Next-generation small molecule inhibitors of clathrin function acutely inhibit endocytosis

• Published in: Structure (London) Vol. 33; no. 5; pp. 878 - 890.e7
• Main Authors: Horatscheck, André, Krauß, Michael, Bulut, Haydar, Chambon, Valerie, Zadah, Massilullah Shafaq, Dransart, Estelle, Peloza, Kimberly, Santos, Karine F., Robertson, Mark J., Prichard, Kate, Miksche, Sandra, Radetzki, Silke, von Kries, Jens-Peter, Wahl, Markus C., McCluskey, Adam, Johannes, Ludger, Haucke, Volker, Nazaré, Marc
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.05.2025
• Subjects: Adaptor Proteins, Vesicular Transport - chemistry; Adaptor Proteins, Vesicular Transport - metabolism; Animals; Binding Sites; cathrin; Clathrin - antagonists & inhibitors; Clathrin - chemistry; Clathrin - metabolism; endocytosis; Endocytosis - drug effects; Humans; Protein Binding; small molecule inhibition; Small Molecule Libraries - chemistry; Small Molecule Libraries - pharmacology; Sulfonamides; Thiazolidines; virus entry; Virus Internalization - drug effects; small molecule inhibition; virus entry; endocytosis; cathrin
• ISSN: 0969-2126; 1878-4186; 1878-4186
• DOI: 10.1016/j.str.2025.02.011

Clathrin-mediated endocytosis (CME) is the predominant endocytic pathway in eukaryotic cells and a major regulator of cell physiology as it facilitates the internalization of receptors, channels, and transporters and viral entry. The clathrin terminal domain acts as a central protein interaction hub within the endocytic protein network. Previously described inhibitors of CME display off-target activities that result in cytotoxicity, providing limitations to their use. We report the development and characterization of next-generation small molecule inhibitors of clathrin terminal domain function. These compounds termed Pitstop 2c and Pitstop 2d occupy the binding site within the clathrin terminal domain for endocytic protein ligands including epsin, resulting in potent inhibition of receptor-mediated endocytosis and reduced entry of vesicular stomatitis virus (VSV) with minimal cytotoxic side effects. Next-generation Pitstops thus provide an improved toolset to address clathrin function in cell physiology with potential applications as inhibitors of virus and pathogen entry. [Display omitted] •Next-generation small molecule inhibitors of clathrin function are presented•Pitstops 2c and 2d inhibit CME but display low cytotoxicity•Pitstops 2c and 2d act by blocking ligand access to the clathrin terminal domain•Pitstops 2c and 2d inhibit early stages of CME and displace epsins from clathrin Horatschek, Krauss et al. report the identification and characterization of next-generation small molecule inhibitors of clathrin function. These molecules provide an improved toolset to address the role of clathrin in cell physiology with potential applications as inhibitors of endocytosis, and of virus and pathogen entry.