title: 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.
authors.affiliation: Department of Pharmacology, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: Brandon M
authors.initials: BM
authors.lastname: Kenwood
authors.affiliation: Department of Pharmacology, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: Janelle L
authors.initials: JL
authors.lastname: Weaver
authors.affiliation: Department of Medicine, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: Amandeep
authors.initials: A
authors.lastname: Bajwa
authors.affiliation: Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: Ivan K
authors.initials: IK
authors.lastname: Poon
authors.affiliation: Department of Pharmacology, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: Frances L
authors.initials: FL
authors.lastname: Byrne
authors.affiliation: Department of Pharmacology, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: Beverley A
authors.initials: BA
authors.lastname: Murrow
authors.affiliation: Department of Chemistry and Virginia Tech Center for Drug Discovery, Virginia Tech, Blacksburg, VA 24061, USA.
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Calderone
authors.affiliation: Department of Medicine, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: Liping
authors.initials: L
authors.lastname: Huang
authors.affiliation: Department of Pharmacology, University of California at San Diego, La Jolla, CA 92093, USA.
authors.forename: Ajit S
authors.initials: AS
authors.lastname: Divakaruni
authors.affiliation: Department of Pharmacology, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: Jose L
authors.initials: JL
authors.lastname: Tomsig
authors.affiliation: University of Tokyo, Tokyo, Japan.
authors.forename: Kohki
authors.initials: K
authors.lastname: Okabe
authors.affiliation: Department of Chemistry, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: Ryan H
authors.initials: RH
authors.lastname: Lo
authors.affiliation: Department of Pharmacology, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: G
authors.initials: G
authors.lastname: Cameron Coleman
authors.affiliation: Department of Chemistry, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: Linda
authors.initials: L
authors.lastname: Columbus
authors.affiliation: Department of Pharmacology, University of Virginia, Charlottesville, VA 22908, USA ; Department of Medicine, University of Virginia, Charlottesville, VA 22908, USA ; Department of Cardiovascular Research Center, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Yan
authors.affiliation: Department of Biomedical Engineering, University of Virginia, Charlottesville, VA 22908, USA ; Department of Cardiovascular Research Center, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Saucerman
authors.affiliation: Department of Biomedical Engineering, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: Jeffrey S
authors.initials: JS
authors.lastname: Smith
authors.affiliation: Department of Biomedical Engineering, University of Virginia, Charlottesville, VA 22908, USA ; Department of Cardiovascular Research Center, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: Jeffrey W
authors.initials: JW
authors.lastname: Holmes
authors.affiliation: Department of Pharmacology, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: Kevin R
authors.initials: KR
authors.lastname: Lynch
authors.affiliation: Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: Kodi S
authors.initials: KS
authors.lastname: Ravichandran
authors.affiliation: University of Tokyo, Tokyo, Japan.
authors.forename: Seiichi
authors.initials: S
authors.lastname: Uchiyama
authors.affiliation: Department of Chemistry and Virginia Tech Center for Drug Discovery, Virginia Tech, Blacksburg, VA 24061, USA.
authors.forename: Webster L
authors.initials: WL
authors.lastname: Santos
authors.affiliation: Seahorse Bioscience, North Billerica, MA 01862, USA.
authors.forename: George W
authors.initials: GW
authors.lastname: Rogers
authors.affiliation: Department of Medicine, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: Mark D
authors.initials: MD
authors.lastname: Okusa
authors.affiliation: Department of Pharmacology, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: Douglas A
authors.initials: DA
authors.lastname: Bayliss
authors.affiliation: Department of Pharmacology, University of Virginia, Charlottesville, VA 22908, USA ; Department of Medicine, University of Virginia, Charlottesville, VA 22908, USA ; Department of Cardiovascular Research Center, University of Virginia, Charlottesville, VA 22908, USA ; Emily Couric Clinical Cancer Center, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: Kyle L
authors.initials: KL
authors.lastname: Hoehn
keywords: ANT, adenine nucleotide translocase; Bioenergetics; CCCP; DNP; ECAR, extracellular acidification rate; FCCP; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; Ischemia; Mitochondria; OCR, oxygen consumption rate; ROS, reactive oxygen species; TCA cycle, tricarboxylic acid cycle; TMPD, N,N,N′,N′-tetramethyl-p-phenylenediamine dihydrochloride; TMRM, tetramethylrhodamine