Screening identified two bisindolylmaleimides as 100 microM inhibitors of the GTPase activity of dynamin I. Focused library approaches allowed development of indole-based dynamin inhibitors called dynoles. 100-Fold in vitro enhancement of potency was noted with the best inhibitor, 2-cyano-3-(1-(2-(dimethylamino)ethyl)-1H-indol-3-yl)-N-octylacrylamide (dynole 34-2), a 1.3 +/- 0.3 microM dynamin I inhibitor. Dynole 34-2 potently inhibited receptor mediated endocytosis (RME) internalization of Texas red-transferrin. The rank order of potency for a variety of dynole analogues on RME in U2OS cells matched their rank order for dynamin inhibition, suggesting that the mechanism of inhibition is via dynamin. Dynoles are the most active dynamin I inhibitors reported for in vitro or RME evaluations. Dynole 34-2 is 15-fold more active than dynasore against dynamin I and 6-fold more active against dynamin mediated RME (IC(50) approximately 15 microM; RME IC(50) approximately 80 microM). The dynoles represent a new series of tools to better probe endocytosis and dynamin-mediated trafficking events in a variety of cells.

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Dynamin I is a GTPase enzyme required for endocytosis and is an excellent target for the design of potential endocytosis inhibitors. Screening of a library of tyrphostins, in our laboratory, against the GTPase activity of dynamin I gave rise to a microM potent lead, 2-cyano-3-(3,4-dihydroxyphenyl)thioacrylamide (1, IC50 70 microM). Our initial investigations suggested that only the dimeric form of 1 displayed dynamin I GTPase inhibitory activity. Subsequent synthetic iterations were based on dimeric analogues and afforded a number of small molecules, low microM potent, inhibitors of dynamin I GTPase, in particular, symmetrical analogues with a minimum of two free phenolic -OHs: catechol-acrylamide (9) (IC50= 5.1 +/- 0.6 microM), its 3,4,5-trihydroxy congener (10) (IC50= 1.7 +/- 0.2 microM), and the corresponding 3-methyl ether (11) (IC50= 9 +/- 3 microM). Increasing the length of the central alkyl spacer from ethyl to propyl (22-24) afforded essentially identical activity with IC50's of 1.7 +/- 0.2, 1.7 +/- 0.2, and 5 +/- 1 microM, respectively. No decrease in activity was noted until the introduction of a hexyl spacer. Our studies highlight the requirement for two free amido NHs with neither the mono-N-methyl (86) nor the bis-N-methyl (87) analogues inhibiting dynamin I GTPase. A similar effect was noted for the removal of the nitrile moieties. However, modest potency was observed with the corresponding ester analogues of 9-11: ethyl ester (90), propyl ester (91), and butyl ester (92), with IC50's of 42 +/- 3, 38 +/- 2, and 61 +/- 2 microM, respectively. Our studies reveal the most potent and promising dynamin I GTPase inhibitor in this series as (22), which is also known as BisT.