Endocytosis is an essential process by which cells internalise extracellular material and is important in various signal transduction pathways. A key enzyme involved in clathrin-mediated endocytosis is dynamin, which induces membrane fission during the final stage of endocytosis. Its distinguishing features include the ability to oligomerise into helices or rings, which effectively enhances the activation of GTP hydrolysis, promoting lipid fission. Dysregulation of critical endocytic signaling pathways has been associated with chronic neuropathic pain disorders, as well as oncogenesis in various cancers. The small molecule, dynole 34-2, have been shown to be efficacious at inhibiting dynamin in vitro, in-cell endocytosis, and in animal disease models such as leukemia and neuropathic pain. However, despite encouraging therapeutic potential, the mechanism and binding site by which dynole 34-2 inhibits dynamin is undefined.
The underlying hypothesis is that dynole 34-2 disrupts dynamin’s ability to oligomerise, thereby impairing GTP hydrolysis. The mechanism of action of dynole 34-2 was identified to be allosteric, whereby its inhibitory action was conformationally locked to the catalytically active two-start helix conformation of dynamin, while the abortive one-start helix, basal and SH3-stimulated ring conformational states were unaffected. Mutagenesis studies targeting an allosteric site on dynamin, Hinge 2, were also employed to elucidate a potential binding site for dynole 34-2. However, all Hinge 2 mutants remained perpetually inhibited by dynole 34-2, suggesting otherwise. Subsequently, an alternative modelling approach based on recently published oligomeric structures of dynamin suggested that the binding site is dynamically induced during higher-order assembly, rather than being solely dependent on residues within monomeric regions.