Drp1/dynamin-related protein 1

Mdivi-1 has an estimated EC50 of 1–10 μM and inhibits Dnm1 GTPase activity in a dose-dependent manner. Mdivi-1 causes the enhanced Hill coefficient of GTP seen in Dnm1 GTP hydrolysis, as well as an increase in the apparent K0.5 of GTP and a decrease in the apparent Vmax of GTP hydrolysis [1]. Following apoptosis induction, cells treated with Mdivi-1 exhibited decreased cytochrome c release and decreased phosphatidylserine exposure, which is consistent with apoptosis inhibition and other studies that used other tactics to disrupt DRP1 activity [2]. In ischemic retina, Mdivi-1 induces apoptotic cell death [3].

mdivi-1 targets the mitochondrial fission DRP, Dnm1, in vivo. GMPPCP-dependent Dnm1 self-assembly is quantitatively blocked by Mdivi-1 at a concentration range that is comparable to its impact on mitochondrial fission in vivo [1]. In the normal mouse retina, Mdivi-1 (50 mg/kg, intraperitoneal injection) dramatically lowers the expression of GFAP protein [3].
Drp1 and GFAP protein expression was significantly increased in the early neurodegenerative events (within 12 hours) of ischemic mouse retina. Mdivi-1 treatment blocked apoptotic cell death in ischemic retina, and significantly increased RGC survival at 2 weeks after ischemia. In the normal mouse retina, Drp1 is expressed in the ganglion cell layer (GCL) as well as the inner plexiform layer, the inner nuclear layer (INL), and the outer plexiform layer (OPL). In the GCL, Drp1 immunoreactivity was strong in RGCs. While Drp1 protein expression was increased in the GCL of vehicle-treated ischemic retina at 12 hours. Mdivi-1 treatment did not change this increase of Drp1 protein expression but significantly decreased GFAP protein expression. Conclusions: These findings suggest that altered Drp1 activity after acute IOP elevation may be an important component of a biochemical cascade leading to RGC death in ischemic retina.[3]

GTP hydrolysis: A continuous regenerative assay was used to measure the GTPase activity of Dnm1 as described (Ingerman and Nunnari, 2005). For determination of the kinetic parameters, GTPase assays were carried out in 25 mM HEPES, 25 mM PIPES, pH 7, 150 mM NaCl, 30 mM imidazole, pH 7.4, 7.5 mM KCl, 5 mM MgCl2, 1 mM phospho(enol)pyruvate (PEP), 20 U/mL pyruvate kinase/lactate dehydrogenase, 4% dimethyl sulfoxide (DMSO), and 600 μM NADH. Chemical inhibitor and GTP concentrations were varied, as specified. GTPase assay reactions were started by addition of approximately 10 μg of purified protein, in freezing buffer, to the GTPase assay reaction buffer containing the small molecule. All GTPase assay reactions were started in a 200 µL volume, of which 150 µL was placed into the well of a 96-well plate. Depletion of NADH, as monitored by reading the A340 of the reaction, was measured every 20 s for a total of 40 min using a SpectraMAX 250 96-well plate reader. Spectrophotometric data were transferred to Excel and the measured steady state depletion of NADH over time was converted to protein activity. K0.5, Km, kcat, Ki, Hill coefficient and other kinetic parameters were calculated numerically, using the Genfit function of Mathcad. [1]

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MOMP assay: Outer membrane permeabilization in isolated mitochondrial was performed as described (Chipuk et al., 2005). Murine heavy membrane fractions (mitochondria) were purified from C57Bl/6 liver, female, less than 3 months, using dounce homogenization and differential centrifugation. For MOMP assays, mitochondria were incubated in mitochondrial isolation buffer (MIB: 200 mM mannitol, 68 mM sucrose, 10 mM HEPES-KOH pH 7.4, 100 mM KCl, 1 mM EDTA, 1 mM EGTA, 0.1% BSA) plus or minus BH3-only proteins or peptides for 90 minutes at RT. For drug inhibition studies, mitochondria were pre-incubated with indicated compounds for 30 minutes prior to the addition of proteins. Reactions were then fractionated into supernatant and pellet by centrifugation at 5,500 x g for 5 minutes, and analyzed by SDSPAGE and western blot with anti-cytochrome c. For MOMP reconstitution studies, heavy membrane fractions were isolated from the livers of polydIdC-treated MxCre bak- / - baxf / - animals. [1]

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LUV permeabilization assay: Large unilamellar vesicle (LUV) release assays were prepared as described (Kuwana et al., Cell 2002). Briefly, lipids were dried and resuspended in buffer containing fluorescein conjugated dextran (10kD) in a water bath sonicator. Unilamelar vesicles were formed by extrusion of the suspension through a 400 nm pore sized filter using an extruder. Unincorporated dextrans were removed by float-up centrifugation in a sucrose gradient. Liposomes were resuspended in buffer and incubated with recombinant proteins (full-length monomeric BAX, Suzuki et al., Cell 2000) and chemicals for 2.5 h at room temperature. The assay mix was filtered through .1µ pore sized membrane and the released dextran was detected as fluorescence in the filtrate. The percentage of release was calculated between the baseline provided by the buffer control and 100 % release obtained by liposomes solubilized in 1% CHAPS. [1]

Yeast actin morphology: Cells were treated with DMSO, 100 μM mdivi-1 or 100 μM mdivi-1 and 200 μM Latrunculin-A. F-actin was visualized with Alexa fluor 488 phalloidin as described (Adams and Pringle, 1991) and imaged using a DeltaVision deconvolution microscope. [1]
Yeast plate growth assay: YPGlycerol plates were topped with 10 ml YPGlycerol containing 1% low melt agar and 75 μM mdivi-1, and cells were spotted 12 hours later using a 48 well pinning device. After pinning cells, plates were incubated at 24°C or 37°C and imaged using an Eagle Eye II imaging system.[1]
mdivi-1 Partially Blocks Cytochrome c Release after Stimulation of Apoptosis with Staurosporine A. HeLa cells were grown in DMEM on cover slips at 37°C with 5% CO2 overnight, then incubated with DMSO, 1μM staurosporine (STS) plus DMSO or 1μm STS plus 50 μm mdivi-1 for four hours. Cytochrome c was visualized by indirect immunofluorescence with anti-cytochrome c and secondary goat anti-mouse Alexa fluor 488 on a Leica confocal microscope. [1]

C57BL/6 mice received injections of mdivi-1 (50 mg/kg) or vehicle, and then transient retinal ischemia was induced by acute IOP elevation. RGC survival was measured after FluoroGold labeling. Drp1 and glial fibrillary acidic protein (GFAP) protein expression and distribution were assessed at 12 hours after ischemia-reperfusion by Western blot and immunohistochemistry. Apoptotic cell death was assessed by TUNEL staining.[3]

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Dissolved in DMSO; 50 mg/kg; i.p. injection

C57BL/6 mice

[1]. Chemical inhibition of the mitochondrial division dynamin reveals its role in Bax/Bak-dependent mitochondrial outer membrane permeabilization. Dev Cell. 2008 Feb;14(2):193-204.

[2]. A chemical inhibitor of DRP1 uncouples mitochondrial fission and apoptosis. Mol Cell. 2008 Feb 29;29(4):409-10.

[3]. A selective inhibitor of drp1, mdivi-1, increases retinal ganglion cell survival in acute ischemic mouse retina. Invest Ophthalmol Vis Sci. 2011 Apr 27;52(5):2837-43.

Mitochondrial fusion and division play important roles in the regulation of apoptosis. Mitochondrial fusion proteins attenuate apoptosis by inhibiting release of cytochrome c from mitochondria, in part by controlling cristae structures. Mitochondrial division promotes apoptosis by an unknown mechanism. We addressed how division proteins regulate apoptosis using inhibitors of mitochondrial division identified in a chemical screen. The most efficacious inhibitor, mdivi-1 (for mitochondrial division inhibitor) attenuates mitochondrial division in yeast and mammalian cells by selectively inhibiting the mitochondrial division dynamin. In cells, mdivi-1 retards apoptosis by inhibiting mitochondrial outer membrane permeabilization. In vitro, mdivi-1 potently blocks Bid-activated Bax/Bak-dependent cytochrome c release from mitochondria. These data indicate the mitochondrial division dynamin directly regulates mitochondrial outer membrane permeabilization independent of Drp1-mediated division. Our findings raise the interesting possibility that mdivi-1 represents a class of therapeutics for stroke, myocardial infarction, and neurodegenerative diseases.[1]

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DRP1, a member of the dynamin family of large GTPases, mediates mitochondrial fission. In a recent issue of Developmental Cell, Cassidy-Stone et al. (2008) identified mdivi-1, a new DRP1 inhibitor that prevents mitochondria division and Bax-mediated mitochondrial outer membrane permeabilization during apoptosis.[2]

C15H10CL2N2O2S

353.22

351.984

C, 51.00; H, 2.85; Cl, 20.07; N, 7.93; O, 9.06; S, 9.08

338967-87-6

3825829

White to light brown solid powder

1.6±0.1 g/cm3

522.5±60.0 °C at 760 mmHg

289 °C

269.8±32.9 °C

0.0±1.4 mmHg at 25°C

1.729

3.85

1

3

2

22

465

0

O=C1N(C2=C(Cl)C=C(Cl)C(OC)=C2)C(S)=NC3=C1C=CC=C3

NZJKEVWTYMOYOR-UHFFFAOYSA-N

InChI=1S/C15H10Cl2N2O2S/c1-21-13-7-12(9(16)6-10(13)17)19-14(20)8-4-2-3-5-11(8)18-15(19)22/h2-7H,1H3,(H,18,22)

3-(2,4-dichloro-5-methoxyphenyl)-2-thioxo-2,3-dihydroquinazolin-4(1H)-one

Mdivi-1; Mitochondrial Division Inhibitor 1; Mdivi 1; 3-(2,4-dichloro-5-methoxyphenyl)-2-sulfanyl-4(3H)-quinazolinone; Mitochondrial division inhibitor 1; Mdivi 1; Mdivi1.

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: ≥ 4 mg/mL (11.32 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 40.0 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: 5% DMSO +40% PEG 300 +ddH2O: 7mg/mL

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Solubility in Formulation 3: 2.5 mg/mL (7.08 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 4: 10 mg/mL (28.31 mM) in 17% Polyethylene glycol 12-hydroxystearate in Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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