Development of Novel Antivirals Targeting Viral RNA Methylation

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PROJECT 4: DEVELOPMENT OF NOVEL ANTIVIRALS TARGETING VIRAL RNA METHYLATION SUMMARY Coronaviruses cap their RNA by coordinated action of two methyltransferases (MTase): Nsp14, which catalyzes N7-guanidine methylation of GTP at the 5′ terminus of viral RNAs, and Nsp16, which forms C2′-O-methyl- ribosyladenine at the subsequent nucleotide. By mimicking mRNA of the host cell, the resulting cap structure is critical for immune evasion, stabilization of viral RNA and efficient translation. While loss of function mutations in the MTase domain of Nsp14 are lethal to SARS-CoV-2, SARS-CoV strains that carry mutant Nsp16 have low virulence, suggesting that targeting of these enzymes, either alone or in combination with other viral proteins, has strong therapeutic potential. In this application, we propose to develop antiviral agents that target MTase activities of Nsp14 and Nsp16. Our approach will assess the potential of RNA capping MTases as a novel target family for development of antiviral agents. Importantly, since both enzymes are conserved across coronaviruses known to infect humans, this approach could provide a footprint for development of pan-coronaviral acting agents. Inhibitors for each of the MTases will be identified using a combination of small molecule discovery approaches: computational docking, fragment linking and merging, and high throughput screening (HTS) to identify novel chemotypes. Enabled by the recent developments in availability of make-on-demand libraries, we propose to use ultra-large library docking to identify candidate inhibitors. Availability of drug-like compounds in in-house small molecule libraries will facilitate inhibitor identification through HTS. Hit compounds will be tested in a series of activity assays and validated using direct binding strategies. Experimentally determined structures of MTases in complex with small molecule inhibitors will be used to guide optimization, aided by access to make-on-demand libraries. The identified inhibitors will be prioritized based on their selectivity against a comprehensive panel of human MTases and antiviral activity in cellular models of SARS-CoV-2 infection. The subsequent medicinal chemistry optimization and assessment of antiviral activity in animal models is expected to result in Optimized Lead compounds, which will be further elaborated by our industry partners (Roche).