Development of an assay for Complex I/Complex III of the respiratory chain using solid supported membranes and its application in mitochondrial toxicity screening in drug discovery.

Membrane-bound transporter proteins are involved in cell signal transduction and metabolism as well as influencing key pharmacological properties such as drug bioavailability. The functional activity of transporters that belong to the group of electrically active membrane proteins can be directly monitored using the solid-supported membrane-based SURFE(2)R™ technology (SURFace Electrogenic Event Reader; Scientific Devices Heidelberg GmbH, Heidelberg, Germany). The method makes use of membrane fragments or vesicles containing transport proteins adsorbed onto solid-supported membrane-covered electrodes and allows the direct measurement of their activity. This technology has been used to develop a robust screening compatible assay for Complex I/Complex III, key components of the respiratory chain in 96-well microtiter plates. The assay was screened against 1,000 compounds from the ComGenex Lead-like small molecule library to ascertain whether mitochondrial liabilities might be an underlying, although undesirable feature of typical commercial screening libraries. Some 105 hits (compounds exhibiting >50% inhibition of Complex I/Complex III activity at 10 µM) were identified and their activities were subsequently confirmed in duplicate, yielding a confirmation rate of 68%. Analysis of the confirmed hits also provided evidence of structure-activity relationships and two compounds from one structural class were further evaluated in dose-response experiments. This study provides evidence that profiling of compounds for potential mitochondrial liabilities, even at an early stage of drug discovery, may be a necessary additional quality filter that should be considered during the compound screening and profiling cascade.

Electrophysiological characterization of ATPases in native synaptic vesicles and synaptic plasma membranes.

Vesicular V-ATPase (V-type H+-ATPase) and the plasma membrane-bound Na+/K+-ATPase are essential for the cycling of neurotransmitters at the synapse, but direct functional studies on their action in native surroundings are limited due to the poor accessibility via standard electrophysiological equipment. We performed SSM (solid supported membrane)-based electrophysiological analyses of synaptic vesicles and plasma membranes prepared from rat brains by sucrose-gradient fractionation. Acidification experiments revealed V-ATPase activity in fractions containing the vesicles but not in the plasma membrane fractions. For the SSM-based electrical measurements, the ATPases were activated by ATP concentration jumps. In vesicles, ATP-induced currents were inhibited by the V-ATPase-specific inhibitor BafA1 (bafilomycin A1) and by DIDS (4,4'-di-isothiocyanostilbene-2,2'-disulfonate). In plasma membranes, the currents were inhibited by the Na+/K+-ATPase inhibitor digitoxigenin. The distribution of the V-ATPase- and Na+/K+-ATPase-specific currents correlated with the distribution of vesicles and plasma membranes in the sucrose gradient. V-ATPase-specific currents depended on ATP with a K0.5 of 51+/-7 microM and were inhibited by ADP in a negatively co-operative manner with an IC50 of 1.2+/-0.6 microM. Activation of V-ATPase had stimulating effects on the chloride conductance in the vesicles. Low micromolar concentrations of DIDS fully inhibited the V-ATPase activity, whereas the chloride conductance was only partially affected. In contrast, NPPB [5-nitro-2-(3-phenylpropylamino)-benzoic acid] inhibited the chloride conductance but not the V-ATPase. The results presented describe electrical characteristics of synaptic V-ATPase and Na+/K+-ATPase in their native surroundings, and demonstrate the feasibility of the method for electrophysiological studies of transport proteins in native intracellular compartments and plasma membranes.