A High-Throughput Screen of a Library of Therapeutics Identifies Cytotoxic Substrates of P-glycoprotein

The ATP-binding cassette transporter P-glycoprotein (P-gp) is known to limit both brain penetration and oral bioavailability of many chemotherapy drugs. Although Food and Drug Administration guidelines require that potential interactions of investigational drugs with P-gp be explored, often this information does not enter into the literature. As such, we developed a high- throughput screen (HTS) to identify substrates of P-gp from a series of chemical libraries, testing a total of 10,804 compounds, most of which have known mechanisms of action. We used the CellTiter-Glo viability assay to test library compounds against parental KB-3-1 human cervical adenocarcinoma cells and the colchicine-selected sub-line KB-8-5-11 that over-expresses P-gp. KB-8-5-11 cells were also tested in the presence of a P-gp inhibitor (tariquidar) to assess reversability of transporter-mediated resistance. Of the tested compounds, a total of 90 P-gp substrates were identified including 55 newly identified P-gp substrates. Substrates were confirmed using an orthogonal killing assay against HEK-293 cells overexpressing P-gp. We confirmed that AT7159 (cyclin-dependent kinase inhibitor); AT9283, (Janus kinase 2/3 inhibitor); ispinesib (kinesin spindle protein inhibitor); gedatolisib (PKI-587, phosphoinositide 3- kinase/mammalian target of rampamycin inhibitor); GSK-690693 (AKT inhibitor); and KW- 2478 (heat shock protein 90 inhibitor) were substrates, and direct ATPase stimulation was assessed. ABCG2 was also found to confer high levels of resistance to AT9283, GSK-690693 and gedatolisib, while ispinesib, AT7519 and KW-2478 were weaker substrates. Combinations of P-gp substrates and inhibitors were assessed to demonstrate on-target synergistic cell killing. These data identify compounds for which oral bioavailability or brain penetration may be affected by P-gp. SIGNIFICANCE STATEMENT The ATP-binding cassette transporter P-glycoprotein (P-gp) is known to be expressed at barrier sites where it acts to limit oral bioavailability and brain penetration of substrates. In order to identify novel compounds that are transported by P-gp, we developed a high-throughput screen using the KB-3-1 cancer cell line and its colchicine selected subline, KB-8-5-11. We screened the Mechanism Interrogation Plate (MIPE) library, the NCATS pharmaceutical collection (NPC), the NCATS Pharmacologically Active Chemical Toolbox (NPACT), and a kinase inhibitor library comprised of 977 compounds, for a total of 10,804 compounds. Of the 10,804 compounds screened, a total of 90 substrates were identified of which 55 were novel. P-gp expression may adversely affect the oral bioavailability or brain penetration of these compounds.