FCCP

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FCCP
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Executive Summary Information

Compound FCCP (carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone)
Toxicities Cytotoxicity
Mechanisms FCCP is a protonophore that depletes the proton gradient of the mitochondrial membrane and uncouples respiration from ATP synthesis. FCCP is preferred as more specific in its activity compared to the common reagent, 2,4-dinitrophenol.
Comments This compound is an MOA standard for inhibition of mitochondrial function.
Feedback Contact Gold Compound Working Group (GCWG)
FCCP
FCCP.png


Identifiers
Leadscope Id LS-52242
CAS 370-86-5
ChemSpider 3213
Pathway DBs
Assay DBs
PubChem CID 3330
Omics DBs
Properties
ToxCast Accepted no
Toxic Effect Cytotoxicity
ToxBank Accepted yes
Approved on 20121023
Toxicities Cytotoxicity


In Vivo Data ? Compound Assessment
Adverse Events ? The related protonophore, 2,4-dinitrophenol, causes a dose-proportional wasting of energy and has been used in humans as a dietary aid. Its dose-limiting toxicity is not deficiency of ATP, but rather fatal hyperthermia caused by the heat generated from wasting energy.

References:

-Simkins S. (1937). "Dinitrophenol and desiccated thyroid in the treatment of obesity: a comprehensive clinical and laboratory study". J Am Med Assoc 108: 2110-2117.
-Tainter ML, Cutting WC, Stockton AB (1934). "Use of Dinitrophenol in Nutritional Disorders : A Critical Survey of Clinical Results". Am J Public Health 24 (10): 1045-1053.

Toxicity Mechanisms ? FCCP is a key tool for measuring mitochondrial (dys)function, appropriate to assessing the effect of other toxicants on mitochondrial function. In many cultured cells, there is sufficient capacity for glycolysis to maintain viability despite inhibition of oxidative phosphorylation. In these cases, treatment with oligomycin, and subsequently with FCCP, rotenone, mixothiazol, and ionophores, can be used to assess capacity for NADH formation, flux through the electron transport chain, and maintenance of the mitochondrial membrane potential. Brand and Nicholls (2011) provide an excellent review of the effects of oligomycin alone and in conjunction with other inhibitors on mitochondrial function.

References:

-Martin D. Brand and David G. Nicholls, "Assessing mitochondrial dysfunction in cells", Biochem. J. (2011) 435:297-312.

A study of the MOA of rotenone in mimicking Parkinson's disease in animal models exemplifies the use of FCCP in elucidating MOA. Rotenone causes cell death in neurons excited with glutamate, and the formation of reactive oxygen species has been cited as causing the cytotoxicity. However, glutamate also induces increased ATP utilization to maintain the ion gradient that is depleted by activation of the glutamate receptor. FCCP has been used as a tool to show that it is loss of capacity for ATP synthesis rather than reactive oxygen species that is cytotoxic. Similar conclusions were reached for MPP+-induced neurotoxicity.

References:

-Nagendra Yadava and David G. Nicholls, "Spare Respiratory Capacity Rather Than Oxidative Stress Regulates Glutamate Excitotoxicity after Partial Respiratory Inhibition of Mitochondrial Complex I with Rotenone", J. Neuroscience (2007) 27:7310 -7317.
-Johnson-Cadwell LI, Jekabsons MB, Wang A, Polster BM, Nicholls DG, "Mild Uncoupling' does not decrease mitochondrial superoxide levels in cultured cerebellar granule neurons but decreases spare respiratory capacity and increases toxicity to glutamate and oxidative stress", J Neurochem. (2007) 101:1619-31.
-Fonck C, Baudry M., "Rapid reduction of ATP synthesis and lack of free radical formation by MPP+ in rat brain synaptosomes and mitochondria", Brain Res. (2003) 975:214-21.

The ATP synthase reaction is close enough to equilibrium so that under conditions of ischemia, e.g. in cardiomyocytes, ATP synthase runs in reverse as an ATPase. Uncouplers such as FCCP mimic this effect so that dispersing the mitochondrial proton gradient not only blocks mitochondrial ATP synthesis but drives ATP synthase in the direction of ATP hydrolysis to actively deplete ATP, accounting for 50-80% of total ATP utilization. Since oligomycin blocks the ATPase activity of complex V, it is protective vs. ischemia or FCCP. Because oligomycin protects only against ATP depletion and not depletion of the proton gradient, it is concluded generally that cytotoxicity of inhibitors of oxidative phosphorylation reflects depletion of ATP and not depletion of the mitochondrial membrane potential.

References:

-Robert B. Jennings, Keith a. Reimer, and Charles Steenbergen, "Effect of Inhibition of the Mitochondrial ATPase on Net Mycocardial ATP in Total Ischemia", J Mol Cell Cardiol (1991) 23:1383-1395.
-A. L. Nieminen, A. K. Saylor, B. Herman, and J. J. Lemasters, "ATP depletion rather than mitochondrial depolarization mediates hepatocyte killing after metabolic inhibition", Am J Physiol Cell Physiol July 1, (1994) 267:C67-C74.
-Gary J. Grover, Karnail S. Atwal, Paul G. Sleph, Feng-Li Wang, Hossain Monshizadegan, Thomas Monticello, and David W. Green, "Excessive ATP hydrolysis in ischemic myocardium by mitochondrial F1F0-ATPase: effect of selective pharmacological inhibition of mitochondrial ATPase hydrolase activity", Am J Physiol Heart Circ Physiol 287: H1747-H1755 (2004).

The physical chemical effect of FCCP on membranes has been characterized in model systems.

The ATP synthase reaction is close enough to equilibrium so that under conditions of ischemia, e.g. in cardiomyocytes, ATP synthase runs in reverse as an ATPase. Uncouplers such as FCCP mimic this effect so that dispersing the mitochondrial proton gradient not only blocks mitochondrial ATP synthesis but drives ATP synthase in the direction of ATP hydrolysis to actively deplete ATP, accounting for 50-80% of total ATP utilization. Since oligomycin blocks the ATPase activity of complex V, it is protective vs. ischemia or FCCP. Because oligomycin protects only against ATP depletion and not depletion of the proton gradient, it is concluded generally that cytotoxicity of inhibitors of oxidative phosphorylation reflects depletion of ATP and not depletion of the mitochondrial membrane potential.

References:

-João P. Monteiro, André F. Martins, Marlene Lúcio, Salette Reis, Carlos F. G. C. Geraldes, Paulo J. Oliveira, and Amália S. Jurado, "Interaction of carbonylcyanide ptrifluoromethoxyphenylhydrazone (FCCP) with lipid membrane systems: a biophysical approach with relevance to mitochondrial uncoupling", J Bioenerg Biomembr (2011) 43:287-298.

FCCP mimics the effects of hypoxia on depleting cellular ion gradients, which results from the inability of mitochondria to supply energy for the maintenance of these gradients. However, FCCP generally inhibits rather than activates the hypoxia-sensitive HIF-1? transcriptional regulation system, which may reflect both loss of ATP for protein synthesis and phosphorylation and increased cellular O2 tension due to inhibition of oxidative phosphorylation.

References:

-K. J. Buckler and R. D. Vaughan-J, "Effects of mitochondrial uncouplers on intracellular calcium, pH and membrane potential in rat carotid body type I cells", Journal of Physiology (1998), 513:819-833.
-K.S. Park, I. Jo, K. Pak, S.W. Bae, H. Rhim, S.H. Suh, J. Park, H. Zhu, I. So, K.W. Kim, "FCCP depolarizes plasma membrane potential by activating proton and Na+ currents in bovine aortic endothelial cells", Pflugers Arch. 443 (2002) 344-352.
-Rusha Thomas and Myoung H. Kim, "Targeting the hypoxia inducible factor pathway with mitochondrial uncouplers", Mol Cell Biochem 296: 35-44, 2007.
-Minh-Son To, Edoardo C. Aromataris, Joel Castro, Michael L. Roberts, Greg J. Barritt, and Grigori Y. Rychkov, "Mitochondrial uncoupler FCCP activates proton conductance but does not block store-operated Ca2+ current in liver cells", Archives of Biochemistry and Biophysics 495 (2010) 152-158.

Low level FCCP inhibits activation of stellate cells by TGF?, suggesting that this compound may be a tool to understand why some but not all hepatic cytotoxins are pro-fibrotic.

References:

-Eduardo L Guimarães , Jan Best, Laurent Dollé, Mustapha Najimi, Etienne Sokal, Leo A van Grunsven, "Mitochondrial uncouplers inhibit hepatic stellate cell activation", BMC Gastroenterology 2012, 12:68.

FCCP is not specific to mitochondrial membranes and will also conduct protons across the plasma membrane. However, disruption of the mitochondrial gradient appears to dominate the cellular effects of FCCP

References:

-Minh-Son To, Edoardo C. Aromataris, Joel Castro, Michael L. Roberts, Greg J. Barritt, and Grigori Y. Rychkov, "Mitochondrial uncoupler FCCP activates proton conductance but does not block store-operated Ca2+ current in liver cells", Archives of Biochemistry and Biophysics 495 (2010) 152-158.

Therapeutic Target ? Uncoupling of respiration was the mechanism rationalizing the use of uncouplers as dietary aids, with uncontrolled wasting of energy leading to weight loss. This mechanism is still under investigation for its therapeutic utility.

References:

-Harper JA, Dickinson K, Brand MD (2001). "Mitochondrial uncoupling as a target for drug development for the treatment of obesity". Obesity reviews : an official journal of the International Association for the Study of Obesity 2 (4): 255-265.



PubMed references

The following resource link will perform a PubMed query for the terms "FCCP" and "mytochondrial function".
FCCP Search

References


PK-ADME ? Compound Assessment
PK parameters ? Not Applicable
Therapeutic window ? Acute administration of 20–50 mg/kg of a related compound, 2,4-dinitrophenol, in humans can be lethal. Since toxicity and efficacy arise from a common biochemical mechanism, the therapeutic window was narrow and had to be carefully titrated when this protonophore was used to treat obesity.

References:

-Hsiao AL, Santucci KA, Seo-Mayer P, et al. (2005). "Pediatric fatality following ingestion of dinitrophenol: postmortem identification of a "dietary supplement"". Clin Toxicol (Phila) 43 (4): 281–285.
-Simkins S. (1937). "Dinitrophenol and desiccated thyroid in the treatment of obesity: a comprehensive clinical and laboratory study". J Am Med Assoc 108: 2110–2117

Metabolically activated ? No

Omics and IC50 Data ? Compound Assessment
Gene expression profiles known. ? References:
-Sabu Kuruvilla, Charles W. Qualls, Jr., Ronald D. Tyler, Sam M. Witherspoon, Gina R. Benavides, Lawrence W. Yoon, Karen Dold, Roger H. Brown, Subbiah Sangiah, and Kevin T. Morgan, “Effects of Minimally Toxic Levels of Carbonyl Cyanide P-(Trifluoromethoxy) Phenylhydrazone (FCCP), Elucidated through Differential Gene Expression with Biochemical and Morphological Correlations”, Toxicological Sciences 73, 348–361 (2003).
-Rusha Thomas and Myoung H. Kim, “Targeting the hypoxia inducible factor pathway with mitochondrial uncouplers”, Mol Cell Biochem 296: 35–44, 2007.
-Eduardo L Guimarães , Jan Best, Laurent Dollé, Mustapha Najimi, Etienne Sokal, Leo A van Grunsven, “Mitochondrial uncouplers inhibit hepatic stellate cell activation”, BMC Gastroenterology 2012, 12:68.
-Robert Tripmacher, Timo Gaber, Ren Dziurla, Thomas Hupl, Kerem Erekul,Andreas Grtzkau, Miriam Tschirschmann, Alexander Scheffold, Andreas Radbruch, Gerd-Rdiger Burmester and Frank Buttgereit, “Human CD4+ T cells maintain specific functions even under conditions of extremely restricted ATP production”, Eur. J. Immunol. 2008. 38: 1631–1642.
-Sandra L. Haenninen, Jarkko J. Ronkainen, Hanna Leskinen, and Pasi Tavi,“Mitochondrial uncoupling downregulates calsequestrin expression and reduces SR Ca2+ stores in cardiomyocytes”, Cardiovascular Research (2010) 88, 75–82.
Proteomics profiles known. ? References:
-De Pauw A, Demine S, Tejerina S, Dieu M, Delaive E, Kel A, Renard P, Raes M, Arnould T., “Mild mitochondrial uncoupling does not affect mitochondrial biogenesis but downregulates pyruvate carboxylase in adipocytes: role for triglyceride content reduction.”, Am J Physiol Endocrinol Metab. 2012; 302:E1123-41
Metabonomics profiles known. ? References:
-Sabu Kuruvilla, et al., “Effects of Minimally Toxic Levels of Carbonyl Cyanide P-(Trifluoromethoxy) Phenylhydrazone (FCCP), Elucidated through Differential Gene Expression with Biochemical and Morphological Correlations”, Toxicological Sciences 73, 348–361 (2003).
-Balcke GU, Kolle SN, Kamp H, Bethan B, Looser R, Wagner S, Landsiedel R, van Ravenzwaay B., “Linking energy metabolism to dysfunctions in mitochondrial respiration--a metabolomics in vitro approach”, Toxicol Lett. (2011) 203:200-9
-Zhdanov AV, Favre C, O'Flaherty L, Adam J, O'Connor R, Pollard PJ, Papkovsky DB., “Comparative bioenergetic assessment of transformed cells using a cell energy budget platform”, Integr Biol (Camb). 2011 Nov;3(11):1135-42.
Fluxomics profiles known. ?
Epigenomics profiles known. ?
Observed IC50 for in vitro cellular efficacy. ? Not Applicable
Observed IC50 for in vitro cellular toxicity studies. ? Excellent review of use in vitro studies of mitochondrial function points out need to titrate FCCP levels to optimized for different cell types and conditions.

References:

-Martin D. Brand and David G. Nicholls, “Assessing mitochondrial dysfunction in cells”, Biochem. J. (2011) 435:297–312.

Depletes plasma membrane proton gradient in HEPG2 cells with IC50 = 7 uM

References:

-Minh-Son To, Edoardo C. Aromataris, Joel Castro, Michael L. Roberts, Greg J. Barritt, and Grigori Y. Rychkov, “Mitochondrial uncoupler FCCP activates proton conductance but does not block store-operated Ca2+ current in liver cells”, Archives of Biochemistry and Biophysics 495 (2010) 152–158.

Commonly used at 5-20 uM in gene expression studies.

References:

-Sabu Kuruvilla, et al., “Effects of Minimally Toxic Levels of Carbonyl Cyanide P-(Trifluoromethoxy) Phenylhydrazone (FCCP), Elucidated through Differential Gene Expression with Biochemical and Morphological Correlations”, Toxicological Sciences 73, 348–361 (2003).
-Rusha Thomas and Myoung H. Kim, “Targeting the hypoxia inducible factorpathway with mitochondrial uncouplers”, (Mol Cell Biochem 296: 35–44, 2007.
-Eduardo L Guimarães , et al. “Mitochondrial uncouplers inhibit hepatic stellate cell activation”,BMC Gastroenterology 2012, 12:68.

Physical Properties ? Compound Assessment
Accepted and listed within the ToxCast/Tox21 program. ? No - Not included in ToxCast Phase I and II Chemicals List
Substance stability. ? Stable in buffer and for at least 24 h in media. Rapidly depleted in cell culture, probably representing partitioning into membranes rather than degradation.

References:

-Sabu Kuruvilla, et al., “Effects of Minimally Toxic Levels of Carbonyl Cyanide P-(Trifluoromethoxy) Phenylhydrazone (FCCP), Elucidated through Differential Gene Expression with Biochemical and Morphological Correlations”, Toxicological Sciences 73, 348–361 (2003).
Soluble in buffer solution at 30 times the in vitro IC50 for toxicity. ?
estimated intrinsic solubility : 1.8862E-3 mg/ml


estimated solubility in pure water at pH 5.52: 2.6427E-3 mg/ml
estimated solubility in water at pH 7.4: 4.59E-2 mg/ml
Calculations performed using ACD/PhysChem v 12.0

Solubility in DMSO 100 times buffer solubility. ? Soluble to 100 mM in DMSO Tocris Bioscience (0453) Product details

Stock solutions in ethanol have been used for gene expression studies

References:

-Sabu Kuruvilla, et al., “Effects of Minimally Toxic Levels of Carbonyl Cyanide P-(Trifluoromethoxy) Phenylhydrazone (FCCP), Elucidated through Differential Gene Expression with Biochemical and Morphological Correlations”, Toxicological Sciences 73, 348–361 (2003).
Vessel binding properties. ?
Vapor pressure. (Non-volatile) ? estimated vapor pressure (25°C): 1.64E-006 mmHg (Calculation performed using EPI Suite v4.10)


Authors of this ToxBank wiki page

David Bower, Egon Willighagen
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