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

Tobie D. Lee, Olivia W. Lee, Kyle R. Brimacombe, Lu Chen, Rajarshi Guha, Sabrina Lusvarghi, Bethilehem G. Tebase, Carleen Klumpp-Thomas, Robert W. Robey, Suresh V. Ambudkar, Min Shen, Michael M. Gottesman, View ORCID ProfileMatthew D. Hall
doi: https://doi.org/10.1101/528992
Tobie D. Lee
National Center for Advancing Translational Sciences (T.D.L., O.W.L, K.R.B, L.C., R.G., C.K.-L., M.S., M.D.H.) National Institutes of Health, Rockville MD
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Olivia W. Lee
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Kyle R. Brimacombe
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Lu Chen
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Rajarshi Guha
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Sabrina Lusvarghi
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Bethilehem G. Tebase
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Carleen Klumpp-Thomas
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Robert W. Robey
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Suresh V. Ambudkar
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Min Shen
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Michael M. Gottesman
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Matthew D. Hall
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  • ORCID record for Matthew D. Hall
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Abstract

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 abbreviations
P-gp
P-glycoprotein
BBB
blood-brain barrier
HTS
high-throughput screen
ABCB1
ATP-binding cassette family member B1
ABCG2
ATP-binding cassette family member G2
NCATS
National Center for Advancing Translational Science
NPC
NCATS Pharmaceutical Collection
MIPE
Mechanism Interrogation Plate
NPACT
NCATS Pharmacologically Active Chemical Toolbox
PhA
pheophorbide A
FTC
Fumitremorgin C
AUC
area under the curve
NAMPT
nicotinamide phosphoribosyltransferase
CDK
cyclin-dependent kinase
PI3K
phosphoinositide-3 kinase
mTOR
mammalian target of rapamycin
JAK
Janus kinase
Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted January 23, 2019.
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A High-Throughput Screen of a Library of Therapeutics Identifies Substrates of P-glycoprotein
Tobie D. Lee, Olivia W. Lee, Kyle R. Brimacombe, Lu Chen, Rajarshi Guha, Sabrina Lusvarghi, Bethilehem G. Tebase, Carleen Klumpp-Thomas, Robert W. Robey, Suresh V. Ambudkar, Min Shen, Michael M. Gottesman, Matthew D. Hall
bioRxiv 528992; doi: https://doi.org/10.1101/528992
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A High-Throughput Screen of a Library of Therapeutics Identifies Substrates of P-glycoprotein
Tobie D. Lee, Olivia W. Lee, Kyle R. Brimacombe, Lu Chen, Rajarshi Guha, Sabrina Lusvarghi, Bethilehem G. Tebase, Carleen Klumpp-Thomas, Robert W. Robey, Suresh V. Ambudkar, Min Shen, Michael M. Gottesman, Matthew D. Hall
bioRxiv 528992; doi: https://doi.org/10.1101/528992

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