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.

Lung Cancer Metabolic reprogramming is a hallmark of metastatic lung cancer cells (A549_3R). In article number http://doi.wiley.com/10.1002/anbr.202200050, Victor Shahin and co‐workers establish that 5‐7 carbon glycols reduce the A549_3R ATP production by interfering with LDH and mitochondria. The cover image reveals a decline in mitochondrial membrane potential of A549_3R when exposed to 1,6‐HD.

Lung cancer is the leading cause of cancer deaths. Nonsmall cell lung cancer (NSCLC) accounts for the vast majority of the histological subtypes and at advanced stage exhibits extremely low survival rate. Certain compounds, such as glycols, inhibit migration and decreased viability of the highly metastatic NSCLC cells A549_3R has been recently shown. The glycols‐induced effects are associated with a parallel reduction of ATP production, detected in A549_3R spheroids embedded in 3D desmoplastic‐like extracellular matrix, following overnight treatment. Herein, selected glycols are tested for their ability to acutely compromise bioenergetics, metabolism, and viability of A549_3R cells. Therefore, 1,5‐pentanediol (1,5‐PD), 1,6‐hexanediol (1,6‐HD), and 1,7‐heptanediol (1,7‐Hept) turn out to be the most effective. They interfere with key enzymes in glycolysis and elicit most or all of the following responses: acute collapse of the critical mitochondrial membrane potential, substantial reduction of ATP, pyruvate and L‐lactate levels, and massive increase in reactive oxygen species production. Moreover, computational docking analysis reveals that the glycols bind to the substrate‐binding residues of lactate dehydrogenase A, which catalyzes the interconversion of pyruvate and lactate and is upregulated in aggressive tumors. Hence, the effective glycols may be promising anticancer compounds, or lead substances, for localized and intratumoral applications.