High-throughput screening (HTS) has been postulated in several quarters to be a contributory factor to the decline in productivity in the pharmaceutical industry. Moreover, it has been blamed for stifling the creativity that drug discovery demands. In this article, we aim to dispel these myths and present the case for the use of HTS as part of a proven scientific tool kit, the wider use of which is essential for the discovery of new chemotypes.
Transient receptor potential (TRP) channels have been found to play important roles in cellular physiology and hold promise as therapeutic targets. These channels activate in response to a variety of chemical or physical stimuli and conduct non-selective cation currents (NSCC). Due to their unique activation properties, application of automated electrophysiology to measure the channel activity has been difficult. Using HEK293 cells stably expressing human TRP channels, hTRPC6 and hTRPA1, we developed and validated a high-throughput Rb(+) efflux assay for NSCC channels. The assay was performed in cell-based 96-well format. A significant increase in Rb(+) efflux can be detected upon channel activation by specific agonists, confirming that both TRPC6 and TRPA1 channels are permeable to Rb(+) ions. The agonists induced Rb(+) efflux can be blocked by known channel blockers and selected compounds from our high-throughput screening (HTS) hits. The assay is suitable for HTS with Z' factors of 0.53 and above. We also tested the Ca(2+) effect on channel activities in this assay. Both TRPC6 and TRPA1 channels were found to be inhibited by increasing the concentration of Ca(2+) in the assay buffer. However, Ca(2+) significantly reduced the potency of allyl isothiocyanate (AITC) on TRPA1 but did not affect the potency of carbochol on TRPC6. Using this assay for secondary confirmation screen, we successfully identified and confirmed the positive hits as TRPC6 inhibitors.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.