors have been reported in the literature over the past decade.…”

“…At three step sequence for the protection of active functionalities with amine, carboxylic acid and hydroxy groups of (2R,3S)-PIS, gave the protected PIS derivative (6) in 64% yield over 3 steps. Thus, a variety of the coupling reagents was employed between the PIS derivative (9) with a large steric hindrance and cinnamic acid, it was difficult to improve the chemical yields of the corresponding ester (10). Deprotection of the Cbz group of 7 by hydrogenolysis and coupling between the resultant amino group and cinnamic acid with DMT-MM/DIPEA successfully proceeded to afford the cinnamoyl derivative (8) in satisfactory yield.…”

Design, synthesis, and biological evaluation of novel dipeptide-type SARS-CoV 3CL protease inhibitors: Structure–activity relationship study

“…Briefly, the kinetic parameters were determined at a constant substrate concentration, and the inhibitor concentrations were varied to assess the K i values [22]. The IC 50 values were determined only for certain potent inhibitors, based on the apparent decrease in the substrate concentration (H-Thr-Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg-Lys-NH 2 ) upon digestion by R188I SARS 3CL pro , as described previously [19,34]. The cleavage reaction was monitored by analytical HPLC, and the cleavage rates were calculated from the decrease in the substrate peak area.…”

Synthesis of Bioactive Natural Products as Protein Inhibitors

“…The stability of the SARS 3CL-R188I mutant protease makes it possible to evaluate the inhibitors quantitatively. 43) A substrate-based peptide aldehyde, Ac-ThrSer-Ala-Val-Leu-Gln-H, was selected as the starting compound, and the optimum side chain structure of the potent inhibitor by both peptide aldehyde synthesis methodology with thioacetal and X-ray crystallographic analyses was found to be tetrapeptide aldehyde, Ac-ThrVal-Cha-His-H, which showed the high inhibitory activity, with an IC 50 value of 98 nM toward SARS 3CL-R188I mutant protease. 44 inhibitor, Ac-Thr-Ser-Ala-Val-Leu-Gln-H, at the P1 and P2 sites with the protease appeared to be remarkably effective, and further modification of the small nonpeptide inhibitor is now underway (Fig.…”