Fragment based discovery of a small molecule RhoGDI2 ligand HR3119 that inhibits cancer cell metastasis

Guanine nucleotide dissociation inhibitors (GDIs) proteins including RhoGDI2 play a central role in regulating the functions of Ras superfamily proteins that are known to be important cancer drug targets. Given the known challenges in directly targeting Ras superfamily proteins with small compounds, targeting GDIs represents a unique opportunity but has met with limited success. Herein, we describe the discovery of the first small compound HR3119 that binds RhoGDI2 in low micromolar affinity (Kd ~ 7 μM) starting from a millimolar binding affinity fragment hit (Kd ~ 0.7 mM) through iterative structure-guided design. We identify that HR3119 surrogates occupy the binding interface between RhoGDI2 and its endogenous ligand Rac1 in a series of co-crystal structures, laying out rich information for future compound design. We further demonstrate that HR3119 disrupts RhoGDI2-Rac1 binding and promotes the degradation of Rac1, as well as engages RhoGDI2 in cells. As an illustration of this novel chemical probe, we show that HR3119 suppressed the invasion and migration of invasive breast cancer cells at a 5 μM concentration and in a concentration-dependent manner. Interestingly, (6R)-HR3119 exhibited a 100-fold binding affinity advantage to that of (6S)-HR3119 (4 μM vs 290 μM) and improved anti-metastatic activity compared to racemic HR3119. This work provides valuable insights into the discovery of small compounds targeting GDIs in terms of methodology, chemistry starting points, compound design, and phenotype studies, underscoring exciting new perspectives in early drug discovery.