Identification and targeting of a novel drug pocket in the tumour angiogenesis regulator RhoJ

Abstract

The Rho GTPase RhoJ is a key regulator of tumour angiogenesis and has been identified as a target for anti-angiogenic drug design. Its high sequence conservation with the related Rho GTPase Cdc42 complicates selective drug discovery. RhoJ has a unique N-terminal extension (Asn2-Lys19) that is absent in Cdc42 and other Rho GTPases. Here, we use molecular dynamics simulations to identify a novel allosteric pocket formed by the RhoJ N-terminus and show that this pocket contributes to interactions with key downstream signalling partners. Structure-based virtual screening of small molecules targeting this pocket identified the antifungal terconazole as a potential binder. Computational docking revealed that terconazole occupies the N-terminal pocket, sharing critical contacts with the interface between RhoJ and PAK3, a key interacting partner. To validate this, we developed an optimized bioluminescence resonance energy transfer (eBRET2)-based biosensor to monitor the interaction between RhoJ and PAK3 in live cells and saw that terconazole disrupted the RhoJ:PAK3 interaction in a dose-dependent manner. Together, our MD results show that the RhoJ N-terminal extension assumes structure on binding to PAK3 and contributes to that interaction, while our cellular biosensor data support targeting this pocket to modulate the RhoJ:PAK3 interaction. The ability of terconazole to bind to the N-terminal pocket and disrupt PAK3 binding provides proof of concept for the specific targeting of RhoJ in the development of novel anti-angiogenic drugs.