biostudies-literatureLiashkovich IDeutsche ForschungsgemeinschaftCoordenação de Aperfeiçoamento de Pessoal de Nível SuperiorMinistry of Education and Science of the Russian Federatione10425https://www.ebi.ac.uk/biostudies/studies/S-EPMC10354767biostudies-literatureUnknown8(4)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 downstBioengineering & translational medicinePitstop-2 and its novel derivative RVD-127 disrupt global cell dynamics and nuclear pores integrity by direct interaction with small GTPases.PMC10354767Finance Code 001SH 167/6‐1075‐15‐2022‐264LI 2157/3‐1SH 167/6‐2SH 167/9‐1Gorelkin PVKolmogorov VStefanello STBulk EKouzel IUHaufe GLiashkovich IMizdal CRDulebo AErofeev AShahin VVidyadharan RfalsePitstop-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 downst2023-01-01T00:00:00Z2023 Jul2026-06-02T12:03:34.307Z2026-04-18T03:11:19.173ZS-EPMC103547673747605910.1002/btm2.10425