Discovery of Stereoselective Targeted Covalent Inhibitors of the RAB27-Effector Protein-Protein Interaction

RAB27A and RAB27B are homologous small GTPases that regulate intracellular vesicle trafficking, orchestrating endocytic and exocytic processes that affect cellular communication, immune responses, and the dynamics of the cellular microenvironment. Through their interactions with effector proteins, RAB27A/B plays roles in tumor metastasis and chronic inflammation. However, pharmacological modulation of their activity faces challenges typical of small GTPases, including a lack of well-defined pockets outside the conserved GTP binding site and large RAB27-effector protein-protein interaction (PPI) surfaces. Here, we present the discovery and development of the first cell-active, rationally designed covalent inhibitors of the RAB27-effector interaction, targeting a non-conserved cysteine residue flanking the PPI interface. An electrophile-first biochemical screen led to a novel class of acrylamide covalent inhibitors, and X-ray crystallography structure-guided design led to optimized inhibitors and probes that enantioselectively target RAB27A/B Cys123 in cells. Potency and selectivity were confirmed through biochemical and cellular assays, including chemical proteomics and phenotype recapitulation in melanocytes, alongside a matched inactive enantioprobe control. In contrast, a previously reported compound, Nexinhib-20, was found to be cytotoxic and to exert its activity through non-selective reactivity. This work provides the first toolbox of cell-active chemical probes for RAB27 that can be used in future studies to shed light on the function of this protein and its potential as a therapeutic target.