title: Identification of new MRP4 inhibitors from a library of FDA approved drugs using a high-throughput bioluminescence screen.
abstract: Multidrug resistance protein 4 (MRP4) effluxes a wide variety of drugs and endogenous signaling molecules from cells and has been proposed as an attractive therapeutic target in several solid tumors, including neuroblastoma and colorectal cancer. MRP4 also regulates the pharmacokinetics of its drug substrates and its absence can increase their tissue penetration. We observed that MRP4 can efflux the bioluminescence substrate d-luciferin, and exploited this phenomenon to develop a robust, high throughput, live cell-based bioluminescent screen to identify new MRP4 inhibitors. We applied this screen to a combined library of 3600 compounds, all of which were either FDA-approved drugs or bioactive compounds with defined mechanisms of action. From the primary screen, 36 compounds effectively inhibited MRP4 (>4-fold increase in bioluminescence), with inhibitors of receptor tyrosine kinases and phosphodiesterases highly over-represented. Selected compounds were tested for their ability to sensitize MRP4-overexpressing cell lines to the MRP4 substrate drugs 6-mercaptopurine and SN-38, with sensitization up to 6.5-fold with the ryanodine receptor antagonist dantrolene. These newly identified MRP4 inhibitors are readily available and are either established drugs or well-characterized bioactive compounds. As such, they should be immediately useful as investigative tools, and suitable for testing both in vitro and in vivo.
authors.affiliation: Children's Cancer Institute Australia, Lowy Cancer Research Centre, Randwick, NSW, Australia. Electronic address: lcheung@ccia.unsw.edu.au.
authors.forename: Leanna
authors.initials: L
authors.lastname: Cheung
authors.affiliation: Children's Cancer Institute Australia, Lowy Cancer Research Centre, Randwick, NSW, Australia. Electronic address: dyu@ccia.unsw.edu.au.
authors.forename: Denise M T
authors.initials: DM
authors.lastname: Yu
authors.affiliation: Children's Cancer Institute Australia, Lowy Cancer Research Centre, Randwick, NSW, Australia. Electronic address: zneiron@hotmail.com.
authors.forename: Zillan
authors.initials: Z
authors.lastname: Neiron
authors.affiliation: Children's Cancer Institute Australia, Lowy Cancer Research Centre, Randwick, NSW, Australia; ACRF Drug Discovery Centre for Childhood Cancer, Lowy Cancer Research Centre, Randwick, NSW, Australia. Electronic address: TFailes@ccia.org.au.
authors.forename: Tim W
authors.initials: TW
authors.lastname: Failes
authors.affiliation: Children's Cancer Institute Australia, Lowy Cancer Research Centre, Randwick, NSW, Australia; ACRF Drug Discovery Centre for Childhood Cancer, Lowy Cancer Research Centre, Randwick, NSW, Australia. Electronic address: GArndt@ccia.org.au.
authors.forename: Greg M
authors.initials: GM
authors.lastname: Arndt
authors.affiliation: Children's Cancer Institute Australia, Lowy Cancer Research Centre, Randwick, NSW, Australia. Electronic address: jfletcher@ccia.unsw.edu.au.
authors.forename: Jamie I
authors.initials: JI
authors.lastname: Fletcher
mesh_terms: D002166:Camptothecin / Q000031:analogs & derivatives / Q000032:analysis / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D017277:Drug Approval; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D000077146:Irinotecan; D008156:Luciferases / Q000032:analysis*; D008163:Luminescent Measurements / Q000379:methods*; D015122:Mercaptopurine / Q000032:analysis / Q000494:pharmacology; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D054852:Small Molecule Libraries / Q000032:analysis* / Q000494:pharmacology; D014481:United States
chemical_list: D002166:Camptothecin; D000077146:Irinotecan; D008156:Luciferases; D015122:Mercaptopurine; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D054852:Small Molecule Libraries; C073492:ABCC4 protein, human
keywords: 6-mercaptopurine (PubChem CID: 667490); ABCC4; Bioluminescence; High-throughput screening; MK-571 (PubChem CID: 5281888); MRP4 inhibitor; Multidrug resistance; SN-38 (PubChem CID: 104842); d-luciferin (PubChem CID: 6800291); dantrolene (PubChem CID: 6604100); estradiol 17-beta-d-glucuronide (PubChem CID: 5281887); glafenine hydrochloride (PubChem CID: 3085326).; indomethacin (PubChem CID: 3715); nalidixic acid (PubChem CID: 3864541); prazosin hydrochloride (PubChem CID: 68546); tyrphostin AG 1478 (PubChem CID: 2051)