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, effectively enhancing the activation of its GTP hydrolysis and 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. However, despite encouraging therapeutic potential, the mechanism by which dynole 34-2 inhibits dynamin is undefined. Our aim is to elucidate the mechanism of action of dynole 34-2 on dynamin inhibition and assess whether its inhibition of cellular endocytosis occurs through an on-target mechanism.
The underlying hypothesis is that dynole 34-2 disrupts dynamin’s ability to oligomerise, subsequently impairing GTP hydrolysis. Initial molecular docking predicted the binding site of dynole 34-2 to a novel allosteric site on dynamin, Hinge 2. However, production and screening of predicted Hinge 2 mutant species of full-length dynamin 1 showed that single point mutations were insufficient at preventing dynole 34-2 inhibition, suggesting an alternative binding mode. Furthermore, our data revealed an allosteric mechanism of action for dynole 34-2 against dynamin, whereby only phospholipid-dependent oligomerisation was impaired, and not basal activity. To validate whether dynole 34-2 inhibits cellular endocytosis by targeting dynamin, we employed a Cellular Thermal Shift Assay (CETSA), which measures changes in protein denaturation in response to drug binding within cells, in which potential targets are subsequently identified by mass spectrometry. Our data from screening the cellular proteome revealed various protein targets whose thermal stability was altered by dynole 34-2.