VDAC, mtDNA release, IFN signaling, neutrophil extracellular traps

The interaction of VBIT-4 with recombinant purified VDAC1, VDAC2, and VDAC3 was analyzed using the MST method. VBIT-4 bound to the three recombinant isoforms with a similar binding affinity, although 3-fold lower than that of VDAC1 purified from rat liver mitochondria.[1]

The VDAC oligomerization inhibitor VBIT-4 decreases mtDNA release, IFN signaling, neutrophil extracellular traps, and disease severity in a mouse model of systemic lupus erythematosus. Thus, VDAC oligomerization inhibition is a potential therapeutic approach for diseases associated with mtDNA release.
VBIT-4 could ameliorate lupus-like symptoms in MpJ-Faslpr mice. VBIT-4 blocked the development of skin lesions and the thickening of the epidermis that accompanies leukocyte infiltration, and suppressed facial and dorsal alopecia without affecting mortality or body weight. VBIT-4 also decreased spleen and lymph node weights. [2]

High-throughput Screening to Identify Inhibitors of VDAC1 Oligomerization The screen was conducted using the cells in a 96-well format for enhancement of BRET2 signals to identify inhibitors of VDAC1 oligomerization. T-REx cells with low VDAC1 levels were transfected to express rVDAC1-GFP2 and rVDAC1-Rluc and seeded at a density of 9,000 cells/well in a 96-well plate. Compounds (1 μl of 2 mm stock solutions) were added to a final concentration of 10 μm in 100 μl (1% final DMSO concentration). The cells were pre-incubated for 1 h with the NCI compounds and then incubated with the apoptosis inducers for an additional 3 h (STS, 1 μm; selenite, 30 μm; As2O3, 60 μm). The tested NCI compounds were dispensed by a robotic system into the 96-well plates. After treatment, the medium was removed and assayed for BRET2 signals as described above. Liquid handling was done with the Tecan (Männedorf, Switzerland) Freedom 150 Robotic & MCA Liquid Handling System, although luciferase luminescence and fluorescence readings were obtained a robot-integrated Tecan Infinite M1000 reader.[1]

Cross-linking Experiments Cells (2.5–3 mg/ml) in PBS were harvested after the appropriate treatment and incubated with the cross-linking reagent EGS, pH 8.3, for 15 min. Samples (60–80 μg of protein) were subjected to SDS-PAGE and immunoblotting using anti-VDAC1 antibodies. Quantitative analysis of immunoreactive VDAC1 dimer, trimer, and multimer bands was performed using FUSION-FX. [1]

Animal model of SLE (systemic lupus erythematosus)
Formulation: VBIT-4 was freshly dissolved in DMSO and diluted in water (final pH 5.0, DMSO 0.05%).
Doses: 20 mg/kg
Administration route: taken with drinking water
Animal model of SLE (systemic lupus erythematosus). All experiments were approved by the ACUC (Animal Care and Use Committee) of the NIH/NHLBI. Female MRL/MpJ-Faslpr/J mice (stock #000485) were used as a model to determine the etiology of systemic lupus erythematosus (SLE). MRL/MpJ mice were used as a control for MRL/MpJ-Faslpr/J mice. All mice were purchased from The Jackson Laboratory. VBIT-4 was freshly dissolved in DMSO and diluted in water (final pH 5.0, DMSO 0.05%). The MRL/MpJ-Faslpr/J mice were treated with a daily freshly diluted dose of VBIT-4 (20 mg/kg) or vehicle water (final pH 5.0, DMSO 0.05%) in drinking water for 5 w, beginning at 11 w of age until euthanasia at 16 w of age. Blood and urine samples were collected when the mice were 16 w of age. Body weight were measured before and after VBIT-4 administration (11 and 16 w of age respectively). Skin, kidney, thymus, and lymph nodes were also collected.[2]

[1] Novel Compounds Targeting the Mitochondrial Protein VDAC1 Inhibit Apoptosis and Protect against Mitochondrial Dysfunction. J Biol Chem. 2016 Nov 25;291(48):24986-25003. doi: 10.1074/jbc.M116.744284. Epub 2016 Oct 13.\nPMID: 27738100;

[2] Science. 2019 Dec 20;366(6472):1531-1536. doi: 10.1126/science.aav4011. Epub 2019 Dec 19.\nPMID: 31857488

[3] Sci Rep. 2020 Dec 16;10(1):22101. doi: 10.1038/s41598-020-79056-w.PMID: 33328613

Apoptosis is thought to play a critical role in several pathological processes, such as neurodegenerative diseases (i.e. Parkinson's and Alzheimer's diseases) and various cardiovascular diseases. Despite the fact that apoptotic mechanisms are well defined, there is still no substantial therapeutic strategy to stop or even slow this process. Thus, there is an unmet need for therapeutic agents that are able to block or slow apoptosis in neurodegenerative and cardiovascular diseases. The outer mitochondrial membrane protein voltage-dependent anion channel 1 (VDAC1) is a convergence point for a variety of cell survival and death signals, including apoptosis. Recently, we demonstrated that VDAC1 oligomerization is involved in mitochondrion-mediated apoptosis. Thus, VDAC1 oligomerization represents a prime target for agents designed to modulate apoptosis. Here, high-throughput compound screening and medicinal chemistry were employed to develop compounds that directly interact with VDAC1 and prevent VDAC1 oligomerization, concomitant with an inhibition of apoptosis as induced by various means and in various cell lines. The compounds protected against apoptosis-associated mitochondrial dysfunction, restoring dissipated mitochondrial membrane potential, and thus cell energy and metabolism, decreasing reactive oxidative species production, and preventing detachment of hexokinase bound to mitochondria and disruption of intracellular Ca2+ levels. Thus, this study describes novel drug candidates with a defined mechanism of action that involves inhibition of VDAC1 oligomerization, apoptosis, and mitochondrial dysfunction. The compounds VBIT-3 and VBIT-4 offer a therapeutic strategy for treating different diseases associated with enhanced apoptosis and point to VDAC1 as a promising target for therapeutic intervention.[1]

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Mitochondrial stress releases mitochondrial DNA (mtDNA) into the cytosol, thereby triggering the type Ι interferon (IFN) response. Mitochondrial outer membrane permeabilization, which is required for mtDNA release, has been extensively studied in apoptotic cells, but little is known about its role in live cells. We found that oxidatively stressed mitochondria release short mtDNA fragments via pores formed by the voltage-dependent anion channel (VDAC) oligomers in the mitochondrial outer membrane. Furthermore, the positively charged residues in the N-terminal domain of VDAC1 interact with mtDNA, promoting VDAC1 oligomerization. The VDAC oligomerization inhibitor VBIT-4 decreases mtDNA release, IFN signaling, neutrophil extracellular traps, and disease severity in a mouse model of systemic lupus erythematosus. Thus, inhibiting VDAC oligomerization is a potential therapeutic approach for diseases associated with mtDNA release.[2]

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The voltage-dependent anion channel 1 (VDAC1) is a key player in mitochondrial function. VDAC1 serves as a gatekeeper mediating the fluxes of ions, nucleotides, and other metabolites across the outer mitochondrial membrane, as well as the release of apoptogenic proteins initiating apoptotic cell death. VBIT-4, a VDAC1 oligomerization inhibitor, was recently shown to prevent mitochondrial dysfunction and apoptosis, as validated in mouse models of lupus and type-2 diabetes. In the present study, we explored the expression of VDAC1 in the diseased myocardium of humans and rats. In addition, we evaluated the effect of VBIT-4 treatment on the atrial structural and electrical remodeling of rats exposed to excessive aldosterone levels. Immunohistochemical analysis of commercially available human cardiac tissues revealed marked overexpression of VDAC1 in post-myocardial infarction patients, as well as in patients with chronic ventricular dilatation\dysfunction. In agreement, rats exposed to myocardial infarction or to excessive aldosterone had a marked increase of VDAC1 in both ventricular and atrial tissues. Immunofluorescence staining indicated a punctuated appearance typical for mitochondrial-localized VDAC1. Finally, VBIT-4 treatment attenuated the atrial fibrotic load of rats exposed to excessive aldosterone without a notable effect on the susceptibility to atrial fibrillation episodes induced by burst pacing. Our results indicate that VDAC1 overexpression is associated with myocardial abnormalities in common pathological settings. Our data also indicate that inhibition of the VDAC1 can reduce excessive fibrosis in the atrial myocardium, a finding which may have important therapeutic implications. The exact mechanism\s of this beneficial effect need further studies.[3]

C21H23CLF3N3O3

457.8738

457.138

C, 55.09; H, 5.06; Cl, 7.74; F, 12.45; N, 9.18; O, 10.48

2086257-77-2

2086257-77-2 (racemate);2086268-69-9 (R-isoemr);2086269-51-2 (S-isomer);

126697666

Light yellow to yellow solid powder

3.9

2

8

7

31

560

0

QYSQXVAEFPWMEM-UHFFFAOYSA-N

InChI=1S/C21H23ClF3N3O3/c22-15-1-3-16(4-2-15)26-20(30)13-18(14-29)28-11-9-27(10-12-28)17-5-7-19(8-6-17)31-21(23,24)25/h1-8,18,29H,9-14H2,(H,26,30)

N-(4-chlorophenyl)-4-hydroxy-3-(4-(4-(trifluoromethoxy)phenyl)piperazin-1-yl)butanamide

VBIT-4; VBIT4; N-(4-chlorophenyl)-4-hydroxy-3-[4-[4-(trifluoromethoxy)phenyl]piperazin-1-yl]butanamide; N-(4-chlorophenyl)-4-hydroxy-3-(4-(4-(trifluoromethoxy)phenyl)piperazin-1-yl)butanamide; N-(4-chlorophenyl)-4-hydroxy-3-{4-[4-(trifluoromethoxy)phenyl]piperazin-1-yl}butanamide; SCHEMBL18641899; QYSQXVAEFPWMEM-UHFFFAOYSA-N; EX-A5330; VBIT 4

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)

DMSO : 90 mg/mL (~200 mM)
Ethanol : 40-90 mg/mL
Water : < 1 mg/mL (Insoluble)

~4.5 mg/ml (9.8 mM) in 5% DMSO: 40% PEG300: 5% Tween 80: 50% ddH2O  (Please use freshly prepared in vivo formulations for optimal results.)