Pitstop-2 and its novel derivative RVD-127 disrupt global cell dynamics and nuclear pores integrity by direct interaction with small GTPases

Clathrin-mediated endocytosis (CME) is an essential cell physiological process of broad biomedical relevance. Since the recent introduction of Pitstop-2 as a potent CME inhibitor, we and others have reported on substantial clathrin-independent inhibitory effects. Herein, we developed and experimentally validated a novel fluorescent derivative of Pitstop-2, termed RVD-127, to clarify Pitstop-2 diverse effects. Using RVD-127, we were able to trace additional protein targets of Pitstop-2. Besides inhibiting CME, Pitstop-2 and RVD-127 proved to directly and reversibly bind to at least two members of the small GTPase superfamily Ran and Rac1 with particularly high efficacy. Binding locks the GTPases in a guanosine diphosphate (GDP)-like conformation disabling their interaction with their downstream effectors. Consequently, overall cell motility, mechanics and nucleocytoplasmic transport integrity are rapidly disrupted at inhibitor concentrations well below those required to significantly reduce CME. We conclude that Pitstop-2 is a highly potent, reversible inhibitor of small GTPases. The inhibition of these molecular switches of diverse crucial signaling pathways, including nucleocytoplasmic transport and overall cell dynamics and motility, clarifies the diversity of Pitstop-2 activities. Moreover, considering the fundamental importance and broad implications of small GTPases in physiology, pathophysiology and drug development, Pitstop-2 and RVD-127 open up novel avenues.

Automated summary

This study investigates the inhibitory effects of Pitstop-2 on clathrin-mediated endocytosis (CME) and its effects on small GTPases. It is demonstrated that Pitstop-2 and its derivative RVD-127 can directly bind to small GTPases, preventing their interaction with downstream effectors and disrupting overall cell motility and nucleocytoplasmic transport integrity. These findings broaden the research scope of Pitstop-2 and have important implications in physiology, pathophysiology, and drug development.