Building a Better Dynasore: The Dyngo Compounds Potently Inhibit Dynamin and Endocytosis

Abstract

Dynamin GTPase activity increases when it oligomerizes either into helices in the presence of lipid templates or into rings in the presence of SH3 domain proteins. Dynasore is a dynamin inhibitor of moderate potency (IC50 ˜ 15 μM in vitro). We show that dynasore binds stoichiometrically to detergents used for in vitro drug screening, drastically reducing its potency (IC50 = 479 μM) and research tool utility. We synthesized a focused set of dihydroxyl and trihydroxyl dynasore analogs called the Dyngo™ compounds, five of which had improved potency, reduced detergent binding and reduced cytotoxicity, conferred by changes in the position and/or number of hydroxyl substituents. The Dyngo compound 4a was the most potent compound, exhibiting a 37‐fold improvement in potency over dynasore for liposome‐stimulated helical dynamin activity. In contrast, while dynasore about equally inhibited dynamin assembled in its helical or ring states, 4a and 6a exhibited >36‐fold reduced activity against rings, suggesting that they can discriminate between helical or ring oligomerization states. 4a and 6a inhibited dynamin‐dependent endocytosis of transferrin in multiple cell types (IC50 of 5.7 and 5.8 μM, respectively), at least sixfold more potently than dynasore, but had no effect on dynamin‐independent endocytosis of cholera toxin. 4a also reduced synaptic vesicle endocytosis and activity‐dependent bulk endocytosis in cultured neurons and synaptosomes. Overall, 4a and 6a are improved and versatile helical dynamin and endocytosis inhibitors in terms of potency, non‐specific binding and cytotoxicity. The data further suggest that the ring oligomerization state of dynamin is not required for clathrin‐mediated endocytosis.

Phosphatidylserine dictates the assembly and dynamics of caveolae in the plasma membrane

“…To determine the impact of budding off and on the plasma membrane, we sought to inhibit dynamin. The treatment of cells with Dyngo-4a TM , a potent inhibitor of dynamin (32), illustrated that the fraction of tracks classified as sub-diffusive and free motions in cells treated with Dyngo-4a TM were very close to that of untreated, with the median diffusion coefficients of 3.0 ϫ 10 Ϫ3 and 1.2 ϫ 10 Ϫ2 m 2 /s, respectively. This indicates that dynamin had minimal impact on the mobility of caveolae with this analysis (Fig.…”

Dynamin triple knockout cells reveal off target effects of commonly used dynamin inhibitors

“…Dynasore, the first reported dynamin inhibitor (Macia et al, 2006), is widely used to investigate the role of dynamin in cellular events. Dyngo-4a, a close structural analog of dynasore, has been increasingly used in recent studies because of its higher potency in dynamin inhibition (Harper et al, 2011;Howes et al, 2010;McCluskey et al, 2013). We reasoned that if the effects of these dynamin inhibitors on cells are mediated through dynamin binding and inhibition, then cells that lack dynamin (i.e.…”

“…Triple KO (TKO) cells obtained from these conditional KO cells upon tamoxifen-induced gene recombination had the same phenotype as dynamin 1 and 2 DKO cells. Surprisingly, dynasore (Macia et al, 2006) and Dyngo-4a (Harper et al, 2011;Howes et al, 2010;McCluskey et al, 2013), two widely used and structurally related small molecule inhibitors of dynamin, still produced a robust impairment of fluidphase endocytosis and peripheral membrane ruffles in TKO cells. Given the property of these drugs to cause these very strong effects even in cells where dynamin is absent, caution is required in the interpretation of their cellular action.…”

Resolving the Sites of HIV Entry: Dynamin Networks Hold the Key

“…For instance, the molecule inhibitor dynole 32a inhibits dynamin activity and promotes disassembly of dynamin helices [68], dyngo-4a and dynasore inhibit GTPase activity of dynamin without disrupting its helical form [69] and ryngo1-23 stimulates the basal GTPase activity by promoting the formation of dynamin rings [49]. Much of the controversy regarding dynamin's role in HIV infection stems from the assumption that the primary role of dynamin networks is to aid in the late stages of endocytosis and that any inhibition of HIV infection thus observed must relate to this singular cellular process.…”