Inhibition of Multidrug Resistance-Linked P-Glycoprotein (ABCB1) Function by 5鈥?Fluorosulfonylbenzoyl 5鈥?Adenosine: Evidence for an ATP Analogue That Interacts with Both Drug-Substrate-and Nucleotide-

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• 作者：Shinobu Ohnuma ; Eduardo Chufan ; Krishnamachary Nandigama ; Lisa M. Miller Jenkins ; Stewart R. Durell ; Ettore Appella ; Zuben E. Sauna ; Suresh V. Ambudkar
• 刊名：Biochemistry
• 出版年：2011
• 出版时间：May 10, 2011
• 年：2011
• 卷：50
• 期：18
• 页码：3724-3735
• 全文大小：1115K
• 年卷期：v.50,no.18(May 10, 2011)
• ISSN：1520-4995

文摘

5鈥?Fluorosulfonylbenzonyl 5鈥?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 (IC₅₀ = 0.21 mM) and the binding of 8-azido[伪-³²P]ATP (IC₅₀ = 0.68 mM). In addition, ¹⁴C-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 [¹²⁵I]iodoarylazidoprazosin (IAAP; IC₅₀ = 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.