VBIT-4

VDAC, mtDNA release, IFN signaling, neutrophil extracellular traps

使用MST方法分析VBIT-4与重组纯化的VDAC1、VDAC2和VDAC3的相互作用。VBIT-4以相似的结合亲和力与三种重组亚型结合，尽管比从大鼠肝线粒体纯化的VDAC1低3倍。[1]

VDAC寡聚化抑制剂VBIT-4在系统性红斑狼疮小鼠模型中降低mtDNA释放、IFN信号传导、中性粒细胞外陷阱和疾病严重程度。因此，VDAC低聚抑制是一种潜在的治疗与mtDNA释放相关疾病的方法
VBIT-4可改善MpJ Faslpr小鼠的狼疮样症状。VBIT-4阻断了皮肤病变的发展和伴随白细胞浸润的表皮增厚，并在不影响死亡率或体重的情况下抑制了面部和背部脱发。VBIT-4还降低了脾脏和淋巴结重量。[2]

鉴定VDAC1低聚抑制剂的高通量筛选使用96孔形式的细胞进行筛选，以增强BRET2信号，从而鉴定VDAC1-低聚抑制剂。转染具有低VDAC1水平的T-REx细胞以表达rVDAC1-GFP2和rVDAC1 Rluc，并以9000个细胞/孔的密度接种在96孔板中。将化合物（1μl的2mm储备溶液）添加到100μl中的最终浓度为10μm（1%的DMSO最终浓度）。将细胞与NCI化合物预孵育1小时，然后与凋亡诱导剂再孵育3小时（STS，1μm；亚硒酸盐，30μm；As2O3，60μm）。通过机器人系统将测试的NCI化合物分配到96孔板中。处理后，除去培养基，并如上所述测定BRET2信号。液体处理是用Tecan（Männedorf，Switzerland）Freedom 150 Robotic&MCA液体处理系统完成的，尽管荧光素酶发光和荧光读数是通过机器人集成的Tecan Infinite M1000阅读器获得的。[1]

交联实验PBS中的细胞（2.5–3 mg/ml）在适当处理后收获，并与交联试剂EGS（pH 8.3）孵育15分钟。使用抗VDAC1抗体对样品（60–80μg蛋白质）进行SDS-PAGE和免疫印迹。使用FUSION-FX对免疫反应性VDAC1二聚体、三聚体和多聚体带进行定量分析。[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

65Ų

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 请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
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