title: Measuring interference of drug-like molecules with the respiratory chain: toward the early identification of mitochondrial uncouplers in lead finding.
abstract: The electron transport chain (ETC) couples electron transfer between donors and acceptors with proton transport across the inner mitochondrial membrane. The resulting electrochemical proton gradient is used to generate chemical energy in the form of adenosine triphosphate (ATP). Proton transfer is based on the activity of complex I-V proteins in the ETC. The overall electrical activity of these proteins can be measured by proton transfer using Solid Supported Membrane technology. We tested the activity of complexes I, III, and V in a combined assay, called oxidative phosphorylation assay (oxphos assay), by activating each complex with the corresponding substrate. The oxphos assay was used to test in-house substances from different projects and several drugs currently available on the market that have reported effects on mitochondrial functions. The resulting data were compared to the influence of the respective compounds on mitochondria as determined by oxygen consumption and to data generated with an ATP depletion assay. The comparison shows that the oxidative phosphorylation assay provides both a rapid approach for detecting interaction of compounds with respiratory chain proteins and information on their mode of interaction. Therefore, the oxphos assay is a useful tool to support structure activity relationship studies by allowing early identification of mitotoxicity and for analyzing the outcome of phenotypic screens that are susceptible to the generation of mitotoxicity-related artifacts.
authors.affiliation: 1 R&D LGCR, Sanofi-Aventis Deutschland GmbH, Frankfurt, Germany .
authors.forename: Ursula
authors.initials: U
authors.lastname: Stock
authors.forename: Hans
authors.initials: H
authors.lastname: Matter
authors.forename: Kerstin
authors.initials: K
authors.lastname: Diekert
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Dörner
authors.forename: Stefan
authors.initials: S
authors.lastname: Dröse
authors.forename: Thomas
authors.initials: T
authors.lastname: Licher
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D015374:Biosensing Techniques / Q000379:methods; D002478:Cells, Cultured; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D045222:Electron Transport Chain Complex Proteins / Q000187:drug effects* / Q000378:metabolism*; D017022:Flow Injection Analysis / Q000379:methods; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects* / Q000502:physiology*; D008928:Mitochondria; D004364:Pharmaceutical Preparations / Q000008:administration & dosage*; D051381:Rats; D013552:Swine
chemical_list: D045222:Electron Transport Chain Complex Proteins; D004364:Pharmaceutical Preparations