Protocol to identify covalent inhibitors targeting RhoA Cys16.

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.