Mahdavi, E. | bioRxiv Science

bioRxiv2025

Deep mutational scanning reveals pharmacologically relevant insights into TYK2 signaling and disease

Tyrosine Kinase 2 (TYK2) is a genetically defined target for autoimmune disease, with first-generation inhibitors showing clinical success in some but not all associated indications. A deeper understanding of TYK2 structure-function, protein-ligand interactions, and the impact of human variants could inform next-generation therapeutics. Here, we applied Deep Mutational Scanning (DMS) to assess >23,000 amino acid substitutions across two TYK2 functions: IFN- signaling and protein abundance. This enabled high-resolution structure-function mapping and the identification of novel allosteric sites. By coupling DMS with inhibitor treatment, we uncovered variants that modulate compound potency. We also show that human variants - both common and rare - that are protective against autoimmune phenotypes reduce TYK2 protein abundance. Together, these findings demonstrate that DMS can prospectively reveal novel druggable sites, clarify structure-activity relationships (SAR), and highlight TYK2 degradation as a potential therapeutic strategy in autoimmunity.

genomics↗

bioRxiv2024

High resolution deep mutational scanning of the melanocortin-4 receptor enables target characterization for drug discovery

Deep Mutational Scanning (DMS) is an emerging method to systematically test the functional consequences of thousands of sequence changes to a protein target in a single experiment. Because of its utility in interpreting both human variant effects and protein structure-function relationships, it holds substantial promise to improve drug discovery and clinical development. However, applications in this domain require improved experimental and analytical methods. To address this need, we report novel 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 MC4Rs function across 18 distinct experimental conditions, resulting in >20 million unique measurements. From this, we identified variants that have unique effects on MC4R-mediated Gs- and Gq-signaling pathways, which could be used to design drugs that selectively bias MC4Rs 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.

synthetic biology↗