Fragment-Based Discovery of a Small-Molecule RhoGDI2 Ligand, HR3119, that Inhibits Cancer Cell Migration

Guanine nucleotide dissociation inhibitors (GDIs) proteins, including RhoGDI2, regulate the functions of Ras superfamily proteins that are known to be important cancer drug targets. Given the challenges in directly targeting Ras superfamily proteins with small molecules, targeting GDIs represents a unique opportunity but has seen limited success. In this work, we discovered <b>HR3119</b> as the first ligand of RhoGDI2 with low-micromolar affinity (<i>K</i>_d = 8 μM) starting from a millimolar binding affinity fragment hit (<i>K</i>_d = 714 μM). <b>HR3119</b> and its derivatives were rationally designed based on a series of ligand-bound RhoGDI2 crystal structures. <b>HR3119</b> occupies the protein-protein interaction interface between RhoGDI2 and its endogenous ligand Rac1 to disrupt RhoGDI2-Rac1 binding. Interestingly, the complex structure suggests that (6<i>R</i>)-<b>HR3119</b> preferentially bound to RhoGDI2 when crystallized with a racemic mixture. The purified (6<i>R</i>)-<b>HR3119</b> demonstrated a nearly 100-fold binding affinity advantage compared to (6<i>S</i>)-<b>HR3119</b>. Finally, (6<i>R</i>)-<b>HR3119</b> engaged with RhoGDI2 in cells and suppressed the migration of aggressive breast cancer cells. Our work provides insights into the discovery of small-molecule compounds targeting RhoGDI2 in terms of methodology, chemistry starting points, compound design, and phenotype studies, underscoring exciting new perspectives in early drug discovery.