Ras homolog family member A (RhoA) is a therapeutic hotspot in many cancers but remains undruggable. Here, we present a protocol for identifying covalent inhibitors targeting RhoA Cys16. We describe steps for the identification of lead compounds by activity-based protein profiling (ABPP) screening and the validation of engagement by mass spectrometry and chemical biology experiments. We then detail procedures for investigating the effects of the compounds on RhoA signaling in cancer cells and mouse colorectal cancer (CRC) models. For complete details on the use and execution of this protocol, please refer to Koo et al. 1 • Detailed steps for identifying covalent inhibitors targeting RhoA Cys16 • Gel-based ABPP for identifying specific covalent ligands for RhoA over Rac1 and Cdc42 • Chemoproteomics and chemical biology experiments to examine target engagement in cancer cells • Molecular and cellular assays to study RhoA-inhibitory and anticancer effects of the ligands Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Ras homolog family member A (RhoA) is a therapeutic hotspot in many cancers but remains undruggable. Here, we present a protocol for identifying covalent inhibitors targeting RhoA Cys16. We describe steps for the identification of lead compounds by activity-based protein profiling (ABPP) screening and the validation of engagement by mass spectrometry and chemical biology experiments. We then detail procedures for investigating the effects of the compounds on RhoA signaling in cancer cells and mouse colorectal cancer (CRC) models.
Koo et al. (Tue,) studied this question.