The novel coronavirus, SARS-CoV-2, has been identified as the causative agent for the current coronavirus disease (COVID-19) pandemic. 3CL protease (3CL pro ) plays a pivotal role in the processing of viral polyproteins. We report peptidomimetic compounds with a unique benzothiazolyl ketone as a warhead group, which display potent activity against SARS-CoV-2 3CL pro . The most potent inhibitor YH-53 can strongly block the SARS-CoV-2 replication. X-ray structural analysis revealed that YH-53 establishes multiple hydrogen bond interactions with backbone amino acids and a covalent bond with the active site of 3CL pro . Further results from computational and experimental studies, including an in vitro absorption, distribution, metabolism, and excretion profile, in vivo pharmacokinetics, and metabolic analysis of YH-53 suggest that it has a high potential as a lead candidate to compete with COVID-19.

We report the design and synthesis of a series of dipeptide-type inhibitors with novel P3 scaffolds that display potent inhibitory activity against SARS-CoV 3CLpro. A docking study involving binding between the dipeptidic lead compound 4 and 3CLpro suggested the modification of a structurally flexible P3 N-(3-methoxyphenyl)glycine with various rigid P3 moieties in 4. The modifications led to the identification of several potent derivatives, including 5c-k and 5n with the inhibitory activities (Ki or IC50) in the submicromolar to nanomolar range. Compound 5h, in particular, displayed the most potent inhibitory activity, with a Ki value of 0.006 μM. This potency was 65-fold higher than the potency of the lead compound 4 (Ki=0.39 μM). In addition, the Ki value of 5h was in very good agreement with the binding affinity (16 nM) observed in isothermal titration calorimetry (ITC). A SAR study around the P3 group in the lead 4 led to the identification of a rigid indole-2-carbonyl unit as one of the best P3 moieties (5c). Further optimization showed that a methoxy substitution at the 4-position on the indole unit was highly favorable for enhancing the inhibitory potency.

We describe here the design, synthesis and biological evaluation of a series of molecules toward the development of novel peptidomimetic inhibitors of SARS-CoV 3CL(pro). A docking study involving binding between the initial lead compound 1 and the SARS-CoV 3CL(pro) motivated the replacement of a thiazole with a benzothiazole unit as a warhead moiety at the P1' site. This modification led to the identification of more potent derivatives, including 2i, 2k, 2m, 2o, and 2p, with IC(50) or K(i) values in the submicromolar to nanomolar range. In particular, compounds 2i and 2p exhibited the most potent inhibitory activities, with K(i) values of 4.1 and 3.1 nM, respectively. The peptidomimetic compounds identified through this process are attractive leads for the development of potential therapeutic agents against SARS. The structural requirements of the peptidomimetics with potent inhibitory activities against SARS-CoV 3CL(pro) may be summarized as follows: (i) the presence of a benzothiazole warhead at the S1'-position; (ii) hydrogen bonding capabilities at the cyclic lactam of the S1-site; (iii) appropriate stereochemistry and hydrophobic moiety size at the S2-site and (iv) a unique folding conformation assumed by the phenoxyacetyl moiety at the S4-site.

Adenylation (A) domains found in all nonribosomal peptide synthetase (NRPS) modules are essential catalytic components and function as gatekeepers to select amino acid building blocks during nonribosomal peptide biosynthesis. Leveraging the strict substrate recognition characteristics of these enzymes, we targeted the development of active site-directed proteomic probes for A domains in NRPSs that enable detection, isolation, identification, and enzymatic characterization of A domains in native proteomes. Here, we describe a general strategy for selective chemical labeling of individual A domains in NRPS enzymes using active site-directed proteomic probes coupled to 5′-O-N-(aminoacyl)sulfamoyladenosine (AMS) scaffold with a clickable benzophenone functionality. These probes selectively target individual A domains in natural product producer proteomes by ligand-directed protein labeling. The data demonstrate that these proteomic tools can greatly facilitate the molecular identification, functional characterization, and profiling of virtually any kind of A domains of NRPS enzymes in complex biological systems.

This work describes the design, synthesis, and evaluation of low-molecular weight peptidic SARS-CoV 3CL protease inhibitors. The inhibitors were designed based on the potent tripeptidic Z-Val-Leu-Ala(pyrrolidone-3-yl)-2-benzothiazole (8; Ki = 4.1 nM), in which the P3 valine unit was substituted with a variety of distinct moieties. The resulting series of dipeptide-type inhibitors displayed moderate to good inhibitory activities against 3CL(pro). In particular, compounds 26m and 26n exhibited good inhibitory activities with Ki values of 0.39 and 0.33 μM, respectively. These low-molecular weight compounds are attractive leads for the further development of potent peptidomimetic inhibitors with pharmaceutical profiles. Docking studies were performed to model the binding interaction of the compound 26m with the SARS-CoV 3CL protease. The preliminary SAR study of the peptidomimetic compounds with potent inhibitory activities revealed several structural features that boosted the inhibitory activity: (i) a benzothiazole warhead at the S1' position, (ii) a γ-lactam unit at the S1-position, (iii) an appropriately hydrophobic leucine moiety at the S2-position, and (iv) a hydrogen bond between the N-arylglycine unit and a backbone hydrogen bond donor at the S3-position.