The Development of Selective Inhibitors to Oncogenic Rac1 Variants - PROJECT SUMMARY Rac1, a member of the Rho GTPase subfamily, regulates essential cellular functions including cytoskeletal arrangement, cell motility, cell cycle progression, and proliferation. Dysregulation of Rac1, such as through the oncogenic P29S mutation or the expression of the alternative splice variant Rac1b, is common in cancer and contributes to tumor progression, metastasis, and drug resistance. While Rac1 gain-of-function mutations, amplification, or increased activity of activating proteins are necessary for driving tumorigenesis, the Rac1b variant alone has also been shown to promote oncogenicity. While Rac1 and Rac1b differ by only a 19- amino acid insertion after Rac1b's Switch II pocket, this alteration changes the Switch I and II dynamics of Rac1b, resulting in changes in cellular activity and promotion of tumorigenesis. Furthermore, distinguishing the roles of these variants in cellular signaling and disease is challenging due to their sequence and structural similarity, coupled with the scarcity of selective chemical tools for effectively targeting these proteins in cellular systems. Current inhibitors of Rac1 reversibly target the conserved GEF binding pockets or compete with GDP/GTP for the conserved Switch I/II region, resulting in compounds with low potency and lack of specificity to Rac1. Given the differences in the dynamics of the Switch I and II pockets among Rac1 variants, I hypothesize that targeting these regions will lead to the development of Rac1 variant selective inhibitors. My preliminary data demonstrates that Switch II pocket inhibitors can exhibit selectivity for Rac1 isoforms, aiding in understanding their distinct roles in cancer progression. For Aim 1, I will utilize structure-based design to optimizing selective and cellular active Switch II pocket inhibitors towards Rac1/Rac1b G12C. To assess the potency and selectivity of these compounds in a cellular system, I will introduce the Rac1/Rac1b G12C mutation in immortalized cancer cell lines via CRISPR Prime editing. To create therapeutics towards oncogenic Rac1 variants, Aim 2 of this proposal focuses on developing covalent inhibitors targeting the conserved cysteine in the Switch I pocket of Rac1 GTPases. This strategy will exploit Rac1 P29S and Rac1b fast-cycling phenotype and low affinity for GTP to create inhibitors that outcompete GTP for binding the Switch I pocket. In conjunction with the Arkin lab, I have identified fragments from a disulfide tethering screen with affinity for the Switch I pocket, which will be optimized for binding Rac1 P29S and Rac1b. Since Rac1 P29S and Rac1b have different Switch I conformations and lower affinity for GTP than Rac1 WT, I hypothesize that these identified covalent Switch I pocket binders will preferentially inhibit GTP binding of these oncogenic variants over Rac1 WT. This project aims to create selective chemical tools to elucidate Rac1 isoforms’ role in oncogenesis and provide therapeutic inhibitors for selectively targeting oncogenic Rac1 variants in cancers. By continuing my training in integrating chemical genetics and cell biological techniques and leveraging the expertise and collaborative environment provided in the Shokat lab, I have the tools to successfully complete this project.
