A high-throughput assay to identify robust inhibitors of dynamin GTPase activity

Abstract

19Clathrin-mediated endocytosis is the major pathway by which cells internalize 20 materials from the external environment. Dynamin, a large multidomain GTPase, is a 21 key regulator of clathrin-mediated endocytosis. It assembles at the necks of invaginated 22 clathrin-coated pits and, through GTP hydrolysis, catalyzes scission and release of 23 clathrin-coated vesicles from the plasma membrane. Several small molecule inhibitors 24 of dynamin's GTPase activity, such as Dynasore and Dyngo-4a, are currently available, 25 although their specificity has been brought into question. Previous screens for these 26 inhibitors measured dynamin's stimulated GTPase activity due to lack of sufficient 27 sensitivity, hence the mechanisms by which they inhibit dynamin are uncertain. We 28 report a highly sensitive fluorescence-based assay capable of detecting dynamin's 29 basal GTPase activity under conditions compatible with high throughput screening. 30Utilizing this optimized assay, we conducted a pilot screen of 8000 compounds and 31 identified several "hits" that inhibit the basal GTPase activity of dynamin-1. Subsequent 32 dose-response curves were used to validate the activity of these compounds. 33Interestingly, we found neither Dynasore nor Dyngo-4a inhibited dynamin's basal 34GTPase activity, although both inhibit assembly-stimulated GTPase activity. This assay 35 provides the basis for a more extensive search for robust dynamin inhibitors. 36 37 peer-reviewed)

Prodrugs of the Archetypal Dynamin Inhibitor Bis‐T‐22

“…Other groups have reported dynasore analogues, [42] some selective serotonin reuptake inhibitors, [43] and high throughput screening hits as dynamin inhibitors. [42,44,45] Of these, the MiTMAB, [27,46] Bis-T [47] and dynole [48] classes have anti-tumour activity that show selectivity for cancer cells with minimal to no effect on non-tumourigenic fibroblasts. These dynamin inhibitors induce apoptosis via the intrinsic pathway, exclusively following cytokinesis failure and polyploidisation.…”

“…Inhibitors from our laboratories span the MiTMAB, [26–28] RTIL, [29] Iminodyn, [30] Pthaladyn, [31] dyngo®, [32] Rhodadyn, [33] Dynoles TM,[34–36] Quinodyn, [37] Naphthaladyn, [38] phenothiazine drugs [39] and Bis‐T‐22, [40,41] compound families. Other groups have reported dynasore analogues, [42] some selective serotonin reuptake inhibitors, [43] and high throughput screening hits as dynamin inhibitors [42,44,45] . Of these, the MiTMAB, [27,46] Bis‐T [47] and dynole [48] classes have anti‐tumour activity that show selectivity for cancer cells with minimal to no effect on non‐tumourigenic fibroblasts.…”

Pyrimidyn‐Based Dynamin Inhibitors as Novel Cytotoxic Agents

“…[75] Other groups have reported dynasore analogues, some selective serotonin reuptake inhibitors and high throughput screening hits as dynamin inhibitors. [76][77][78] Of these, the MiTMAB, [43,51] Bis-T56 and dynole [41] classes have anti-tumour activity that show selectivity for cancer cells with minimal to no effect on non-tumourigenic fibroblasts. These dynamin inhibitors induce apoptosis via the intrinsic pathway, exclusively following cytokinesis failure and polyploidisation.…”