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.

Development and characterization of mitochondrial membrane affinity chromatography columns derived from skeletal muscle and platelets for the study of mitochondrial transmembrane proteins

“…Figure 2C shows that oligomycin A inhibits the hydrolysis of on-column ATP. Further, a series of concentrations of oligomycin A in the mobile phase were run on the MMAC-Muscle column and an IC 50 of 40.2 ± 13.5 nM (Figure 3A) with a maximum reduction of ~60% in ATP hydrolysis was observed, p<0.001, which is consistent with previously reported IC 50 values [20]. While it was clearly demonstrated that hydrolytic activity of F-ATPase/ATP synthase activity was present, a series of concentrations of ADP [1.56 uM to 8 mM] were run on the MMAC-Muscle column to determine whether the ATP synthase activity was also retained.…”

Antiinfectives targeting enzymes and the proton motive force

“…Although targeting the PMF might be expected to be purely mitotoxic, Stock et al (26) have shown that compounds with logP > 6 have generally low mitotoxicity, which is due, they proposed, to low membrane permeability attributable to accumulation in lipophilic membranes.…”

A Solid Supported Membrane-Based Technology for Electrophysical Screening of B0AT1-Modulating Compounds

“…17 However, early generations of SSM-based electrophysiology devices offered only a limited degree of automation and sample output, and thus were not applicable for large-scale profiling purposes. 18–20 The current generation of SSM-based devices, the Surfe 2 r 96SE, an automated electrophysiology-based high-throughput instrument, allows simultaneous measurement of up to 96 sensors as well as buffer exchange and optional automated sensor preparation with the pipetting unit, significantly increasing assay performance—a prerequisite for medium- to high-throughput approaches.…”