Pitstop 2d

Pitstop 2d is a clathrin ( Clathrin ) terminal domain (TD) inhibitor with an IC₅₀ of 1.7 μM. Pitstop 2d occupies the Clathrin box binding site within the clathrin TD, blocking the binding of endocytic protein ligands such as Epsin to clathrin. Pitstop 2d inhibits clathrin-mediated endocytosis ( Endocytosis ) and the cellular entry of vesicular stomatitis virus ( VSV ). Pitstop 2d has low cytotoxicity, does not disrupt the nuclear permeability barrier, and does not inhibit clathrin-independent endocytosis. Pitstop 2d can be used in studies related to endocytosis and vesicular stomatitis virus infection ^[1].

In Vitro, Pitstop 2d potently inhibits the interaction between purified clathrin terminal domain and amphiphysin B/C domain in cell-free ELISA assays, with an IC₅₀ of 1.7 μM^[1]. Pitstop 2d (25-100 μM; 8-20 h) exhibits only extremely low cytotoxicity in HEK293 and Cos7 cells^[1]. Pitstop 2d (30 μM; 15 min) does not disrupt the nuclear permeability barrier in HeLa cells^[1]. Pitstop 2d (13-45 μM; 15 min preincubation followed by 15 min transferrin uptake; 4 h washout) inhibits clathrin-mediated transferrin endocytosis in HeLa, HEK293T and Cos7 cells, with an IC₅₀ of 13 μM in HeLa cells, and its inhibitory effect is reversible after washout^[1]. Pitstop 2d significantly reduces the proportion of dynamic clathrin-coated pits in Cos7 cells expressing eGFP-clathrin light chain α, shifting CCP dynamics toward long-lived structures^[1]. Pitstop 2d interferes with clathrin dynamics in the perinuclear region of Cos7 cells expressing eGFP-clathrin light chain α, reducing the maximum fluorescence recovery rate after photobleaching to 37.6%^[1]. Pitstop 2d (45 μM; 15 min preincubation followed by 60 min dextran uptake) does not inhibit fluid-phase endocytosis of Alexa488-labeled dextran in HeLa cells^[1]. Pitstop 2d binds to the clathrin box site of the purified clathrin terminal domain in an extended conformation, forming a specific and stable interaction^[1]. Pitstop 2d (100 μM; 1 h) reduces the binding level between clathrin and Epsin 1 in Cos7 cells expressing eGFP-clathrin light chain α, while increasing the binding level between clathrin and SNX9, but has no effect on most other early-acting endocytic proteins^[1]. Pitstop 2d (15 min) increases the total number of clathrin-coated structures on the plasma membrane of BS-C-1 cells, causes the accumulation of shallow clathrin-coated pits, but does not alter the diameter of free clathrin-coated vesicles^[1]. Pitstop 2d (20 μM; 15 min preincubation followed by 1 h virus infection) inhibits over 60% of VSV entry into Vero-E6 cells, confirming that clathrin-mediated endocytosis (CME) is the primary pathway for efficient VSV cellular entry^[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.