Discovery of Novel Inhibitors of Cellular Efflux by High-Content Screening with a Fluorescent Mimic of Taxol

Abstract

Fluorescence-based assays play key roles in drug discovery and development. These assays are widely used due to the widespread availability of fluorescent probes and highly sensitive detection platforms. This method is a mainstay of high-throughput drug screening (HTS) campaigns, where simple and inexpensive assays are preferred for scalability and repeatability. This approach can identify novel chemotypes that may lead to new methods to treat disease. To develop a new phenotypic assay for drug discovery, we investigated a fluorescent mimic of the anticancer drug Taxol, termed Pacific Blue-Gly-taxol (PBGT). This molecular probe binds cellular microtubules and is a highly sensitive substrate of the cellular efflux transporter P-glycoprotein (P-gp). When HeLa cervical carcinoma cells are cotreated with PBGT (1 μM) and the P-gp inhibitor verapamil (25 μM), cellular fluorescence increases by ~ 10-fold as analyzed by confocal microscopy or flow cytometry. Because of the simplicity and sensitivity of this assay of P-gp activity, we envisioned that it could be optimized in a 96-well plate format to provide a useful method to investigate cellular efflux mediated by this protein transporter. To provide a proof of concept, 1584 diverse compounds obtained from the National Cancer Institute (NCI) were screened using automated pipetting and flow cytometry. The primary screen yielded more than 23 hit compounds with equivalent or of higher activity than verapamil (25 μM). Among these hits, we identified diarylureas that do not appear to associate directly with P-gp but rather disrupt the typical rod-like structure of mitochondria. These compounds may inhibit P-gp indirectly by affecting mitochondria or via a target that additionally affects this organelle. These results demonstrate that PBGT is a highly sensitive probe for discovery of inhibitors of P-gp and may allow identification of alternative mechanisms of inhibition of this major drug transporter.