PT - JOURNAL ARTICLE AU - Tobie D. Lee AU - Olivia W. Lee AU - Kyle R. Brimacombe AU - Lu Chen AU - Rajarshi Guha AU - Sabrina Lusvarghi AU - Bethilehem G. Tebase AU - Carleen Klumpp-Thomas AU - Robert W. Robey AU - Suresh V. Ambudkar AU - Min Shen AU - Michael M. Gottesman AU - Matthew D. Hall TI - A High-Throughput Screen of a Library of Therapeutics Identifies Substrates of P-glycoprotein AID - 10.1101/528992 DP - 2019 Jan 01 TA - bioRxiv PG - 528992 4099 - http://biorxiv.org/content/early/2019/01/23/528992.short 4100 - http://biorxiv.org/content/early/2019/01/23/528992.full AB - The ATP-binding cassette transporter P-glycoprotein (P-gp) is known to limit brain penetration 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 transfected with 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. This data will be of use in determining understanding how chemotherapeutic agents will cross the blood-brain barrier.Non-standard abbreviationsP-gpP-glycoproteinBBBblood-brain barrierHTShigh-throughput screenABCB1ATP-binding cassette family member B1ABCG2ATP-binding cassette family member G2NCATSNational Center for Advancing Translational ScienceNPCNCATS Pharmaceutical CollectionMIPEMechanism Interrogation PlateNPACTNCATS Pharmacologically Active Chemical ToolboxPhApheophorbide AFTCFumitremorgin CAUCarea under the curveNAMPTnicotinamide phosphoribosyltransferaseCDKcyclin-dependent kinasePI3Kphosphoinositide-3 kinasemTORmammalian target of rapamycinJAKJanus kinase