High resolution deep mutational scanning enables target characterization for drug discovery

The high cost and failure rates of disease therapies mean that approaches are desperately needed to make drug discovery and development cheaper, faster, and more likely to succeed. Deep Mutational Scanning (DMS), holds substantial promise for leveraging the functional effects of sequence variation to improve drug discovery and clinical development, but its use in such applications is underexplored. Herein, we report novel experimental and analytical DMS methods to precisely and quantitatively interrogate disease-relevant mechanisms, protein-ligand interactions, and assess predicted response to drug treatment. Using these methods, we performed a DMS of the Melanocortin 4 Receptor (MC4R), a G protein-coupled receptor (GPCR) implicated in obesity and an active target of drug development efforts. We assessed the effects of >6,600 single amino acid substitutions on MCR's function across 18 distinct experimental conditions, resulting in >20 million unique measurements. From this, we identified variants that have unique effects on MC4R-mediated G-alpha-s and G-alpha-q signaling pathways, which could be used to design drugs that selectively bias MC4R's activity. We also identified pathogenic variants that are likely amenable to a corrector therapy. Finally, we functionally characterized structural relationships that distinguish the binding of peptide versus small molecule ligands, which could guide compound optimization. Collectively, these results demonstrate that DMS is a powerful method to empower drug discovery and development.