Inhibition mechanism and antiviral activity of an α-ketoamide based SARS-CoV-2 main protease inhibitor

Abstract

SARS-CoV-2 has demonstrated extraordinary ability to evade antibody immunity by antigenic drift. Small molecule drugs may provide effective therapy while being part of a solution to circumvent SARS-CoV-2 immune escape. In this study we report an α-ketoamide based peptidomimetic inhibitor of SARS-CoV-2 main protease (Mpro), RAY1216. Enzyme inhibition kinetic analysis established that RAY1216 is a slow-tight inhibitor with aKi of 8.6 nM; RAY1216 has a drug-target residence time of 104 min compared to 9 min of PF-07321332 (nirmatrelvir), the antiviral component in Paxlovid, suggesting that RAY1216 is approximately 12 times slower to dissociate from the protease-inhibitor complex compared to PF-07321332. Crystal structure of SARS-CoV-2 Mpro:RAY1216 complex demonstrates that RAY1216 is covalently attached to the catalytic Cys145 through the α-ketoamide warhead; more extensive interactions are identified between bound RAY1216 and Mproactive site compared to PF-07321332, consistent with a more stable acyl-enzyme inhibition complex for RAY1216. In cell culture and human ACE2 transgenic mouse models, RAY1216 demonstrates comparable antiviral activities towards different SARS-CoV-2 virus variants compared to PF-07321332. Improvement in pharmacokinetics has been observed for RAY1216 over PF-07321332 in various animal models, which may allow RAY1216 to be used without ritonavir. RAY1216 is currently undergoing phase III clinical trials (https://clinicaltrials.gov/ct2/show/NCT05620160) to test real-world therapeutic efficacy against COVID-19.

Discovery and Mechanism Study of SARS-CoV-2 3C-like Protease Inhibitors with a New Reactive Group

“…In addition, this work also provides a kind of nonpeptidomimetic skeleton structures that we believe will be useful for the development of 3CL pro inhibitors. We compared the complex structure of 3CL pro with 3a with the structures of the 3CL pro in complex with Nirmatrelvir ( 1 ), , SIM0417 ( 2 ), and RAY1216 ( 4 ), which are approved for the treatment of COVID-19. As shown in Figure S9, unlike the three peptidomimetics, Nirmatrelvir, SIM0417, and RAY1216, the tricyclic group of 3a occupied the S1′ and S2 sites.…”

“…The representative inhibitors are summarized in Table . For example, Nirmatrelvir ( 1 ) , and SIM0417 ( 2 ), which are already in clinical use, both use nitrile groups as electrophilic warheads; N3 ( 3 ) employs a classic covalent warhead, Michael receptor warhead; Thiohemiketal formed by the nucleophilic attack of the catalytic cysteine onto the warhead α-ketoamide of RAY1216 ( 4 ) can interact with 3CL pro through more interactions, comparing with Michael receptor and nitrile groups (Table ). In addition, a total of 12 warheads including aldehyde (11a, 5 ), − bisulfite (GC376, 6 ), − alkyne ( 7 ), substituted-ketone (Jun9-62-2R, 8 ), − vinyl sulfonamide (14a, 9 ), ester (7d, 10 ), − pyrogallol (baicalein, 11 ), selenium/sulfer (ebselen, 12 ), ,− urea (carmofur, 13 ), , β-lactams ( 14 ), dithiocarbamate ( 15 ), and thiadiazole ( 16 ) were also reported (Table ).…”

“… a Warheads include nitrile, ,, Michael receptor, ,− α-ketoamide, ,, aldehyde, − bisulfite, − alkyne, substituted-ketone, − vinyl sulfonamide, ester, − pyrogallol, selenium/sulfer, ,− urea, , β-lactam, dithiocarbamate, and thiadiazole . The representative molecule is selected for each type of warhead to display the two-dimensional (2D) structure.…”

“…A global pandemic, coronavirus disease 2019 (COVID-19), caused by the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has far-reaching implications for human lives. , So far, several drugs have been approved or are conditionally approved for treating this disease. These drugs on the market include Remdesivir, Molnupiravir, Nirmatrelvir, , Ensitrelvir, Azvudine, , VV116, SIM0417, and RAY1216 . Among them, Remdesivir, Molnupiravir, Azvudine, and VV116 are the inhibitors of the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2, whereas Ensitrelvir, Nirmatrelvir, SIM0417, and RAY1216 were designed for 3-chymotrypsin-like protease (3CL pro , also referred to as the main protease, M pro ) of SARS-CoV-2.…”

A potent and broad‐spectrum neutralizing nanobody for SARS‐CoV‐2 viruses, including all major Omicron strains

“…2,[6][7][8] Although with reduced protection efficiency of most antibodies and vaccines for new Omicron variants, [9][10][11][12][13] neutralizing antibody treatment still protects many infected people from severe symptoms and death. Although efficacious drugs have been developed to treat COVID-19, [14][15][16][17][18][19] developing new broad-spectrum antibodies against emerging coronavirus strains remains urgent and necessary. 20 Compared to conventional antibodies, nanobodies (Nbs) have many advantages, including high thermostability, great production efficiency, low cost, and easy engineering.…”