Pitstop 2c is a clathrin terminal domain inhibitor with an IC50 of 1.8 μM. Pitstop 2c blocks the binding of epsin 1 to the clathrin terminal domain and impairs epsin 1-mediated membrane curvature formation at the early stage of clathrin-mediated endocytosis (CME). Pitstop 2c enhances the clathrin-independent endocytosis of galectin-3 and alters its intracellular distribution. Pitstop 2c causes mild defects in lysosomal acidification, as well as a slight increase in mitochondrial number but a reduction in average mitochondrial size. Pitstop 2c can be used in studies related to vesicular stomatitis virus infection [1].
In Vitro, Pitstop 2c inhibits clathrin terminal domain-amphiphysin binding in vitro with an IC50 of 1.8 μM[1]. Pitstop 2c (25-100 μM; 8-20 h) exhibits low cytotoxicity in HEK293 cells[1]. Pitstop 2c (25-100 μM; 8-20 h) exhibits low cytotoxicity in Cos7 cells[1]. Pitstop 2c (30 μM; 15 min) does not disrupt the nuclear permeability barrier in HeLa cells[1]. Pitstop 2c (30 μM; 20 min) inhibits transferrin CME in HeLa cells with an IC50 of 13 μM, and nearly completely blocks uptake[1]. Pitstop 2c (45 μM) reversibly inhibits transferrin CME in HEK293T cells[1]. Pitstop 2c (45 μM) reversibly inhibits transferrin CME in Cos7 cells[1]. Pitstop 2c (low, non-toxic dose; 15 min pre-treatment, 5 min imaging) perturbs CCP dynamics in Cos7 cells stably expressing eGFP-clathrin light chain α, reducing the fraction of dynamic coated pits[1]. Pitstop 2c (30 μM; 20 min) impairs clathrin dynamics in the perinuclear region of Cos7 cells stably expressing eGFP-clathrin light chain α, reducing maximal fluorescence recovery to 44.4%[1]. Pitstop 2c (45 μM; 15 min) does not impair clathrin-independent fluid-phase endocytosis of dextran in HeLa cells[1]. Pitstop 2c (15 min pre-incubation, 2 min galectin-3 internalization) enhances clathrin-independent endocytosis of galectin-3 and alters its subcellular distribution in HeLa cells[1]. Pitstop 2c (100 μM; 1 h) displaces Epsin 1 from clathrin and increases clathrin association with SNX9 in Cos7 cells stably expressing eGFP-clathrin light chain α[1]. Pitstop 2c (four-fold molar excess relative to clathrin terminal domain) binds to the clathrin box site of the clathrin terminal domain in a distinct U-shaped conformation, forming specific interactions with residues R64 and K96[1]. Pitstop 2c (20 μM; 15 min pre-incubation, 1 h viral infection) inhibits VSV entry into Vero-E6 cells by >60%[1].
References:
[1]. Horatscheck A, et al. Next-generation small molecule inhibitors of clathrin function acutely inhibit endocytosis. Structure (London, England: 1993). 2025 May 01;33(5):878-890.e7.
In Vitro, Pitstop 2c inhibits clathrin terminal domain-amphiphysin binding in vitro with an IC50 of 1.8 μM[1]. Pitstop 2c (25-100 μM; 8-20 h) exhibits low cytotoxicity in HEK293 cells[1]. Pitstop 2c (25-100 μM; 8-20 h) exhibits low cytotoxicity in Cos7 cells[1]. Pitstop 2c (30 μM; 15 min) does not disrupt the nuclear permeability barrier in HeLa cells[1]. Pitstop 2c (30 μM; 20 min) inhibits transferrin CME in HeLa cells with an IC50 of 13 μM, and nearly completely blocks uptake[1]. Pitstop 2c (45 μM) reversibly inhibits transferrin CME in HEK293T cells[1]. Pitstop 2c (45 μM) reversibly inhibits transferrin CME in Cos7 cells[1]. Pitstop 2c (low, non-toxic dose; 15 min pre-treatment, 5 min imaging) perturbs CCP dynamics in Cos7 cells stably expressing eGFP-clathrin light chain α, reducing the fraction of dynamic coated pits[1]. Pitstop 2c (30 μM; 20 min) impairs clathrin dynamics in the perinuclear region of Cos7 cells stably expressing eGFP-clathrin light chain α, reducing maximal fluorescence recovery to 44.4%[1]. Pitstop 2c (45 μM; 15 min) does not impair clathrin-independent fluid-phase endocytosis of dextran in HeLa cells[1]. Pitstop 2c (15 min pre-incubation, 2 min galectin-3 internalization) enhances clathrin-independent endocytosis of galectin-3 and alters its subcellular distribution in HeLa cells[1]. Pitstop 2c (100 μM; 1 h) displaces Epsin 1 from clathrin and increases clathrin association with SNX9 in Cos7 cells stably expressing eGFP-clathrin light chain α[1]. Pitstop 2c (four-fold molar excess relative to clathrin terminal domain) binds to the clathrin box site of the clathrin terminal domain in a distinct U-shaped conformation, forming specific interactions with residues R64 and K96[1]. Pitstop 2c (20 μM; 15 min pre-incubation, 1 h viral infection) inhibits VSV entry into Vero-E6 cells by >60%[1].
References:
[1]. Horatscheck A, et al. Next-generation small molecule inhibitors of clathrin function acutely inhibit endocytosis. Structure (London, England: 1993). 2025 May 01;33(5):878-890.e7.