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

Shinobu Ohnuma, Eduardo Chufan, Krishnamachary Nandigama, Lisa M.Miller Jenkins, Stewart R. Durell, Ettore Appella, Zuben E. Sauna, Suresh V. Ambudkar

Research output: Contribution to journalArticle

12 Citations (Scopus)

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 (IC50= 0.21 mM) and the binding of 8-azido[α-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.

Original languageEnglish
Pages (from-to)3724-3735
Number of pages12
JournalBiochemistry
Volume50
Issue number18
DOIs
Publication statusPublished - 2011 May 10

ASJC Scopus subject areas

  • Biochemistry

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