Nonpeptidic Irreversible Inhibitors of SARS-CoV-2 Main Protease with Potent Antiviral Activity.

SARS-CoV-2 infections pose a high risk for vulnerable patients. In this study, we designed benzoic acid halopyridyl esters bearing a variety of substituents as irreversible inhibitors of the main viral protease (Mpro). Altogether, 55 benzoyl chloro/bromo-pyridyl esters were synthesized, with broad variation of the substitution pattern on the benzoyl moiety. A workflow was employed for multiparametric optimization, including Mpro inhibition assays of SARS-CoV-2 and related pathogenic coronaviruses, the duration of enzyme inhibition, the compounds' stability versus glutathione, cytotoxicity, and antiviral activity. Several compounds showed IC50 values in the low nanomolar range, kinact/Ki values of >100,000 M-1 s-1 and high antiviral activity. High-resolution X-ray cocrystal structures indicated an important role of ortho-fluorobenzoyl substitution, forming a water network that stabilizes the inhibitor-bound enzyme. The most potent antiviral compound was the p-ethoxy-o-fluorobenzoyl chloropyridyl ester (PSB-21110, 29b, MW 296 g/mol; EC50 2.68 nM), which may serve as a lead structure for broad-spectrum anticoronaviral therapeutics.

Intramuscular vaccination against SARS-CoV-2 transiently induces neutralizing IgG rather than IgA in the saliva.

The mucosal immunity is crucial for restricting SARS-CoV-2 at its entry site. Intramuscularly applied vaccines against SARS-CoV-2 stimulate high levels of neutralizing Abs in serum, but the impact of these intramuscular vaccinations on features of mucosal immunity is less clear. Here, we analyzed kinetic and functional properties of anti-SARS-CoV-2 Abs in the saliva after vaccination with BNT162b2. We analyzed a total of 24 healthy donors longitudinally for up to 16 months. We found that specific IgG appeared in the saliva after the second vaccination, declined thereafter and reappeared after the third vaccination. Adjusting serum and saliva for the same IgG concentration revealed a strong correlation between the reactivity in these two compartments. Reactivity to VoCs correlated strongly as seen by ELISAs against RBD variants and by live-virus neutralizing assays against replication-competent viruses. For further functional analysis, we purified IgG and IgA from serum and saliva. In vaccinated donors we found neutralizing activity towards authentic virus in the IgG, but not in the IgA fraction of the saliva. In contrast, IgA with neutralizing activity appeared in the saliva only after breakthrough infection. In serum, we found neutralizing activity in both the IgA and IgG fractions. Together, we show that intramuscular mRNA vaccination transiently induces a mucosal immunity that is mediated by IgG and thus differs from the mucosal immunity after infection. Waning of specific mucosal IgG might be linked to susceptibility for breakthrough infection.

Discovery and Crystallographic Studies of Trisubstituted Piperazine Derivatives as Non-Covalent SARS-CoV-2 Main Protease Inhibitors with High Target Specificity and Low Toxicity.

The continuous spread of SARS-CoV-2 calls for more direct-acting antiviral agents to combat the highly infectious variants. The main protease (Mpro) is an promising target for anti-SARS-CoV-2 drug design. Here, we report the discovery of potent non-covalent non-peptide Mpro inhibitors featuring a 1,2,4-trisubstituted piperazine scaffold. We systematically modified the non-covalent hit MCULE-5948770040 by structure-based rational design combined with multi-site binding and privileged structure assembly strategies. The optimized compound GC-14 inhibits Mpro with high potency (IC50 = 0.40 µM) and displays excellent antiviral activity (EC50 = 1.1 µM), being more potent than Remdesivir. Notably, GC-14 exhibits low cytotoxicity (CC50 > 100 µM) and excellent target selectivity for SARS-CoV-2 Mpro (IC50 > 50 µM for cathepsins B, F, K, L, and caspase 3). X-ray co-crystal structures prove that the inhibitors occupy multiple subpockets by critical non-covalent interactions. These studies may provide a basis for developing a more efficient and safer therapy for COVID-19.

Small-Molecule Thioesters as SARS-CoV-2 Main Protease Inhibitors: Enzyme Inhibition, Structure-Activity Relationships, Antiviral Activity, and X-ray Structure Determination.

The main protease (Mpro, 3CLpro) of SARS-CoV-2 is an attractive target in coronaviruses because of its crucial involvement in viral replication and transcription. Here, we report on the design, synthesis, and structure-activity relationships of novel small-molecule thioesters as SARS-CoV-2 Mpro inhibitors. Compounds 3w and 3x exhibited excellent SARS-CoV-2 Mpro inhibition with kinac/Ki of 58,700 M-1 s-1 (Ki = 0.0141 µM) and 27,200 M-1 s-1 (Ki = 0.0332 µM), respectively. In Calu-3 and Vero76 cells, compounds 3h, 3i, 3l, 3r, 3v, 3w, and 3x displayed antiviral activity in the nanomolar range without host cell toxicity. Co-crystallization of 3w and 3af with SARS-CoV-2 Mpro was accomplished, and the X-ray structures showed covalent binding with the catalytic Cys145 residue of the protease. The potent SARS-CoV-2 Mpro inhibitors also inhibited the Mpro of other beta-coronaviruses, including SARS-CoV-1 and MERS-CoV, indicating that they might be useful to treat a broader range of coronaviral infections.