Discovery of a molecular glue inhibitor that stabilises a non-productive Kalirin-Rac1 complex

Abstract

Rho guanosine triphosphatases (GTPases) are molecular-switches implicated in neurodegenerative diseases, yet targeting them through competitive inhibition remains a challenge due to their high affinity for guanine nucleotides. Guanine nucleotide exchange factors (GEFs) catalyse GDP-to-GTP nucleotide exchange to activate GTPases, providing an alternative opportunity for GTPase modulation. Herein, we describe a complex-targeted strategy to inhibit nucleotide exchange with covalent molecular glues that engage the Kalirin-Rac1 GEF-GTPase complex at the nucleotide binding site are sequester the GEF Kalirin. Fragment hits were identified through XChem and in silico screening, and a fragment merging approach resulted in the generation of covalent inhibitors RS-009 and MC-278. Multiple analyses demonstrate our compounds inhibit nucleotide exchange both through competition with the nucleotides and by stabilising a ternary inhibitor-Rac1-Kalirin complex, thereby trapping Kalirin in a non-productive state and reducing GEF turnover. Biochemical selectivity screening and cellular activity-based protein profiling (ABPP) show that selectivity can be achieved across distinct GEF-GTPase complexes, which may result in improved spatiotemporal control over targeting the GTPase alone. This work provides evidence for targeting GTPase signalling via stabilization of the GEF-GTPase complex in a unique covalent molecular glue mechanism and provides the basis of a chemical probe or therapeutic.