Endocytosis is an essential process by which cells internalise extracellular material. It is important in multiple signal transduction pathways. A key enzyme involved in clathrin-mediated endocytosis is dynamin, which facilitates membrane fission during the final stage of endocytosis. Its distinguishing features include the ability to oligomerise to helices or rings, which coordinate striking activation of its GTP hydrolysis for lipid fission. Pharmacological inhibition of endocytosis alters key signalling pathways, which have been implicated in oncogenesis in various cancers and in neuropathic pain modulation. Therefore, endocytosis modulation is a viable therapeutic strategy. The small molecule, dynole 34-2, is efficacious at inhibiting dynamin in vitro, in-cell endocytosis, and in animal disease models such as leukemia. 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. The underlying hypothesis is that it binds a unique allosteric site that modulates dynamin oligomerisation. To determine how it affects dynamin oligomerisation and GTP hydrolysis, we compared wild-type dynamin with a truncated dynamin construct lacking oligomerisation capabilities, then employed GTPase assays and sedimentation assays to identify successful oligomerisation. We found that dynole 34-2 only impaired phospholipid-stimulated dynamin GTPase activity and oligomerisation, without effect on the basal GTPase activity of assembly-incompetent. Michaelis-Menten kinetic analyses revealed an inhibition mode that was non-competitive with the GTP binding site. Computational modelling studies were employed, which predicted binding to an unstructured flexible region of dynamin called Hinge 2. To validate the key predicted contact residues, site-directed mutagenesis studies of nine Hinge 2 residues has been performed and activity studies are currently underway. The data show a unique allosteric mechanism of action of dynole on dynamin’s ability to oligomerise, thereby preventing oligomerisation-dependent GTPase activity, but not basal activity. The data also identifies a potential novel drug binding site on dynamin that will guide future modelling studies in the design of more potent analogues.