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

CCCP prevents STING, TBK1, and IRF3 from being phosphorylated by interfering with their ability to bind to each other. STING translocation to the perinuclear region is not inhibited by CCCP, but it does interfere with the activation of STING and its downstream signaling molecules, TBK1 and IRF3. In addition to inducing mitochondrial fission, CCCP hinders the response between STING and TBK1. It's significant to note that STING activity was restored upon knockdown of the mitochondrial fission regulator Drp1, suggesting that CCCP inhibits the DMXAA-triggered STING signaling dye by causing the protonophore CCCP to lose its membrane potential. When RAW264.7 cells and MEFs are treated with DMXAA, CCCP dramatically reduces IFN-β production [1]. To detect mitosis, 1 μM CCCP is enough. There was minimal induction of mitosis in cells treated with 10 μM CCCP, the dose used to induce mitophagy. Due to mitochondrial avoidance binding to inward movement proteins, mitosis mechanistically requires the positioning of failed mitochondria at the cell periphery [4].

Apply CCCP and PPEF at the same dose of 3 mg/kg.bw each. There was a 1-log decrease in the bacterial burden in both situations. However, a 6 log10 decrease in bacterial counts was noted when 3 mg/kg.bw of PPEF and 3 mg/kg.bw of CCCP were combined. The combination treatment's increased antibacterial activity was confirmed by the created model [2]. Tc-MIBI signal in 99mSD scaffolds administered CCCP (4 mg/kg peritoneal) The 31P Seattle spectroscopic signal indicated a reduction in ATP concentration in scaffolds treated CCCP, as did the 99mTc-MIBI signal. We examined the isotope activity in the separated heart tissue of the scaffold that was supplied CCCP in order to determine whether CCCP decreased 99mTc-MIBI. The 99mTc-MIBI signal in the heart of the CCCP group was substantially lower than the heart rate 180 minutes after the injection of 99mTc-MIBI [3].

Anti-Biofilm activity assay [2]
Biofilm inhibition assays was performed by seeding 100 μL of bacterial suspension (~108 CFU) into wells of 96 well plates in the presence of 1XMIC PPEF, 1XMICCCCP, 1XMIC PPEF +1/2XMIC CCCP and 1XMIC CCCP +1/2XMIC PPEF for 24 h whereas, for preformed biofilm eradication assay, 100 μL bacterial suspensions (~108 CFU) was first allowed to form biofilm for 24 h at 37 °C in static condition and then the formed biofilm was incubated with 1XMIC PPEF, 1/2XMIC CCCP, 1XMIC PPEF + 1/2XMIC CCCP for 24 h at 37 °C. In both the cases biofilm mass was evaluated by Crystal violet staining method.

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Checkerboard assay[2]
Synergistic effect of combination of PPEF and CCCP was determined by Checker board method as described previously. In the present study we have taken the combination as follows 1/2XMIC of CCCP fixed and two fold serial dilutions of PPEF similarly 1/2XMIC of PPEF fixed and two fold serial dilutions of CCCP. Minimal Inhibitory Concentration was determined using Tecan Micro-plate Reader at 600 nm. The effect of combination was defined as per the FIC index, whereby FIC = FIC (PPEF) + FIC (CCCP), where FIC (PPEF) is the MIC of PPEF in the combination/MIC of PPEF alone, and FIC (CCCP) is the MIC of CCCP in the combination/MIC of CCCP alone. Interpretation of FIC; antagonistic if FIC > 4.0, indifference if FIC > 1 and ≤4, additive if FIC > 0.5 and ≤1 and synergistic if FIC ≤ 0.540,41. hr> Time-kill assay[2]
To evaluate the effect of PPEF, CCCP and combination of PPEF and CCCP on bacterial growth, a time-response growth curve was constructed according to the standards of the NCCLS42. 1 mL bacterial suspensions at a cell density of 107 CFU mL−1 were exposed to PPEF (1XMIC), CCCP (0.5XMIC) and combination of PPEF (1XMIC) and CCCP (0.5XMIC). In the control tube equal volume of sterile miliQ water was added. These cultures were incubated at 37 °C with constant stirring at 200 rpm. Broth aliquots were collected at different time points, serially diluted in saline solution, plated on MH agar media and grown for 18 h at 37 °C to determine the total CFUs in each culture. Percentage cell recovered was calculated by dividing CFU calculated from treated over the CFU calculated from untreated cells at respective time points.

IFN-β induction[1]
MEFs (5 × 105), Raw264.7 cells (1 × 106), and HeLa cells stable expressing STING (1.5 × 105) were stimulated with DMXAA (100 μg/ml) for 2 or 3 h, or transfected with c-di-GMP (5 μM), cGAMP (5 μg/ml), or poly (dA:dT) (2 μg/ml) for 6 h. CCCP (50 μM) was co-treated with DMXAA (100 μg/ml), or treated for the last 5 h in case of treatment of c-di-GMP or poly (dA:dT).[1]

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Measurement of mitochondrial membrane potential[1]
Wild-type and Drp1−/− MEFs (1 × 105) cells were stimulated with CCCP (50 μM) for 1 h. Cells were stained with TMRM (100 nM) for 30 min, and then analyzed by flow cytometry.

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In vitro cytotoxicity Assay[2]
The cell viability of HEK293T and NIH/3T3 cells against PPEF, CCCP and their combination were assessed by Clonogenic survival assay. Both the cells were seeded at a density of 400 cells per well in a six-well flat bottom Corning® costar® cell culture plate. After 20 h, each compounds were added at 0.5, 2, 8 and 32 μg/mL concentrations. For combined treatment of PPEF and CCCP, 0.5 μg/ml and 2 μg/ml concentration of each were chosen. After subsequent treatment of 24 h, the drugs were removed, washed and the cells were allowed to grow further for 10 days to form colonies. The colonies were stained with 0.5% crystal violet and counted manually.

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

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

Solubility in Formulation 1: ≥ 2.5 mg/mL (12.22 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 25.0 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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 (Please use freshly prepared in vivo formulations for optimal results.)