Substrate Specificity Profiling and Identification of a New Class of Inhibitor for the Major Protease of the SARS Coronavirus^,

Abstract

Severe acute respiratory syndrome (SARS) is an emerging infectious disease associated with a high rate of mortality. The SARS-associated coronavirus (SARS-CoV) has been identified as the etiological agent of the disease. Although public health procedures have been effective in combating the spread of SARS, concern remains about the possibility of a recurrence. Various approaches are being pursued for the development of efficacious therapeutics. One promising approach is to develop small molecule inhibitors of the essential major polyprotein processing protease 3Clpro. Here we report a complete description of the tetrapeptide substrate specificity of 3Clpro using fully degenerate peptide libraries consisting of all 160,000 possible naturally occurring tetrapeptides. The substrate specificity data show the expected P1-Gln P2-Leu specificity and elucidate a novel preference for P1-His containing substrates equal to the expected preference for P1-Gln. These data were then used to develop optimal substrates for a high-throughput screen of a 2000 compound small-molecule inhibitor library consisting of known cysteine protease inhibitor scaffolds. We also report the 1.8 A X-ray crystal structure of 3Clpro bound to an irreversible inhibitor. This inhibitor, an alpha,beta-epoxyketone, inhibits 3Clpro with a k3/Ki of 0.002 microM(-1) s(-1) in a mode consistent with the substrate specificity data. Finally, we report the successful rational improvement of this scaffold with second generation inhibitors. These data provide the foundation for a rational small-molecule inhibitor design effort based upon the inhibitor scaffold identified, the crystal structure of the complex, and a more complete understanding of P1-P4 substrate specificity.

Steady-State and Pre-Steady-State Kinetic Evaluation of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) 3CLpro Cysteine Protease: Development of an Ion-Pair Model for Catalysis

“…Crystallographic studies have shown that the inhibitor forms a single covalent complex with the active site cysteine residue Cys145 (Jay Davies, unpublished results). A similar irreversible peptide mimetic inactivator has been shown to form a single covalent adduct with Cys145 of 3CL pro (27). Isothermal titration calorimetry with PF-596162 was used to support the existence of a single binding site per monomer and to determine the fraction of the active sites occupied ( Figure 2C,D).…”

“…Inactivation studies with the cysteine-reactive, weakly electrophilic inactivator PF-596162 showed a 1:1 inactivation stoichiometry. Another irreversible inhibitor has been shown to specifically modify the catalytic cysteine residue of 3CL pro (27). The pH dependence of the pseudo-second-order rate constant of inactivation of 3CL pro (k obs /[IA]) with iodoacetamide is shown in Figure 7.…”

“…P4-P2′ residues were reported to be indispensable for effective cleavage while P3′-P7′ are not essential but have an effect on cleavage efficiency (37). An additional study of the 3CL pro substrate specificity of P 1 -P 4 substrate positions found that the P 1 glutamine can be replaced by a histidine residue, the P 2 leucine residue is obligate, and the P 3 /P 4 positions are more tolerant of variation (27). Although peptides 5 and 6 have a P1′ mimetic and no P2′, they are good substrates.…”

How Prepared Are We to Control Severe Acute Respiratory Syndrome in Future

“…Virtual screening (Plewczynski et al, 2007;Mukherjee et al, 2008;Nguyen et al, 2011) or a high-throughput screening of small molecule libraries have identified inhibitorsincluding an anti-HIV agent and serotonin antagonist, cinanserin (Blanchard et al, 2004;Kao et al, 2004;Wu et al, 2004a;Chen et al, 2005). Other 3CL protease inhibitors identified so far belongto categories such as plant derived phenolic or flavonoid compounds (Lin et al, 2005;Nguyen et al, 2012), active site, nonactive site or competitive inhibitors (Kaeppler et al, 2005;Lee et al, 2005;Du et al, 2007;Ryu et al, 2010), ketones or ester based inhibitors (Goetz et al, 2007;Zhang et al, 2007;Ghosh et al, 2008;Shao et al, 2008;Verschueren et al, 2008;Zhang et al, 2008), modified peptidomimetic inhibitors (Ghosh et al, 2007), metal conjugated inhibitors (Lee et al, 2007;…”

Recent Advances in Targeting Viral Proteases for the Discovery of Novel Antivirals

“…Similarly, most rational approaches with respect to the design of SARS-CoV M pro inhibitors mainly focus on the optimization of a scaffold privileged to covalent inhibition by the attachment of electrophilic groups. Next to the abovementioned halomethyl ketones, , -unsaturated esters acting as Michael acceptors, and aldehydes, the arsenal of appropriate reactive warheads attached to substrate-based or peptidomimetic inhibitors has been extended to phthalhydrazide moieties [122], epoxyketones [123] and aziridines [124]. Interestingly, an amide derivative (31, Fig.…”