A potent macrocyclic peptide binder enables effector-free crystallization of the GTPase RAB27A

Abstract RAB27A is a member of a highly conserved family of small GTPases that regulate intracellular vesicle trafficking. Multiple studies have linked RAB27A cellular activity to exosome-mediated modulation of the tumour microenvironment and inflammatory signalling, highlighting it as an attractive target for drug discovery. However, identifying structurally validated and selective inhibitors of the RAB27A-effector interaction has proven challenging, partly because wild-type RAB27A crystallizes only in complex with effector proteins such as Slp2a. Using mRNA display, we identified macrocyclic peptide ligands that target the RAB27A effector-binding interface. Optimisation of initial hits led to IMP2660 , a nanomolar-affinity RAB27A binder that effectively disrupts RAB27A-effector interactions. IMP2660 enabled determination of a 2.1 Å structure of active-state RAB27A bound to a noncovalent macrocyclic peptide in the absence of an effector, revealing structural features of the effector-binding surface and providing a platform for future structure-guided ligand development.