A high-throughput screen of the NIH molecular libraries sample collection and subsequent optimization of a lead dipeptide-like series of severe acute respiratory syndrome (SARS) main protease (3CLpro) inhibitors led to the identification of probe compound ML188 (16-(R), (R)-N-(4-(tert-butyl)phenyl)-N-(2-(tert-butylamino)-2-oxo-1-(pyridin-3-yl)ethyl)furan-2-carboxamide, Pubchem CID: 46897844). Unlike the majority of reported coronavirus 3CLpro inhibitors that act via covalent modification of the enzyme, 16-(R) is a non-covalent SARS-CoV 3CLpro inhibitor with moderate MW and good enzyme and antiviral inhibitory activity. A multi-component Ugi reaction was utilized to rapidly explore structure activity relationships within S1′, S1, and S2 enzyme binding pockets. The X-ray structure of SARS-CoV 3CLpro bound with 16-(R) was instrumental in guiding subsequent rounds of chemistry optimization. 16-(R) provides an excellent starting point for the further design and refinement of 3CLpro inhibitors that act by a non-covalent mechanism of action.

Background: 3CLpro protease is required for coronaviral polyprotein processing and is only active as a dimer. Results: MERS-CoV 3CLpro is a weakly associated dimer requiring ligand binding for dimer formation. Conclusion: Ligand-induced dimerization is a key mechanism for regulating the enzymatic activity of MERS-CoV 3CL

Herein we report the discovery and SAR of a novel series of SARS-CoV 3CLpro inhibitors identified through the NIH Molecular Libraries Probe Production Centers Network (MLPCN). In addition to ML188, ML300 represents the second probe declared for 3CLpro from this collaborative effort. The X-ray structure of SARS-CoV 3CLpro bound with a ML300 analog highlights a unique induced-fit reorganization of the S2-S4 binding pockets leading to the first sub-micromolar non-covalent 3CLpro inhibitors retaining a single amide bond.

Human coronaviruses (CoVs) such as severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV) cause epidemics of severe human respiratory disease. A conserved step of CoV replication is the translation and processing of replicase polyproteins containing 16 nonstructural protein domains (nsp's 1 to 16). The CoV nsp5 protease (3CLpro; Mpro) processes nsp's at 11 cleavage sites and is essential for virus replication. CoV nsp5 has a conserved 3-domain structure and catalytic residues. However, the intra-and intermolecular determinants of nsp5 activity and their conservation across divergent CoVs are unknown, in part due to challenges in cultivating many human and zoonotic CoVs. To test for conservation of nsp5 structure-function determinants, we engineered chimeric betacoronavirus murine hepatitis virus (MHV) genomes encoding nsp5 proteases of human and bat alphacoronaviruses and betacoronaviruses. Exchange of nsp5 proteases from HCoV-HKU1 and HCoV-OC43, which share the same genogroup, genogroup 2a, with MHV, allowed for immediate viral recovery with efficient replication albeit with impaired fitness in direct competition with wild-type MHV. Introduction of MHV nsp5 temperature-sensitive mutations into chimeric HKU1 and OC43 nsp5 proteases resulted in clear differences in viability and temperature-sensitive phenotypes compared with MHV nsp5. These data indicate tight genetic linkage and coevolution between nsp5 protease and the genomic background and identify differences in intramolecular networks regulating nsp5 function. Our results also provide evidence that chimeric viruses within coronavirus genogroups can be used to test nsp5 determinants of function and inhibition in common isogenic backgrounds and cell types. C oronaviruses (CoVs) are enveloped, positive-strand RNA viruses that infect a wide range of animal hosts. Human CoVs cause illnesses including the common cold and severe acute respiratory syndrome (SARS) as well as the recently identified Middle East respiratory syndrome (MERS) associated with infection of a novel coronavirus (1). Coronaviruses are members of the order Nidovirales, family Coronaviridae, and subfamily Coronavirinae. Among the viruses in Coronavirinae, four main genera have recently been designated (2): alphacoronaviruses, which contain human coronavirus 229E (HCoV-229E) and HCoV-NL63; betacoronaviruses, containing human coronaviruses SARS-CoV, HKU1, MERS-CoV, and OC43; and gammacoronaviruses and deltacoronaviruses, from which no current human coronaviruses have been identified. The betacoronavirus murine hepatitis virus (MHV) is a well-established model for the study of coronavirus replication and pathogenesis. The MHV genome is 32 kb in length and encodes 7 genes (Fig. 1A) (3-5). An essential step of CoV replication is the translation of the ORF1ab replicase polyproteins and the subsequent processing of up to 16 nonstructural proteins (nsp's 1 to 16), including the nsp12 RNA-dependent RNA polymerase (3,5,6). CoV nsp5 protease (3CLpro; Mpro) med...

Zika virus (ZIKV) is a significant global health threat, as infection has been linked to serious neurological complications, including microcephaly. Using a human stem cell-derived neural progenitor model system, we find that a critical cellular quality control process called the nonsense-mediated mRNA decay (NMD) pathway is disrupted during ZIKV infection. Importantly, disruption of the NMD pathway is a known cause of microcephaly and other neurological disorders. We further identify an interaction between the capsid protein of ZIKV and up-frameshift protein 1 (UPF1), the master regulator of NMD, and show that ZIKV capsid targets UPF1 for degradation. Together, these results offer a new mechanism for how ZIKV infection can cause neuropathology in the developing brain.