dynamin (IC50 = 15 μM); HSV-1/2

Dynasore inhibits dynamin1, dynamin2, and mitochondrial dynamin Drp1 GTPase activity, but not that of other small GTPases. When added, dynasore quickly prevents the creation of coated vesicles, thereby acting as a powerful inhibitor of the known dynamin-dependent endocytic pathway. This effect is seen within seconds. G-shaped, semi-formed pits and O-shaped, completely formed pits, which are trapped during pinch-off, are the two types of coated pit intermediates that accumulate during dynasore processing [1]. Human fetal neurons, astrocytes, primary reproductive tract cells, and epithelial and neural cells are all susceptible to HSV-1 and HSV-2 infection. Dynasore prevents these infections. When given eight hours after virus entrance, dynasore prevents newly generated viral proteins from leaving the nucleus and increases the amount of viral capsids that reach the nuclear pore [2]. Dynasore inhibits the rise in left ventricular end-diastolic pressure brought on either ischemia or reperfusion. Moreover, dynamicsore decreases infarct size and cardiac troponin I efflux upon reperfusion. Dynasore increased cardiomyocyte viability and survival in adult mouse cardiomyocytes grown under oxidative stress [3].

After spinal cord injury (SCI) in rats, dynasore dramatically reduced motor dysfunction at 3, 7, and 10 days. By preventing the activation of pathways leading to mitochondrial apoptosis and astrocyte proliferation in rat neurons following spinal cord damage, Dynasore greatly improves motor function [4].

ATP Measurement[3]
A luminescence assa was used to quantify cardiomyocyte and Hela cell ATP content. Briefly, after Dynasore treatment and H2O2 exposure, cardiomyocytes were lysed and ATP content was measured in the cell lysates. Meanwhile, in a separate set of wells following same experimental protocol, surviving cardiomyocytes were counted using a TBE assay. Cellular ATP per single live cardiomyocyte was then calculated for each treatment condition. Similar procedures were applied to cultured non-stressed Hela cells treated with control or Dynasore.

Isolation and Culture of Adult Mouse Cardiomyocytes[3]
Mouse ventricular myocytes were isolated from male adult C6/Black mouse (8∼12 weeks; Charles River) after dissociation with collagenase II (2 mg/ml) using a previously described method. After dissociation, cardiomyocytes were plated on laminin-precoated 35 mm2 culture dishes at a density of ∼1,500/mm2 and maintained in a humidified atmosphere of 5% CO2 at 37°C. After 1 hour of plating, cardiomyocytes were replenished with fresh medium (serum supplemented or depleted) and subjected to 2 hours of drug treatment (Dynasore or vehicle) followed by oxidative stress (30 µM H2O2 for 35 min). For ATP supplement experiments, the cells were treated with 3 mM ATP for 30 min before exposure to H2O2.

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Live-cell Mitochondria Imaging with Spinning Disc Confocal Microscopy[3]
HeLa cells were maintained in DMEM supplemented with 10% FBS and 100 µg/ml Normocin. Cells were maintained in a humidified atmosphere of 5% CO2 at 37°C. Cells were seeded at a density of 7 × 104 cells/cm2 and allowed to adhere overnight. Cells were then transduced with Organelle Lights™ Mito-RFP *BacMam 1.0. Twenty-four hours after transduction, cells were pretreated with either control or 1 µM Dynasore for 1 hour before being exposed to normal conditions or 200 µM H2O2 for 15 minutes. Before and after exposure to H2O2, cells were imaged using a Nikon Ti inverted microscope, Yokogowa CSU-X1 spinning disk confocal unit with 568-nm DPSS laser source, and a high resolution Cool SNAP HQ2 camera. Images were acquired at 400 ms exposure per frame and automatically processed using a bas relief filter to highlight edges.

Langendorff-Perfused Heart[3]
Male C6/Black mice (8∼12 weeks) were anesthetized with isoflurane (flow 3%) and 100% O2 in an anesthesia chamber and anti-coagulated with heparin (50 IU, i.p.). After cervical dislocation, hearts were rapidly excised, mounted on a Langendorff apparatus and perfused retrogradely at a constant rate of 2.6 ml/min with oxygenated Krebs-Henseleit buffer containing (mmol/L): NaCl 118, NaHCO3 24, CaCL2.2H2O 2.5, KCL 4.7, KH2PO4 1.2, MgSO4-7H2O 1.2, Glucose 11, EDTA 0.5, adjusted to a pH of 7.4. The apparatus was water-jacketed for temperature control to maintain a core temperature of the heart at 37°C. The buffer passed through a membranous “lung” made of SilasticTM Medical Grade Tubing, which was gassed continuously with 95% O2-5% CO2. Fine platinum electrodes were placed on the right atrium and apex of the left ventricle to record the electrocardiogram and heart rate throughout the experiment. A Millar MIKRO-TIP catheter transducer was inserted into the left ventricle from the left atrium to measure left ventricular pressure. Left ventricular end diastolic pressure (LVEDP), left ventricular end systolic pressure (LVESP) and heart rate were monitored and recorded continuously using PowerLab system. Left ventricular developed pressure (LVDP) was calculated by subtracting LVEDP from LVESP. Hearts were paced at 360 bpm with bipolar electrodes attached to the right atrium, using stimuli delivered from a stimulator.
After the initial 15 min stabilization, hearts were excluded from further study if they exhibited one or more of the following exclusion criteria: LVEDP higher than 20 mmHg; LVDP less than 50 mmHg; intrinsic heart rate less than 280 bpm or irregular; or aortic regurgitation. The volume of the perfusate was reduced to 200 ml and allowed to recirculate. The hearts were then randomized to one of the following two treatment groups: Dynasore group (n = 8, added into the recirculating perfusate in stepwise fashion to reach a final concentration of 1 µM within 120 min of recirculation) or DMSO control group (n = 8, added in a similar manner of Dynasore). Hearts were then subjected to 30 min of global ischemia followed by 1 hour of reperfusion. Pacing was initiated after stabilization except during ischemia and was reinitiated 2 min after reperfusion.

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In the Dynasore groups, the rats are given dynasore immediately at a dose of 1, 10, or 30 mg/kg through intraperitoneal injection after SCI, while the rats in the sham and SCI groups receive DMSO (same volume as dynasore groups) through intraperitoneal injection

Rats

[1]. Dynasore, a cell-permeable inhibitor of dynamin. Dev Cell. 2006 Jun;10(6):839-50.

[2]. Dynasore disrupts trafficking of herpes simplex virus proteins. J Virol. 2015 Jul;89(13):6673-84.

[3]. Dynasore protects mitochondria and improves cardiac lusitropy in Langendorff perfused mouse heart. PLoS One. 2013 Apr 15;8(4):e60967.

[4]. Dynasore Improves Motor Function Recovery via Inhibition of Neuronal Apoptosis and Astrocytic Proliferation after Spinal Cord Injury in Rats. Mol Neurobiol. 2016 Nov 7.

[5]. Discovery of the migrasome, an organelle mediating release of cytoplasmic contents during cell migration. Cell Res. 2015 Jan;25(1):24-38.

Dynasore is a carbohydrazide resulting from the formal condensation of the hydrazone moiety of 3,4-dihydroxybenzaldehyde hydrazone with the carboxy group of 3-hydroxy-2-naphthoic acid. It is a cell-permeable, reversible noncompetitive inhibitor of the GTPase activity of dynamin 1 and 2 and Drp1 (mitochondrial), while exhibiting no significant effect against two other small GTPases, MxA and Cdc42. It has a role as an EC 3.6.5.5 (dynamin GTPase) inhibitor. It is a member of catechols, a member of naphthols, a hydrazide and a hydrazone. It is functionally related to a 3-Hydroxy-2-naphthoate.

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Dynamin is essential for clathrin-dependent coated vesicle formation. It is required for membrane budding at a late stage during the transition from a fully formed pit to a pinched-off vesicle. Dynamin may also fulfill other roles during earlier stages of vesicle formation. We have screened about 16,000 small molecules and have identified 1, named here dynasore, that interferes in vitro with the GTPase activity of dynamin1, dynamin2, and Drp1, the mitochondrial dynamin, but not of other small GTPases. Dynasore acts as a potent inhibitor of endocytic pathways known to depend on dynamin by rapidly blocking coated vesicle formation within seconds of dynasore addition. Two types of coated pit intermediates accumulate during dynasore treatment, U-shaped, half formed pits and O-shaped, fully formed pits, captured while pinching off. Thus, dynamin acts at two steps during clathrin coat formation; GTP hydrolysis is probably needed at both steps.[1]

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Dynasore, a small-molecule inhibitor of the GTPase activity of dynamin, inhibits the entry of several viruses, including herpes simplex virus (HSV), but its impact on other steps in the viral life cycle has not been delineated. The current study was designed to test the hypothesis that dynamin is required for viral protein trafficking and thus has pleiotropic inhibitory effects on HSV infection. Dynasore inhibited HSV-1 and HSV-2 infection of human epithelial and neuronal cells, including primary genital tract cells and human fetal neurons and astrocytes. Similar results were obtained when cells were transfected with a plasmid expressing dominant negative dynamin. Kinetic studies demonstrated that dynasore reduced the number of viral capsids reaching the nuclear pore if added at the time of viral entry and that, when added as late as 8 h postentry, dynasore blocked the transport of newly synthesized viral proteins from the nucleus to the cytosol. Proximity ligation assays demonstrated that treatment with dynasore prevented the colocalization of VP5 and dynamin. This resulted in a reduction in the number of viral capsids isolated from sucrose gradients. Fewer capsids were observed by electron microscopy in dynasore-treated cells than in control-treated cells. There were also reductions in infectious progeny released into culture supernatants and in cell-to-cell spread. Together, these findings suggest that targeting dynamin-HSV interactions may provide a new strategy for HSV treatment and prevention. Importance: HSV infections remain a global health problem associated with significant morbidity, particularly in neonates and immunocompromised hosts, highlighting the need for novel approaches to treatment and prevention. The current studies indicate that dynamin plays a role in multiple steps in the viral life cycle and provides a new target for antiviral therapy. Dynasore, a small-molecule inhibitor of dynamin, has pleiotropic effects on HSV-1 and HSV-2 infection and impedes viral entry, trafficking of viral proteins, and capsid formation.[2]

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Background: Heart failure due to diastolic dysfunction exacts a major economic, morbidity and mortality burden in the United States. Therapeutic agents to improve diastolic dysfunction are limited. It was recently found that Dynamin related protein 1 (Drp1) mediates mitochondrial fission during ischemia/reperfusion (I/R) injury, whereas inhibition of Drp1 decreases myocardial infarct size. We hypothesized that Dynasore, a small noncompetitive dynamin GTPase inhibitor, could have beneficial effects on cardiac physiology during I/R injury. Methods and results: In Langendorff perfused mouse hearts subjected to I/R (30 minutes of global ischemia followed by 1 hour of reperfusion), pretreatment with 1 µM Dynasore prevented I/R induced elevation of left ventricular end diastolic pressure (LVEDP), indicating a significant and specific lusitropic effect. Dynasore also decreased cardiac troponin I efflux during reperfusion and reduced infarct size. In cultured adult mouse cardiomyocytes subjected to oxidative stress, Dynasore increased cardiomyocyte survival and viability identified by trypan blue exclusion assay and reduced cellular Adenosine triphosphate(ATP) depletion. Moreover, in cultured cells, Dynasore pretreatment protected mitochondrial fragmentation induced by oxidative stress. Conclusion: Dynasore protects cardiac lusitropy and limits cell damage through a mechanism that maintains mitochondrial morphology and intracellular ATP in stressed cells. Mitochondrial protection through an agent such as Dynasore can have clinical benefit by positively influencing the energetics of diastolic dysfunction.[3]

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Spinal cord injury (SCI) is a common and devastating central nervous system insult which lacks efficient treatment. Our previous experimental findings indicated that dynamin-related protein 1 (Drp1) mediates mitochondrial fission during SCI, and inhibition of Drp1 plays a significant protective effect after SCI in rats. Dynasore inhibits GTPase activity at both the plasma membrane (dynamin 1, 2) and the mitochondria membrane (Drp1). The aim of the present study was to investigate the beneficial effects of dynasore on SCI and its underlying mechanism in a rat model. Sprague-Dawley rats were randomly assigned to sham, SCI, and 1, 10, and 30 mg dynasore groups. The rat model of SCI was established using an established Allen's model. Dynasore was administered via intraperitoneal injection immediately. Results of motor functional test indicated that dynasore ameliorated the motor dysfunction greatly at 3, 7, and 10 days after SCI in rats (P < 0.05). Results of western blot showed that dynasore has remarkably reduced the expressions of Drp1, dynamin 1, and dynamin 2 and, moreover, decreased the Bax, cytochrome C, and active Caspase-3 expressions, but increased the expressions of Bcl-2 at 3 days after SCI (P < 0.05). Notably, the upregulation of proliferating cell nuclear antigen (PCNA) and glial fibrillary acidic protein (GAFP) are inhibited by dynasore at 3 days after SCI (P < 0.05). Results of immunofluorescent double labeling showed that there were less apoptotic neurons and proliferative astrocytes in the dynasore groups compared with SCI group (P < 0.05). Finally, histological assessment via Nissl staining demonstrated that the dynasore groups exhibited a significantly greater number of surviving neurons compared with the SCI group (P < 0.05). This neuroprotective effect was dose-dependent (P < 0.05). To our knowledge, this is the first study to indicate that dynasore significantly enhances motor function which may be by inhibiting the activation of neuronal mitochondrial apoptotic pathway and astrocytic proliferation in rats after SCI.[4]

C18H14N2O4

322.31

322.095

C, 67.08; H, 4.38; N, 8.69; O, 19.85

304448-55-3

135533054

Light brown to brown solid powder

1.36±0.1 g/cm3

1.665

4.06

4

5

3

24

470

0

C1=CC=C2C=C(C(=CC2=C1)C(=O)N/N=C/C3=CC(=C(C=C3)O)O)O

SYNDQCRDGGCQRZ-VXLYETTFSA-N

InChI=1S/C18H14N2O4/c21-15-6-5-11(7-17(15)23)10-19-20-18(24)14-8-12-3-1-2-4-13(12)9-16(14)22/h1-10,21-23H,(H,20,24)/b19-10+

(E)-N-(3,4-dihydroxybenzylidene)-3-hydroxy-2-naphthohydrazide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.76 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (7.76 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)