PROJECT 5: INHIBITING VIRAL MACRODOMAINS USING STRUCTURE-BASED DESIGN SUMMARY Viral macrodomains counter the host immune response by removing ADP-ribosylation modifications from host proteins, with important consequences for interferon and many other signaling pathways. Previous experiments in cell and animal models that use wild type and catalytically dead SARS-CoV and chikungunya virus (CHIKV) macrodomains validate these proteins as potential drug targets. However, no inhibitors exist for viral macrodomains. Using an integration of high throughput X-ray based fragment screen and docking, we have identified over 200 binders to the SARS-CoV-2 macrodomain (Mac1). Our subsequent assays guided the development of the first structurally characterized tool compounds that are more potent than the substrate ADP- ribose. In addition, we have leveraged the chemical knowledge to uncover additional scaffolds by docking. In this proposal, we advance compounds through a structure-based design approach, using biochemical assays against peptide binding and catalytic function. To identify starting points against the CHIKV macrodomain, we will perform a new X-ray crystallography-based fragment screen and perform an Ultra-large docking campaign (Screening Core). Our plan is to design compounds with low potency against human macrodomains and to leverage medicinal chemistry to drive potency against viral macrodomains. Our early activities will identify multiple alternative scaffolds with high potency in vitro to progress to target engagement in cells. Our cell-based aims will use cellular thermal shift and immunofluorescence of interferon treated cells as early markers of target engagement (Proteomics Core, In Vitro Virology Core). We will identify biomarkers by comparing RNAseq, proteomics, phosphoproteomics and ADP-Ribosylation AP-MS of compound treated cells to mutant macrodomains (both as a transgene and in the context of virus). During these activities, we will continue addressing aspects of permeability, off target effects, and other liabilities with the Medicinal Chemistry Core. We will progress to animal models compounds with cellular effects on replication with validated target engagement. Lead molecules with validated target engagement and minimal pharmacokinetic liabilities will allow us to test the effectiveness of candidate molecules in animal models of SARS-CoV-2 and CHIKV (In Vivo Virology Core). Based on previous studies in animal models with catalytically inactive macrodomain mutant viruses, we will prioritize molecules that drop viral load by at least 100-fold. Our work will generate a target package for further development by our industrial partner, Roche. The lessons of targeting SARS-CoV-2 and CHIKV macrodomains will be applicable to developing future agents against macrodomains in other viruses and those implicated in human disease.