Kinase assays

For analysis of the kinetics of inhibition, dynamin I at a final concentration of 17 nM was incubated with GTPase buffer containing PS (2 μg/mL) and varying amounts of GTP (50–250 μM) in the presence of 4a at a concentration range between 0.5 and 6 μM. The reaction was stopped after 30 min by addition of EDTA (0.5 mM, pH7.4).

Cell lines

U2OS cell lines

Preparation method

Soluble in DMSO. General tips for obtaining a higher concentration: Please warm the tube at 37℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Reacting condition

1~100 μM

Applications

Dyngo-4a potently inhibited Tfn endocytosis with IC50 values of 5.7±1.0 μM, approximating the activity of the most potent small molecule endocytosis inhibitors previously reported.

Animal models

Female CD-1 mice

Dosage form

30 mg/kg, intraperitoneal injection

Application

Mice injected with Dyngo-4a took significantly longer to exhibit clear signs of botulism which indicates that Dyngo-4a pretreatment provides significant protection against botulism.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

Target: DynI (brain); DynI (rec); DynII (rec)

IC50: 0.38 μM; 1.1 μM; 2.3 μM

Dyngo-4a is a potent, novel dynamin inhibitor with IC50 values of 0.38 μM, 1.1 μM, and 2.3 μM for DynI (brain), DynI (rec), and DynII (rec), respectively [1]. Dynamin is a large GTPase enzyme that severs as membrane-bound clathrin-coated vesicles [1].

In vitro: Dyngo-4a (100 μM) caused a reduction in β-catenin levels in human colon carcinoma RKO cells [2]. In addition, Dyngo-4a prevented the uptake of BoNT/A-Hc and blocked BoNT/A-induced SNAP25 cleavage in cultured hippocampal neurons [3]. Dyngo-4a is potent, reversible endocytosis inhibitor in non-neuronal cells. Dyngo-4a inhibited synaptic vesicle endocytosis (SVE) in synaptosomes and neurons with an IC50 value of 26.8 μM [1].

In vivo: Dynamin is a potential therapeutic target for counteracting botulism. Dyngo-4a (30 mg/kg, intraperitoneal injection) treatment suppressed BoNT/A-induced muscle paralysis at the rat hemidiaphragm and significantly delayed the onset of botulism in an in vivo murine model [3].

References:
1.  McCluskey A, Daniel JA, Hadzic G, Chau N, Clayton EL, Mariana A, et al. Building a better dynasore: the dyngo compounds potently inhibit dynamin and endocytosis. Traffic. 2013;14(12):1272-89.
2.  Gagliardi M, Hernandez A, McGough IJ, Vincent JP. Inhibitors of endocytosis prevent Wnt/Wingless signalling by reducing the level of basal beta-catenin/Armadillo. J Cell Sci. 2014;127(Pt 22):4918-26.
3.  Harper CB, Martin S, Nguyen TH, Daniels SJ, Lavidis NA, Popoff MR, et al. Dynamin inhibition blocks botulinum neurotoxin type A endocytosis in neurons and delays botulism. J Biol Chem. 2011;286(41):35966-76.