Cysteines Introduced into Extracellular Loops 1 and 4 of Human P-Glycoprotein That Are Close Only in the Open Conformation Spontaneously Form a Disulfide Bond That Inhibits Drug Efflux and ATPase Activity

Loo, Tip W.; Clarke, David M.

doi:10.1074/jbc.m114.583021

Cited by 13 publications

(11 citation statements)

References 59 publications

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“…Cys-less P-gp mutants containing pairs of cross-linkable cysteines in the TM segments (A80C(TM1)/R741C(TM7), I299C(TM5)/F770C(TM8), and T333C(TM6)/L975C(TM12)), intracellular loops (ICLs) (L175C(ICL1)/N820C(ICL3), A259C(ICL2)/W803C(ICL3), and A266C(ICL2)/F1086C(NBD2)), or the NBDs (C431(NBD1)/L1176C(NBD2), L531C(NBD1)/C1074(NBD2), and P517C(NBD1)/I1050C(NBD2)) were transiently expressed in HEK 293 cells at reduced temperature (30 °C) to promote maturation. Cells expressing mutant A80C(TM1)/R741C(TM7) were washed with PBS containing 10 m m dithiothreitol before membrane preparation because the mutant cross-links spontaneously ( 29 ). Membranes were prepared and samples were incubated at 0 °C (A80C/R741C, A259C/W803C, I299C/F770C, and A266C/F1086C) or 20 °C (L175C/N820C, C431/L1176C, and L521C/C1074) for 10 min in the presence or absence of 0.5 m m copper phenanthroline (oxidant to promote disulfide bond formation) in the presence or absence of 0.25 μ m (for TM segment cysteine mutants) or 1 μ m tariquidar (for the ICL and NBD cysteine mutants).…”

Section: Methodsmentioning

confidence: 99%

Mapping the Binding Site of the Inhibitor Tariquidar That Stabilizes the First Transmembrane Domain of P-glycoprotein

Loo

Clarke

2015

Journal of Biological Chemistry

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ABC (ATP-binding cassette) transporters are clinically important because drug pumps like P-glycoprotein (P-gp, ABCB1) confer multidrug resistance and mutant ABC proteins are responsible for many protein-folding diseases such as cystic fibrosis. Identification of the tariquidar-binding site has been the subject of intensive molecular modeling studies because it is the most potent inhibitor and corrector of P-gp. Tariquidar is a unique P-gp inhibitor because it locks the pump in a conformation that blocks drug efflux but activates ATPase activity. In silico docking studies have identified several potential tariquidar-binding sites. Here, we show through cross-linking studies that tariquidar most likely binds to sites within the transmembrane (TM) segments located in one wing or at the interface between the two wings (12 TM segments form 2 divergent wings). We then introduced arginine residues at all positions in the 12 TM segments (223 mutants) of P-gp. The rationale was that a charged residue in the drug-binding pocket would disrupt hydrophobic interaction with tariquidar and inhibit its ability to rescue processing mutants or stimulate ATPase activity. Arginines introduced at 30 positions significantly inhibited tariquidar rescue of a processing mutant and activation of ATPase activity. The results suggest that tariquidar binds to a site within the drug-binding pocket at the interface between the TM segments of both structural wings. Tariquidar differed from other drug substrates, however, as it stabilized the first TM domain. Stabilization of the first TM domain appears to be a key mechanism for high efficiency rescue of ABC processing mutants that cause disease.

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Section: Methodsmentioning

confidence: 99%

Mapping the Binding Site of the Inhibitor Tariquidar That Stabilizes the First Transmembrane Domain of P-glycoprotein

Loo

Clarke

2015

Journal of Biological Chemistry

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“…Indeed, the assumption that restraining TMH motions can impair, at least in part, the conformational changes leading to efflux was corroborated by three almost simultaneous experiments. Loo and Clarke inserted cysteine residues at specific positions in two extracellular loops, ECL1 (A80C) and ECL4 (R741C), to discover that a spontaneous disulfide bond was formed, which inhibited not only protein maturation but also its verapamil‐stimulated ATPase activity if rescued in the presence of a drug substrate . Kodan et al were able to inhibit both basal‐ and Rhodamine 6G‐stimulated ATPase activities by clamping together the extracellular TMH2 and TMH6 portions of a homologous transporter (CmABCB1) with a specifically tailored cyclic peptide, aCAP, with 46‐fold more affinity to the transporter than the substrate itself .…”

Section: Drug Recognitionmentioning

confidence: 99%

About P‐glycoprotein: a new drugable domain is emerging from structural data

Ferreira

Bonito

Ferreira

et al. 2017

WIREs Comput Mol Sci

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P‐glycoprotein (P‐gp) has been considered an important molecular target in the reversal of multidrug resistance (MDR). As such, the development of P‐gp modulators able to restore drug sensitivity in resistant cells is still considered one of the most promising strategies for overcoming MDR. Since the identification of the P‐gp's role in MDR, several studies have been performed in order to develop effective P‐gp modulators and understand the efflux mechanism. However, no efflux modulator is still clinically available for treating multidrug‐resistant cancers. Nevertheless, recent experimental studies suggest that MDR can be surpassed by targeting a specific region within the ABC transporter structure rather than the polyspecific drug‐binding pocket. This article will focus on the information available about this new target region and on a brief overview of which scaffolds would be suitable for modulating P‐gp at this new location. WIREs Comput Mol Sci 2017, 7:e1316. doi: 10.1002/wcms.1316 This article is categorized under: Structure and Mechanism > Computational Biochemistry and Biophysics

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“…The major members of the ABC transporters leading to MDR in cancer cells include ABCB1 (P-glycoprotein, P-gp) and ABCG2 (breast cancer resistance protein, BCRP, mitoxantrone resistance protein, MXR, and placenta-specific ABC transporter, ABCP), which mediate the substrate agents effluxing out of the cells against a concentration gradient coupled to the energy of ATP hydrolysis (9). ABCB1, coded by ABCB1 gene, is composed of two homologous halves, each containing six transmembrane domains and an ATP binding/utilization domain, separated by a flexible polypeptide linker (10). ABCB1 renders MDR in cancer cells by transporting a wide spectrum of compounds including the vinca alkaloids [vinblastine and vincristine (VCR)], the anthracyclines [doxorubicin (DOX) and daunorubicin], epipodophyllotoxins, taxanes, and some tyrosine kinase inhibitors (TKI) out of the cells (11,12).…”

Section: Introductionmentioning

confidence: 99%

Osimertinib (AZD9291) Enhanced the Efficacy of Chemotherapeutic Agents in ABCB1- and ABCG2-Overexpressing Cells In Vitro, In Vivo, and Ex Vivo

Chen

et al. 2016

Molecular Cancer Therapeutics

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The overexpression of ATP-binding cassette (ABC) transporters has been proved to be a major trigger for multidrug resistance (MDR) in certain types of cancer. In our study, we investigated whether osimertinib (AZD9291), a third-generation irreversible tyrosine kinase inhibitor of both activating EGFR mutations and resistance-associated T790M point mutation, could reverse MDR induced by ABCB1 and ABCG2 in vitro, in vivo, and ex vivo. Our results showed that osimertinib significantly increased the sensitivity of ABCB1-and ABCG2-overexpressing cells to their substrate chemotherapeutic agents in vitro and in the model of ABCB1-overexpressing KBv200 cell xenograft in nude mice. Mechanistically, osimertinib increased the intracellular accumulations of doxorubicin (DOX) and Rhodamine 123 (Rho 123) by inhibiting the efflux function of the transporters in ABCB1-or ABCG2-overexpressing cells but not in their parental sensitive cells. Furthermore, osimertinib stimulated the ATPase activity of both ABCB1 and ABCG2 and competed with the [ 125 I] iodoarylazidoprazosin photolabeling bound to ABCB1 or ABCG2, but did not alter the localization and expression of ABCB1 or ABCG2 in mRNA and protein levels nor the phosphorylations of EGFR, AKT, and ERK. Importantly, osimertinib also enhanced the cytotoxicity of DOX and intracellular accumulation of Rho 123 in ABCB1-overexpressing primary leukemia cells. Overall, these findings suggest osimertinib reverses ABCB1-and ABCG2-mediated MDR via inhibiting ABCB1 and ABCG2 from pumping out chemotherapeutic agents and provide possibility for cancer combinational therapy with osimertinib in the clinic.

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Cysteines Introduced into Extracellular Loops 1 and 4 of Human P-Glycoprotein That Are Close Only in the Open Conformation Spontaneously Form a Disulfide Bond That Inhibits Drug Efflux and ATPase Activity

Cited by 13 publications

References 59 publications

Mapping the Binding Site of the Inhibitor Tariquidar That Stabilizes the First Transmembrane Domain of P-glycoprotein

Mapping the Binding Site of the Inhibitor Tariquidar That Stabilizes the First Transmembrane Domain of P-glycoprotein

About P‐glycoprotein: a new drugable domain is emerging from structural data

Osimertinib (AZD9291) Enhanced the Efficacy of Chemotherapeutic Agents in ABCB1- and ABCG2-Overexpressing Cells In Vitro, In Vivo, and Ex Vivo

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