High Throughput Gramicidin-Based Fluorescence Assay to Screen for Small Molecules' Bilayer-Perturbing Potential

Abstract

Many small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter lipid bilayer properties. Such changes in bilayer properties can lead to altered membrane protein function due to the hydrophobic coupling between the host bilayer and its embedded proteins. Amphiphile-induced changes in lipid bilayer properties may, therefore, provide a mechanistic basis for the off target effects of drugs and other biologically active molecules. We have previously developed electrophysiological assays for changes in lipid bilayer properties, as sensed by bilayer-spanning proteins, using the channels formed by the linear gramicidins as probes. Gramicidin channels are mini-proteins formed by the transbilayer dimerization of two non-conducting subunits; they are sensitive to changes in their membrane environment, which renders them excellent probes for monitoring changes in bilayer properties. We now report a fluorescence assay for detecting changes in bilayer properties, using the linear gramicidins as probes. The assay is based on measuring the time course of fluorescence quenching from fluorophore-loaded large unilamellar vesicles, due to the entry of a quencher through the gramicidin channels. The fluorescence method presented is scalable and suitable for both mechanistic studies and high-throughput screening of small molecules for bilayer-perturbing, and potential off-target, effect. To illustrate the validity and power of this approach, we have tested compounds with bilayer-modifying effects that previously have been characterized using the electrophysiological (single-channel) gramicidin approach. We find that the methods are in good agreement. We also have undertaken a systematic study of the bilayer-perturbing effect of short- and intermediate-chain length alcohols (methanol through n-octanol, isopropanol, 2-butanol and tert-butanol) as well the fluorinated alcohols (trifluoroethyl alcoho, hexafluoroisopropanol and nonafluoro-tert-butyl alcohol). These compounds alter lipid bilayers properties at the concentrations at which they alter membrane protein function.