Voltage - dependent anion channel 1 (VDAC1), 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]

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- VBIT - 4 can inhibit VDAC1 oligomerization, preventing apoptosis - associated mitochondrial dysfunction. It can restore dissipated mitochondrial membrane potential, reduce reactive oxidative species production, and prevent the detachment of hexokinase bound to mitochondria and the disruption of intracellular Ca2 + levels in various cell lines induced by different apoptosis inducers [1]
- VBIT - 4 rapidly inhibits the loss of ATP, which is caused by the translocation of VDAC1 to the plasma membrane due to VDAC1 overexpression, in PlVDAC1 - expressing INS - 1 cells

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]

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- VBIT - 4 can alleviate lupus - like symptoms in MPJ - Faslpr mice. It can block the development of skin lesions and the thickening of the epidermis accompanied by leukocyte infiltration, suppress facial and dorsal alopecia, and reduce spleen and lymph node weights without affecting mortality or body weight. It also decreases mtDNA release, type I interferon (IFN) signaling, and neutrophil extracellular traps, thus reducing the severity of the disease [2]
- VBIT - 4 can attenuate the atrial fibrotic load of rats exposed to excessive aldosterone, but has no significant effect on the susceptibility to atrial fibrillation episodes induced by burst pacing

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]

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High - throughput screening was carried out using cells in 96 - well plates. 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. 1 μl of 2 mM VBIT - 4 stock solution was added to a final concentration of 10 μM in 100 μl (with a final DMSO concentration of 1%). The cells were pre - incubated with VBIT - 4 for 1 h, and then incubated with apoptosis inducers (STS, 1 μM; selenite, 30 μM; As2O3, 60 μM) for another 3 h to detect the change of BRET2 signal, so as to screen VDAC1 oligomerization inhibitors [2]

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]

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VBIT - 4 was used to treat MPJ - Faslpr mice to observe the change of lupus - like symptoms, and was used to treat rats with excessive aldosterone to observe the change of atrial fibrosis [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.

[2] VDAC oligomers form mitochondrial pores to release mtDNA fragments and promote lupus-like disease. Science. 2019 Dec 20;366(6472):1531-1536.

[3] VDAC1 in the diseased myocardium and the effect of VDAC1-interacting compound on atrial fibrosis induced by hyperaldosteronism. Sci Rep. 2020 Dec 16;10(1):22101.

Apoptosis is considered to play a crucial role in various pathological processes, such as neurodegenerative diseases (e.g., Parkinson's disease and Alzheimer's disease) and various cardiovascular diseases. Although the mechanisms of apoptosis are well elucidated, effective therapeutic strategies to halt or even slow this process remain lacking. Therefore, there is an urgent need for therapeutic drugs capable of blocking or slowing apoptosis in neurodegenerative and cardiovascular diseases. The mitochondrial outer membrane protein voltage-dependent anion channel 1 (VDAC1) is a convergence point for various cell survival and death signals, including apoptosis. Recently, we demonstrated that VDAC1 oligomerization is involved in mitochondrial-mediated apoptosis. Therefore, VDAC1 oligomerization is an important target for designing drugs to regulate apoptosis. In this study, using high-throughput compound screening and medicinal chemistry methods, we developed compounds that directly interact with VDAC1 and inhibit VDAC1 oligomerization, while also inhibiting apoptosis induced by various pathways and in various cell lines. These compounds can protect cells from apoptosis-related mitochondrial dysfunction, restore dissipated mitochondrial membrane potential, thereby maintaining cellular energy and metabolism, reducing reactive oxygen species production, and preventing hexokinase from detaching from mitochondria and disrupting intracellular Ca2+ levels. Therefore, this study describes novel candidate drugs with well-defined mechanisms of action involving the inhibition of VDAC1 oligomerization, apoptosis, and mitochondrial dysfunction. Compounds VBIT-3 and VBIT-4 provide a therapeutic strategy for treating a variety of diseases associated with enhanced apoptosis and point to VDAC1 as a promising therapeutic target. [1]

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Mitochondrial stress releases mitochondrial DNA (mtDNA) into the cytosol, triggering a type I interferon (IFN) response. Increased mitochondrial outer membrane permeability is essential for mtDNA release, which has been extensively studied in apoptotic cells, but its role in living cells is little known. We found that oxidatively stressed mitochondria release short mtDNA fragments through pores formed by voltage-dependent anion channel (VDAC) oligomers on the mitochondrial outer membrane. In addition, positively charged residues in the N-terminal domain of VDAC1 interact with mtDNA, promoting VDAC1 oligomerization. The VDAC oligomerization inhibitor VBIT-4 reduced mitochondrial DNA (mtDNA) release, interferon (IFN) signaling, neutrophil extracellular trap (NETs) formation, and disease severity in a mouse model of systemic lupus erythematosus. Therefore, inhibiting VDAC oligomerization is a potential approach to treat diseases associated with mtDNA release. [2] Voltage-dependent anion channel 1 (VDAC1) is a key player in mitochondrial function. As a “gatekeeper,” VDAC1 mediates the flow of ions, nucleotides, and other metabolites across the outer mitochondrial membrane and the release of pro-apoptotic proteins that initiate apoptosis. VBIT-4 is a VDAC1 oligomerization inhibitor that has recently been shown to prevent mitochondrial dysfunction and apoptosis in mouse models of lupus and type 2 diabetes. In this study, we investigated the expression of VDAC1 in diseased myocardium in humans and rats. In addition, we evaluated the effects of VBIT-4 treatment on atrial structure and electrical remodeling in rats exposed to excessive aldosterone levels. Immunohistochemical analysis of commercially available human heart tissue showed that VDAC1 expression was significantly increased in heart tissues of patients after myocardial infarction and patients with chronic ventricular dilatation dysfunction. Consistent with this, VDAC1 was significantly increased in ventricular and atrial tissues of rats exposed to myocardial infarction or excessive aldosterone. Immunofluorescence staining showed that VDAC1 was distributed in a punctate pattern, which is a typical feature of mitochondrial localization. Finally, VBIT-4 treatment reduced the degree of atrial fibrosis in rats exposed to excessive aldosterone, but had no significant effect on susceptibility to rapid pacing-induced atrial fibrillation. Our results suggest that VDAC1 overexpression is associated with myocardial abnormalities under common pathological conditions. Our data also suggest that inhibiting VDAC1 can reduce excessive fibrosis of atrial myocardium, a finding that may have important therapeutic implications. The exact mechanism of this beneficial effect needs further investigation. [3] Apoptosis plays a key role in many pathological processes, such as neurodegenerative diseases and cardiovascular diseases. VDAC1 on the outer mitochondrial membrane is a convergence point for various cell survival and death signals. VDAC1 oligomerization is involved in mitochondrial-mediated apoptosis, therefore VDAC1 is a potential target for regulating apoptosis, while VBIT-4 provides a strategy for treating diseases associated with excessive apoptosis. [1][2]

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

ClC1C([H])=C([H])C(=C([H])C=1[H])N([H])C(C([H])([H])C([H])(C([H])([H])O[H])N1C([H])([H])C([H])([H])N(C2C([H])=C([H])C(=C([H])C=2[H])OC(F)(F)F)C([H])([H])C1([H])[H])=O

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.)