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. 2008:438:77-93.
doi: 10.1016/S0076-6879(07)38006-3.

Use of dynasore, the small molecule inhibitor of dynamin, in the regulation of endocytosis

Tom Kirchhausen  1 , Eric Macia, Henry E Pelish
Affiliations

Use of dynasore, the small molecule inhibitor of dynamin, in the regulation of endocytosis

Tom Kirchhausen et al. Methods Enzymol. 2008.

Abstract

The large GTPase dynamin is essential for clathrin-dependent coated-vesicle formation. Dynasore is a cell-permeable small molecule that inhibits the GTPase activity of dynamin1, dynamin2 and Drp1, the mitochondrial dynamin. Dynasore was discovered in a screen of approximately 16,000 compounds for inhibitors of the dynamin2 GTPase. Dynasore is a noncompetitive inhibitor of dynamin GTPase activity and blocks dynamin-dependent endocytosis in cells, including neurons. It is fast acting (seconds) and its inhibitory effect in cells can be reversed by washout. Here we present a detailed synthesis protocol for dynasore, and describe a series of experiments used to analyze the inhibitory effects of dynasore on dynamin in vitro and to study the effects of dynasore on endocytosis in cells.

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Figures

Figure 6.1
Figure 6.1
(A) Chemical structure of dynasore. (B) Effect of dynasore on clathrin-coated structures. The figure shows representative images of clathrin-coated structures of cells treated with DMSO (vehicle) or with 80 µM dynasore. The upper and lower left panels illustrate the appearance of endocytic coated pits and coated vesicles; the upper and lower right panels show the appearance of “U” and “O” shape-coated pits associated with the plasma membrane in cells treated with dynasore. The gray arrows highlight the extent of the constriction states observed upon treatment with dynasore. (From Macia, E., Ehrlich, M., Massol, R., Boucrot, E., Brunner, C., and Kirchhausen, T. (2006). Dynasore, a cell-permeable inhibitor of dynamin. Dev. Cell 10, 839–850.)
Figure 6.2
Figure 6.2
Strategy for the chemical genetics discovery approach. The reverse chemical genetics approach was used to discover dynasore. Dynasore is cell permeable and interferes with all functions known to be associated with dynamin. (From Macia, E., Ehrlich, M., Massol, R., Boucrot, E., Brunner, C., and Kirchhausen, T. (2006). Dynasore, a cell-permeable inhibitor of dynamin. Dev. Cell 10, 839–850; and Newton, A. J., Kirchhausen, T., Murthy, V.N. (2006). Inhibition of dynamin completely blocks compensatory synaptic vesicle endocytosis. Proc. Natl. Acad. Sci. USA 103, 17955–17960.)
Figure 6.3
Figure 6.3
Schemefor the synthesis of dynasore. (a) H4N2, CH3OH, 65°, 43% yield. (b) 3,4-dihydroxy-benzaldehyde, CH3CO2H, CH3CH2OH, 78°, 85% yield.
Figure 6.4
Figure 6.4
Determination of kinetics parameters for the effects of dynasore on the GTPase activity of dynamin. Effect of 40 µM dynasore on the rate of GTPase hydrolysis of 0.2 mM Dyn2ΔPRD determined at ambient temperature. The control experiment was done in the presence of 1% DMSO (vehicle) Initial rates of GTP hydrolysis were determined for different concentrations of GTP (S).
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