CCCP

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CCCP (carbonyl cyanide m-chlorophenyl hydrazine) is widely used uncoupler of oxidative phosphorylation.

The uncoupler of oxidative phosphorylation blocks ATP synthesis by collapsing the proton motive force.

In vitro: A genetic β-galactoside reporter system with a disk diffusion assay on MacConkey Lactose agar petri plates to monitor maintenance of the bacteriophage λ prophage state and viral induction in Escherichia coli K-12. It presented evidence that the phage λ major lytic promoters, pL and pR, are activated via cells containing the reporters following exposure to the energy poison CCCP. Requirement of expression of the λ lytic promoters in response to CCCP is host RecA function and an auto-cleavable CI repressor, as does SOS induction of the λ prophage occurring by a DNA damage-dependent pathway. CCCP-mediated activation of the λ lytic promoters required λ Cro function. CCCP does not modulate an sfi-lacZ SOS reporter [1]. The uncoupler CCCP blocks oxidative phosphorylation by damaging the proton gradient. An anion CCCP can bind a proton. but CCCP with a delocalized negative charge allows it to cross the lipid bilayer in the unprotonated form, while weak acids cross the hydrophobic membrane only when protonated. Many protons can be transported by one molecule of CCCP across the inner membrane. It is described that a genetic reporter system to monitor maintenance of the λ prophage state and viral induction, and evidence is presented that it activates the major leftward and rightward lytic promoters of the λ prophage that cells are exposed to the energy poison CCCP.

In vivo: So far, no study in vivo has been conducted.

Clinical trial: So far, no clinical study has been conducted.

Reference:
[1].  Thomason LC, Court DL. Evidence that bacteriophage λ lysogens may induce in response to the proton motive force uncoupler CCCP. FEMS Microbiol Lett. 2016 Feb;363(3).

p62 prevents carbonyl cyanide m-chlorophenyl hydrazine (CCCP)-induced apoptotic cell death by activating Nrf2.[Pubmed:26208452]

Biochem Biophys Res Commun. 2015 Sep 4;464(4):1139-1144.

Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) is a mitochondrial depolarizing agent that induces reactive oxygen species (ROS)-mediated cell death. The Nrf2-Keap1 pathway is crucial for the elimination of ROS in stressed cells. However, the molecular mechanism underlying the regulation of the Nrf2-Keap1 pathway in CCCP-induced cell death is unknown. In this study, we demonstrated that CCCP promotes Keap1 degradation, and thereby activates Nrf2. This CCCP-mediated Keap1 degradation is partly dependent on autophagy. Moreover, CCCP-induced Keap1 degradation is mainly reliant on p62, which functions as an adaptor protein during selective autophagy. Lack of p62 blocked CCCP-induced Keap1 degradation and inhibited Nrf2 activation, and thereby increased the accumulation of ROS. Ablation of p62 increased the susceptibility of cells to oxidative stress. These results indicate that p62 plays an important role in protecting cells against oxidative stress through Keap1 degradation-mediated Nrf2 activation.

Melanoma differentiation-associated gene 5 is involved in the induction of stress granules and autophagy by protonophore CCCP.[Pubmed:26351918]

Biol Chem. 2016 Jan;397(1):67-74.

The eukaryotic cell has evolved a variety of stress responses against external stimuli, such as innate immunity, the formation of stress granules (SGs), and autophagy. We previously demonstrated that the innate immune adaptor IFN-beta promoter stimulator 1 (IPS-1) plays an essential role in the formation of dsRNA-induced SGs, indicating a connection between SG formation and innate immunity. In this study, it was further demonstrated that melanoma differentiation-associated gene 5 (MDA5), an innate immune sensor, is involved in SG formation induced by carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial protonophore. MDA5 knockdown had no significant impact on the phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha) triggered by CCCP, and MDA5 itself was not recruited to SGs, suggesting that the regulation of MDA5 in the SG response occurs downstream of eIF2alpha. Furthermore, the depletion of MDA5 or G3BP1 led to reduced autophagy in CCCP-stimulated cells, implying that the regulatory effect of MDA5 with respect to autophagy depends on its role in SG formation. This study uncovered an unexpected role of the innate immune protein MDA5 in SG formation and autophagy triggered by the protonophore CCCP, further supporting a correlation between different stress responses.

Evidence that bacteriophage lambda lysogens may induce in response to the proton motive force uncoupler CCCP.[Pubmed:26705574]

FEMS Microbiol Lett. 2016 Feb;363(3). pii: fnv244.

We describe a genetic beta-galactoside reporter system using a disk diffusion assay on MacConkey Lactose agar petri plates to monitor maintenance of the bacteriophage lambda prophage state and viral induction in Escherichia coli K-12. Evidence is presented that the phage lambda major lytic promoters, pL and pR, are activated when cells containing the reporters are exposed to the energy poison carbonyl cyanide m-chlorophenyl hydrazine, CCCP. This uncoupler of oxidative phosphorylation inhibits ATP synthesis by collapsing the proton motive force. Expression of the lambda lytic promoters in response to CCCP requires host RecA function and an autocleavable CI repressor, as does SOS induction of the lambda prophage that occurs by a DNA damage-dependent pathway. lambda Cro function is required for CCCP-mediated activation of the lambda lytic promoters. CCCP does not induce an sfi-lacZ SOS reporter.

Carbonyl Cyanide m-Chlorophenylhydrazine (CCCP) Reverses Resistance to Colistin, but Not to Carbapenems and Tigecycline in Multidrug-Resistant Enterobacteriaceae.[Pubmed:28261184]

Front Microbiol. 2017 Feb 14;8:228.

Background: Carbapenems (CAR), colistin (CST), and tigecycline (TGC) alone or in combination therapy has become the last-resort antibiotics for treating infections caused by multidrug resistant (MDR) bacteria. However, resistance to these reserve antibiotics are increasingly being reported worldwide. Hence, the quest to find other agents that will synergistically restore the efficacy of these antibiotics have increased. Methods: Sixty-three clinical Enterobacteriaceae isolates comprising of Klebsiella pneumoniae (n = 24), Enterobacter spp. (n = 15), Serratia marcescens (n = 12), Citrobacter freundii (n = 8), Escherichia coli (n = 2), and K. oxytoca/michiganensis (n = 2) with known carbapenem resistance mechanisms and undescribed CST and TGC resistance mechanisms were subjected to broth microdilution and meropenem (MEM) disc synergy test in the presence and absence of carbonyl cyanide m-chlorophenylhydrazine (CCCP), a H(+) conductor (protonophore). Results and conclusions: Susceptibility to MEM, imipenem (IMP), CST, and TGC was found in only 2, 0, 17, and 9 isolates respectively. Addition of CCCP reversed resistance to CST, TGC, IMP, and MEM in 44, 3, 0, and 0 isolates respectively; CST had the highest mean minimum inhibitory concentration (MIC) fold change (193.12; p < 0.0001) post CCCP compared to that of MEM (1.70), IMP (1.49) and TGC (1.16). Eight isolates tested positive for the MEM-CCCP disc synergy test. We concluded that CCCP reverse CST resistance in CST-resistant Enterobacteriaceae. Although CCCP is an experimental agent with no therapeutic value clinically, further studies are necessary to decipher the mechanisms underlying the CST-CCCP synergy to inform the development of adjuvants that could be therapeutically effective in CST-resistant infections.