Combining deep mutational scanning and next-generation protein sequencing to harness dominant protein variants to develop DNA repair inhibitors

The most clinically effective DNA repair enzyme inhibitors not only inhibit but also convert the target itself into a therapeutic agent. For example, topoisomerase poisons block the completion of the topoisomerase reaction resulting in a genotoxic DNA-protein adduct. Similar mechanisms underlie clinically relevant PARP inhibitors and other emerging DNA repair inhibitors. The challenge is identifying which repair proteins can be converted into gain-of-function therapeutic agents and how to elicit a gain-of-function phenotype. We developed an approach, Mutational Target Mapping, that uses deep mutational scanning to identify dominant missense variants that define structure-function relationships in drug targets that can direct therapeutic development. Dominant missense protein variants can be used to model the behavior of a potential drug to validate mechanisms of action and define genetic dependencies. Combining this platform with benchtop Next-Generation Protein Sequencing on the Quantum-Si Platinum® instrument, we can identify, track, and characterize the phenotypic effects of dominant missense variants in cells and biochemical assays. We applied this approach to several therapeutic targets discovering key residues in orthosteric and allosteric sites that can be mutated to elicit a dominant phenotype. Here, we describe our complementary genetic (high throughput screens) and proteomic (benchtop protein sequencing) approaches to find and model the behavior of therapeutics targeting DNA repair enzymes.