m-Cl-CCP (CCCP)   中文名称: 羰基氰酯-3-氯苯基腙

oxidative phosphorylation (OXPHOS); STING; TBK1; IRF3; IFN-β[1]

CCCP 通过干扰 STING、TBK1 和 IRF3 相互结合的能力来防止它们被磷酸化。 CCCP 不会抑制 STING 易位到核周区域，但它会干扰 STING 及其下游信号分子 TBK1 和 IRF3 的激活。除了诱导线粒体裂变外，CCCP 还会阻碍 STING 和 TBK1 之间的反应。值得注意的是，敲除线粒体裂变调节因子 Drp1 后，STING 活性恢复，这表明 CCCP 通过导致质子载体 CCCP 失去其膜电位来抑制 DMXAA 触发的 STING 信号染料。当 RAW264.7 细胞和 MEF 用 DMXAA 处理时，CCCP 会显着减少 IFN-β 的产生 [1]。为了检测有丝分裂，1 μM CCCP 就足够了。用 10 μM CCCP（用于诱导线粒体自噬的剂量）处理的细胞中，对有丝分裂的诱导作用极小。由于线粒体避免与向内运动蛋白结合，有丝分裂在机制上需要将失败的线粒体定位在细胞外围[4]。

CCCP 和 PPEF 的施用剂量相同，均为 3 mg/kg.bw。在这两种情况下，细菌负荷均减少了 1 个对数。然而，当 3 mg/kg.bw 的 PPEF 和 3 mg/kg.bw 的 CCCP 组合时，细菌计数减少了 6 log10。创建的模型证实了联合治疗增加的抗菌活性[2]。施用 CCCP（4 mg/kg 腹膜）的 99mSD 支架中的 Tc-MIBI 信号 31P 西雅图光谱信号表明，经 CCCP 处理的支架中 ATP 浓度降低，99mTc-MIBI 信号也是如此。我们检查了 CCCP 提供的支架的分离心脏组织中的同位素活性，以确定 CCCP 是否降低 99mTc-MIBI。 CCCP组心脏中的99mTc-MIBI信号明显低于注射99mTc-MIBI后180分钟的心率[3]。

抗生物膜活性测定[2]
生物膜抑制化验是由播种100μL的细菌悬液(~ 108 CFU)井的96孔板的1 xmic PPEF, 1 xmic CCCP,1 xmic PPEF + 1/2XMIC CCCP和1 xmic CCCP + 1/2XMIC PPEF 24 h而根除预制生物膜试验,100μL细菌悬浮液(~ 108 CFU)首次允许形成生物膜为24小时37°C在静态条件下,然后形成生物膜是孵化1 xmic PPEF, 1/2XMIC挺,1XMIC PPEF + 1/2XMIC CCCP在37°C下放置24小时。两例患者均采用结晶紫染色法测定生物膜质量。

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Checkerboard assay[2]
PPEF与CCCP组合的协同效应如前文所述采用Checker board法确定。在本研究中，我们采用以下组合:固定的CCCP 1/2XMIC和PPEF的2倍串联稀释，同样固定的PPEF 1/2XMIC和CCCP的2倍串联稀释。在600 nm处用Tecan微板阅读器测定最小抑菌浓度。 根据FIC指数定义联合效应，其中FIC = FIC (PPEF) + FIC (CCCP)，其中FIC (PPEF)为PPEF在联合中的MIC /单独PPEF的MIC, FIC (CCCP)为CCCP在联合中的MIC /单独CCCP的MIC。 FIC的解释;FIC > 4.0为拮抗，FIC > 1且≤4为无差异，FIC > 0.5且≤1为可加性，FIC≤0.540,41.

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Time-kill测定[2]
为了评价PPEF、CCCP以及PPEF和CCCP联合使用对细菌生长的影响，按照NCCLS42标准构建了时间响应生长曲线。将细胞密度为107 CFU mL−1的1 mL菌悬液暴露于PPEF (1XMIC)、CCCP (0.5XMIC)以及PPEF (1XMIC)和CCCP (0.5XMIC)的组合中。在对照管中加入等体积的无菌水。这些培养物在37°C下孵育，在200 rpm下不断搅拌。在不同时间点收集肉汤等分，在生理盐水溶液中连续稀释，在MH琼脂培养基上接种，在37℃下培养18 h，以测定每种培养物的总cfu。通过在各自的时间点将处理后的CFU除以未处理细胞的CFU来计算细胞恢复的百分比。

干扰素-β诱导[1]
mef (5 × 105)、Raw264.7细胞(1 × 106)和稳定表达STING (1.5 × 105)的HeLa细胞分别用DMXAA (100 μg/ml)刺激2或3 h，或用c-di-GMP (5 μg/ml)、cGAMP (5 μg/ml)或poly (dA:dT) (2 μg/ml)转染6 h， CCCP (50 μM)与DMXAA (100 μg/ml)共处理，或在c-di-GMP或poly (dA:dT)处理的最后5 h处理。[1]

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线粒体膜电位的测定[1]
用CCCP (50 μM)刺激野生型和Drp1−/−MEFs (1 × 105)细胞1 h，用TMRM (100 nM)染色30 min，然后流式细胞术分析。[1]

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体外细胞毒性试验[2]
采用克隆生存法测定HEK293T和NIH/3T3细胞对PPEF、CCCP及其联合作用的细胞活力。两种细胞均以每孔400个细胞的密度接种于六孔平底康宁®costar®细胞培养板中。20 h后，分别以0.5、2、8、32 μg/mL的浓度加入。PPEF与CCCP联合治疗时，浓度分别为0.5 μg/ml和2 μg/ml。后续处理24 h后，取出药物，洗涤，细胞继续生长10天形成菌落。菌落用0.5%结晶紫染色，人工计数。

Neutropenic Thigh Infection Model in Balb/c mice[2]
Female Balb/c mice n = 6, per dosing group weighing 20–25 g were rendered neutropenic with 2 intraperitoneal injections of cyclophosphamide 150 mg/kg.bw and 100 mg/kg.bw on 4 days and 1 day prior to bacterial infection. 0.1 mL of the 106 CFU/mL bacterial suspension was injected into right posterior thigh muscle. After 2 h post-infection mice were treated with PPEF (3 mg/kg.bw), CCCP (3 mg/kg.bw) and in combination PPEF + CCCP (3 mg/kg.bw + 3 mg/kg.bw) dissolved in 0.1 mL sterile water by single bolus intravenous injection. Twenty-four hours after antibacterial administration, the mice were humanely sacrificed. Right thigh muscles from each mouse were aseptically collected, homogenized and serially diluted and processed for quantitative cultures.

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In this study, researchers analyzed (99m)Tc-MIBI signals in Sprague-Dawley (SD) rat hearts perfused with carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler known to reduce the mitochondrial membrane potential. (99m)Tc-MIBI signals could be used to detect changes in the mitochondrial membrane potential with sensitivity comparable to that obtained by two-photon laser microscopy with the cationic probe tetramethylrhodamine ethyl ester (TMRE). They also measured (99m)Tc-MIBI signals in the hearts of SD rats administered CCCP (4 mg/kg intraperitoneally) or vehicle. (99m)Tc-MIBI signals decreased in rat hearts administered CCCP, and the ATP content, as measured by (31)P magnetic resonance spectroscopy, decreased simultaneously. Next, researchers administered (99m)Tc-MIBI to Dahl salt-sensitive rats fed a high-salt diet, which leads to hypertension and heart failure. The (99m)Tc-MIBI signal per heart tissue weight was inversely correlated with heart weight, cardiac function, and the expression of atrial natriuretic factor, a marker of heart failure, and positively correlated with the accumulation of labeled fatty acid analog. The (99m)Tc-MIBI signal per liver tissue weight was lower than that per heart tissue weight.[3]

Metabolism / Metabolites
Organic nitriles are converted into cyanide ions through the action of cytochrome P450 enzymes in the liver. Cyanide is rapidly absorbed and distributed throughout the body. Cyanide is mainly metabolized into thiocyanate by either rhodanese or 3-mercaptopyruvate sulfur transferase. Cyanide metabolites are excreted in the urine. (L96)

Toxicity Summary
Organic nitriles decompose into cyanide ions both in vivo and in vitro. Consequently the primary mechanism of toxicity for organic nitriles is their production of toxic cyanide ions or hydrogen cyanide. Cyanide is an inhibitor of cytochrome c oxidase in the fourth complex of the electron transport chain (found in the membrane of the mitochondria of eukaryotic cells). It complexes with the ferric iron atom in this enzyme. The binding of cyanide to this cytochrome prevents transport of electrons from cytochrome c oxidase to oxygen. As a result, the electron transport chain is disrupted and the cell can no longer aerobically produce ATP for energy. Tissues that mainly depend on aerobic respiration, such as the central nervous system and the heart, are particularly affected. Cyanide is also known produce some of its toxic effects by binding to catalase, glutathione peroxidase, methemoglobin, hydroxocobalamin, phosphatase, tyrosinase, ascorbic acid oxidase, xanthine oxidase, succinic dehydrogenase, and Cu/Zn superoxide dismutase. Cyanide binds to the ferric ion of methemoglobin to form inactive cyanmethemoglobin. (L97)

[1]. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) suppresses STING-mediated DNA sensing pathway through inducing mitochondrial fission. Biochem Biophys Res Commun. 2017 Aug 30. pii: S0006-291X(17)31704-7.

[2]. Synergistic efficacy of Bisbenzimidazole and Carbonyl Cyanide 3-Chlorophenylhydrazonecombination against MDR bacterial strains. Sci Rep. 2017 Mar 17;7:44419.

[3]. Measurement of technetium-99m sestamibi signals in rats administered a mitochondrial uncoupler and in a rat model of heart failure. PLoS One. 2015 Jan 16;10(1):e0117091.

[4]. PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65. Open Biol. 2012 May;2(5):120080.

[5]. Mitocytosis, a migrasome-mediated mitochondrial quality-control process. Cell. 2021 May 27;184(11):2896-2910.e13.

CCCP is a member of the class of monochlorobenzenes that is benzene substituted by 2-(1,3-dinitrilopropan-2-ylidene)hydrazinyl and chloro groups at positions 1 and 3, respectively. It is a mitochondrial depolarizing agent that induces reactive oxygen species mediated cell death. It has a role as a geroprotector, an antibacterial agent and an ionophore. It is a nitrile, a hydrazone and a member of monochlorobenzenes. It is functionally related to a hydrazonomalononitrile.
Carbonyl cyanide m-chlorophenyl hydrazone is a chemical compound of cyanide.
A proton ionophore. It is commonly used as an uncoupling agent and inhibitor of photosynthesis because of its effects on mitochondrial and chloroplast membranes.

C9H5CLN4

204.617

204.02

C, 52.83; H, 2.46; Cl, 17.33; N, 27.38

555-60-2

555-60-2;

2603

Yellow to brown solid powder

1.26 g/cm3

318.3ºC at 760 mmHg

170-175 °C (dec.)

146.3ºC

1.611

2.228

71.97

1

4

2

14

300

0

UGTJLJZQQFGTJD-UHFFFAOYSA-N

InChI=1S/C9H5ClN4/c10-7-2-1-3-8(4-7)13-14-9(5-11)6-12/h1-4,13H

Carbonyl cyanide 3-chlorophenylhydrazone

CCCP; Mesoxalonitrile 3-chlorophenylhydrazone; Carbonyl cyanide 3-chlorophenylhydrazone; (3-Chlorophenyl)hydrazonomalononitrile; Carbonyl cyanide m-chlorophenylhydrazone; Carbonylcyanide-3-chlorophenylhydrazone; Carbonyl cyanide m-chlorophenyl hydrazone; m-Chlorophenyl carbonylcyanide hydrazone;

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 : ~50 mg/mL (~244.36 mM)
H2O : < 0.1 mg/mL

配方 1 中的溶解度: ≥ 2.5 mg/mL (12.22 mM) (饱和度未知) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80+，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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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；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。