Identification of a novel mitochondrial uncoupler that does not depolarize the plasma membrane

Abstract

Dysregulation of oxidative phosphorylation is associated with increased mitochondrial reactive oxygen species production and some of the most prevalent human diseases including obesity, cancer, diabetes, neurodegeneration, and heart disease. Chemical 'mitochondrial uncouplers' are lipophilic weak acids that transport protons into the mitochondrial matrix via a pathway that is independent of ATP synthase, thereby uncoupling nutrient oxidation from ATP production. Mitochondrial uncouplers also lessen the proton motive force across the mitochondrial inner membrane and thereby increase the rate of mitochondrial respiration while decreasing production of reactive oxygen species. Thus, mitochondrial uncouplers are valuable chemical tools that enable the measurement of maximal mitochondrial respiration and they have been used therapeutically to decrease mitochondrial reactive oxygen species production. However, the most widely used protonophore uncouplers such as carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and 2,4-dinitrophenol have off-target activity at other membranes that lead to a range of undesired effects including plasma membrane depolarization, mitochondrial inhibition, and cytotoxicity. These unwanted properties interfere with the measurement of mitochondrial function and result in a narrow therapeutic index that limits their usefulness in the clinic. To identify new mitochondrial uncouplers that lack off-target activity at the plasma membrane we screened a small molecule chemical library. Herein we report the identification and validation of a novel mitochondrial protonophore uncoupler (2-fluorophenyl){6-[(2-fluorophenyl)amino](1,2,5-oxadiazolo[3,4-e]pyrazin-5-yl)}amine, named BAM15, that does not depolarize the plasma membrane. Compared to FCCP, an uncoupler of equal potency, BAM15 treatment of cultured cells stimulates a higher maximum rate of mitochondrial respiration and is less cytotoxic. Furthermore, BAM15 is bioactive in vivo and dose-dependently protects mice from acute renal ischemic-reperfusion injury. From a technical standpoint, BAM15 represents an effective new tool that allows the study of mitochondrial function in the absence of off-target effects that can confound data interpretation. From a therapeutic perspective, BAM15-mediated protection from ischemia-reperfusion injury and its reduced toxicity will hopefully reignite interest in pharmacological uncoupling for the treatment of the myriad of diseases that are associated with altered mitochondrial function.

SR4 Uncouples Mitochondrial Oxidative Phosphorylation, Modulates AMP-dependent Kinase (AMPK)-Mammalian Target of Rapamycin (mTOR) Signaling, and Inhibits Proliferation of HepG2 Hepatocarcinoma Cells

“…SR4 Increases Mitochondrial ROS Production-Previous studies have shown that some protonophore uncouplers can increase ROS production, whereas others do not (23,38,45). We assessed the ROS production in HepG2 cells using MitoSox TM Red fluorescence.…”

“…Mitochondrial swelling was measured in non-respiring mouse liver mitochondria incubated in buffered isotonic potassium acetate in the presence of valinomycin as described (37,38). Briefly, isolated liver mitochondria were added to 1 ml of isotonic acetate buffer (145 mM potassium acetate, 5 mM TrisHCl, 0.5 mM EDTA, 5 M valinomycin, and 1 M rotenone, pH 7.4) in a cuvette at a final concentration of 0.5 mg/ml mitochondrial protein.…”

“…Just before initiation of the XF assay, cells were washed twice with 200 l/well MAS buffer, permeabilized with 1.0 nM XF PMP (Seahorse Bioscience), and simultaneously provided 4 mM ADP, 10 mM succinate, and 1 M rotenone to obtain state 3 respiration, as described previously (38). The plate was immediately placed into the XF96 instrument, and the assay was initiated.…”

Sphingosine 1-Phosphate Receptor-1 Enhances Mitochondrial Function and Reduces Cisplatin-Induced Tubule Injury

“…54 TKPTS cells were seeded in Seahorse 24-well tissue culture plates at a density of 2.5310 4 cells/well and allowed to adhere for 24 hours. Primary cultures of TECs from kidney were grown to confluence on Seahorse 24-well tissue culture plates, as described above.…”

“…FCCP was used at two doses and monitored over time because it has a narrow dosing range in which it induces the maximal rate of mitochondrial respiration. 54 OCR was measured in 3-minute periods of time (over a total period of 2 hours). Basal mitochondrial respiration, ATP-linked respiration, proton leak (non-ATP-linked oxygen consumption), maximal respiration, nonmitochondrial respiration, reserve respiratory capacity, respiratory control ratio, and coupling efficiency were determined in whole cells according to Brand et al 29 Three to four wells were used for each experimental group.…”