Moreover, the ability of low concentrations of the inhibitors to significantly restore growth indicated the potential of this assay for development into a sensitive screen for M2 inhibitors. Progress in finding new inhibitors targeting drug resistant M2 channels has been slow but recent advances in understanding the structure and BML 111 dynamic properties of the M2 channel in a lipid bilayer environment, and the interaction of amantadine with the channel have spurred structure-based drug design and virtual screening efforts. Several new inhibitors were described in the last few years, many closely resembling amantadine. However, inhibitors targeting drug resistant M2 mutants have not been reported. A notable exception is the spiroadamantane amine shown in Fig. 2 that is active against WT, L26F and V27A M2. A robust and sensitive high-throughput screening assay would enable sampling of an expanded chemical diversity repertoire and guide SAR studies. In this study, we modified an assay described by Krystal et al and optimized it for high-throughput screening, notably by expressing M2 in a yeast strain with defective drug efflux systems, and by simplifying and automating assay conditions. The Arecaidine propargyl ester tosylate resulting growth restoration assay is robust, highly sensitive, quantitative, cost-effective and technically simple. We found that the yeast growth inhibition was greatest for WT, giving rise to a good Z�� factor for screening. Because a successful drug should inhibit WT M2 as well as amantadine-resistant mutated forms, we use WT in the primary screen followed by testing of active compounds against mutants in secondary assays. Our screen of over 250,000 pure chemicals and semi-purified fractions from natural extracts identified only 21 active compounds. This 0.008% hit rate is 10- to 100-fold lower than typically encountered in screens for other targets. The assay shows high selectivity for M2 channel inhibitors as further testing of the 21 active compounds revealed no false positives. In positive readout cell-based assays, high selectivity is imparted largely by the requirement for active compounds to both inhibit their target and not hamper cell growth, effectively eliminating many non-selective active chemicals. However, since we have observed higher hit rates in yeast growth restoration assays with other targets, the very low hit rate and the paucity of chemical classes identified indicates that M2 is not easily inhibited by the drug-like synthetic chemicals assembled in commercially available libraries. Natural products are known to encompass a much wider range of biologically relevant chemical space, so we were surprised that our large-scale screen of semi-purified fractions obtained from natural extracts did not yield a single hit. We do not believe this is due to technical factors such as testing of the fractions at too low a concentration to reveal activity.