title: Inhibition of multidrug resistance-linked P-glycoprotein (ABCB1) function by 5'-fluorosulfonylbenzoyl 5'-adenosine: evidence for an ATP analogue that interacts with both drug-substrate-and nucleotide-binding sites.
abstract: 5'-Fluorosulfonylbenzonyl 5'-adenosine (FSBA) is an ATP analogue that covalently modifies several residues in the nucleotide-binding domains (NBDs) of several ATPases, kinases, and other proteins. P-glycoprotein (P-gp, ABCB1) is a member of the ATP-binding cassette (ABC) transporter superfamily that utilizes energy from ATP hydrolysis for the efflux of amphipathic anticancer agents from cancer cells. We investigated the interactions of FSBA with P-gp to study the catalytic cycle of ATP hydrolysis. Incubation of P-gp with FSBA inhibited ATP hydrolysis (IC(50 )= 0.21 mM) and the binding of 8-azido[α-(32)P]ATP (IC(50) = 0.68 mM). In addition, (14)C-FSBA cross-links to P-gp, suggesting that FSBA-mediated inhibition of ATP hydrolysis is irreversible due to covalent modification of P-gp. However, when the NBDs were occupied with a saturating concentration of ATP prior to treatment, FSBA stimulated ATP hydrolysis by P-gp. Furthermore, FSBA inhibited the photo-cross-linking of P-gp with [(125)I]iodoarylazidoprazosin (IAAP; IC(50) = 0.17 mM). As IAAP is a transport substrate for P-gp, this suggests that FSBA affects not only the NBDs but also the transport-substrate site in the transmembrane domains. Consistent with these results, FSBA blocked efflux of rhodamine 123 from P-gp-expressing cells. Additionally, mass spectrometric analysis identified FSBA cross-links to residues within or nearby the NBDs but not in the transmembrane domains, and docking of FSBA in a homology model of human P-gp NBDs supports the biochemical studies. Thus, FSBA is an ATP analogue that interacts with both the drug-binding and ATP-binding sites of P-gp, but fluorosulfonyl-mediated cross-linking is observed only at the NBDs.
authors: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4256, USA.
authors: Shinobu
authors: S
authors: Ohnuma
authors: Eduardo
authors: E
authors: Chufan
authors: Krishnamachary
authors: K
authors: Nandigama
authors: Lisa M Miller
authors: LM
authors: Jenkins
authors: Stewart R
authors: SR
authors: Durell
authors: Ettore
authors: E
authors: Appella
authors: Zuben E
authors: ZE
authors: Sauna
authors: Suresh V
authors: SV
authors: Ambudkar
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry*; D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000737:chemistry*; D000345:Affinity Labels / Q000494:pharmacology*; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods; D009711:Nucleotides / Q000737:chemistry; D011485:Protein Binding; D021381:Protein Transport
chemical_list: C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000345:Affinity Labels; D003432:Cross-Linking Reagents; D009711:Nucleotides; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D000255:Adenosine Triphosphate; D000241:Adenosine