Title: Mapping Structure-Activity Relationships of Chemical Inhibitors via Genome-Editing Determining the target and mechanism of action of small molecules is a critical challenge in chemical biology and drug discovery. Identifying structure-activity relationships (SAR) that connect the structure of small molecules with target binding and downstream biological activities is essential for target validation and lead compound optimization. While medicinal chemistry enables considerable chemical variation of small molecules for this purpose, approaches to generate reciprocal SAR by systematically varying the protein target structure are less prevalent. Nonetheless, approaches that vary protein structure could be incredibly informative, as the identification of mutant alleles that enhance or suppress chemical inhibitors may not only validate the target of a drug but also provide SAR that could prove critical for understanding binding interactions, the molecular mechanism of drug action, and target biology. Moreover, combining genetic variants and chemical variants, such as protein mutants with libraries of small molecules, may dramatically increase the possible combinations of structural permutations that may be tested at the interface of these molecules. Although high-throughput mutagenesis of proteins is challenging, new genome-editing technologies provide the improved capability to systematically alter protein sequence in situ by directly manipulating the endogenous coding sequence. In this application, the development of new technology is proposed to interrogate small molecule SAR by systematically altering the protein target structure using genome-editing and evaluating these mutants in chemical suppressor screening using different selection criteria. Mutations that enhance or suppress the selection imposed by specific small molecules will provide structural information on the small molecule's binding site, generating information that pairs a genetic variant with a chemical variant. By evaluating a large pool of protein target mutants against a collection of small molecule variants, this pairwise information can be integrated across the protein target in a high-throughput fashion to generate maps of SAR. To demonstrate its feasibility, CRISPR- SAR mapping was first successfully applied to investigate the mechanism of lysine-specific demethylase 1 (LSD1) pharmacological inhibitors. This initial application led to the discovery of new facets of LSD1 function, highlighting the utility of CRISPR-SAR mapping in elucidating target biology. Several strategies to improve the precision, scope, and utility of CRISPR-SAR mapping as well as future applications are subsequently presented. The innovative and early-stage nature of this proposal as well as its potential to transform chemical biology and pathological mechanisms makes it well suited for the NIH Director's New Innovator Award.
Deciphering functional relationships between chemical inhibitors and their biological targets is essential for target validation, lead compound optimization, and for understanding drug mechanism of action as well as target biology. To interrogate these relationships directly in cells on large-scale, we will develop new approaches that combine chemical suppressor screening with genome-editing to mutagenize target proteins in a pooled fashion. The high-throughput biochemistry developed here will provide unprecedented perspectives and deep insight into protein structure and function as they relate to small molecule mechanism of action that could prove critical for elucidating disease mechanisms and developing novel small molecule therapeutic modulators.