Location of contact residues in pharmacologically distinct drug binding sites on P-glycoprotein

Mittra, Rituparna; Pavy, Megan; Subramanian, Nandhitha; George, Anthony; O’Mara, Megan L.; Kerr, Ian D.; Callaghan, Richard

doi:10.1016/j.bcp.2016.10.002

Cited by 29 publications

(33 citation statements)

References 42 publications

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“…obtained from five independent observations. rate of ATP hydrolysis and the degree of stimulation by nicardipine were similar to values published by our laboratories (Taylor et al, 2001;Mittra et al, 2017). Moreover, this confirms the integrity of the purified P-gp preparations and enables their use to characterize interaction with noscapine and its derivatives.…”

Section: Mcf7supporting

confidence: 86%

“…In general, substrates for transport tend to stimulate the ATPase activity of P-gp and display tight coupling between binding sites and NBDs (Muller et al, 1996;Orlowski et al, 1996). Inhibitors, or modulators, have been demonstrated to bind to P-gp and either stimulate (nicardipine) or inhibit (tariquidar) the overall rate of ATP hydrolysis (Martin et al, 1999;Mittra et al, 2017). Whole-cell transport assays have demonstrated that [ 3 H]-tariquidar is a nontransported substrate for P-gp.…”

Section: Potency Stimulationmentioning

confidence: 99%

“…The synergistic interaction between noscapine derivatives and vinblastine raises the issue of where the two compounds interact on P-gp. Despite the availability of several structures for P-gp (Aller et al, 2009;Alam et al, 2018;Kim and Chen, 2018), the location of drug-binding sites remains elusive, although biochemical approaches are beginning to shed detailed information (Loo et al, 2009;Mittra et al, 2017). Thus, it…”

Section: Potency Stimulationmentioning

confidence: 99%

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Overcoming P-Glycoprotein–Mediated Drug Resistance with Noscapine Derivatives

et al. 2018

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The antitussive agent noscapine has been shown to inhibit the proliferation of cancer cells by disruption of tubulin dynamic. However, the efficacy of several anticancer drugs that inhibit tublin dynamics (vinca alkaloids and taxanes) is reduced by the multidrug resistance phenotype. These compounds are substrates for P-glycoprotein (P-gp)-mediated extrusion from cells. Consequently, the antiproliferative activity of noscapine and a series of derivatives was measured in drug-sensitive and drugresistant cells that overexpress P-gp. None of the noscapine derivatives displayed lower potency in cells overexpressing P-gp, thereby suggesting a lack of interaction with this pump. However, the cellular efflux of a fluorescent substrate by P-gp was potently inhibited by noscapine and most derivatives. Further investigation with purified, reconstituted P-gp demonstrated that inhibition of P-gp function was due to direct interaction of noscapine derivatives with the transporter. Moreover, coadministration of vinblastine with two of the noscapine derivatives displayed synergistic inhibition of proliferation, even in P-gp-expressing resistant cell lines. Therefore, noscapine derivatives offer a dual benefit of overcoming the significant impact of P-gp in conferring multidrug resistance and synergy with tubulin-disrupting anticancer drugs.

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Section: Mcf7supporting

confidence: 86%

Section: Potency Stimulationmentioning

confidence: 99%

Section: Potency Stimulationmentioning

confidence: 99%

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Overcoming P-Glycoprotein–Mediated Drug Resistance with Noscapine Derivatives

et al. 2018

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“…However, several binding sites on the MDR1 molecule have been proposed (44,45), making the issue of MDR1-mediated DDIs more intricate. Therefore, two model MDR1 substrates, Hoechst 33342 and rhodamine 123, specific for two different binding sites, were employed in our study.…”

Section: Figmentioning

confidence: 99%

MDR1 and BCRP Transporter-Mediated Drug-Drug Interaction between Rilpivirine and Abacavir and Effect on Intestinal Absorption

Reznicek

Čečková

Ptackova

et al. 2017

Antimicrob Agents Chemother

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Rilpivirine (TMC278) is a highly potent nonnucleoside reverse transcriptase inhibitor (NNRTI) representing an effective component of combination antiretroviral therapy (cART) in the treatment of HIV-positive patients. Many antiretroviral drugs commonly used in cART are substrates of ATP-binding cassette (ABC) and/or solute carrier (SLC) drug transporters and, therefore, are prone to pharmacokinetic drug-drug interactions (DDIs). The aim of our study was to evaluate rilpivirine interactions with abacavir and lamivudine on selected ABC and SLC transporters in vitro and assess its importance for pharmacokinetics in vivo. Using accumulation assays in MDCK cells overexpressing selected ABC or SLC drug transporters, we revealed rilpivirine as a potent inhibitor of MDR1 and BCRP, but not MRP2, OCT1, OCT2, or MATE1. Subsequent transport experiments across monolayers of MDCKII-MDR1, MDCKII-BCRP, and Caco-2 cells demonstrated that rilpivirine inhibits MDR1-and BCRP-mediated efflux of abacavir and increases its transmembrane transport. In vivo experiments in male Wistar rats confirmed inhibition of MDR1/BCRP in the small intestine, leading to a significant increase in oral bioavailability of abacavir. In conclusion, rilpivirine inhibits MDR1 and BCRP transporters and may affect pharmacokinetic behavior of concomitantly administered substrates of these transporters, such as abacavir.KEYWORDS drug transporter, rilpivirine, abacavir, oral bioavailability, pharmacokinetics, drug-drug interactions, ABC transporters R ilpivirine is a novel nonnucleoside reverse transcriptase inhibitor (NNRTI) approved by the FDA (in 2011) and the European Medicines Agency (EMA; in 2012) (1, 2) for treatment of antiretroviral therapy-naive patients (3, 4). In addition, rilpivirine shows fewer adverse effects than efavirenz and represents an important component of combination antiretroviral therapy (cART), particularly in the treatment of HIV-1-infected patients with viral loads of less than 100,000 copies/ml (5).Many antiretroviral drugs commonly used in cART are substrates or inhibitors of ABC (ATP-dependent) and/or SLC (solute carrier) drug transporters (6-9). Their coadministration with other compounds interacting with these transporters can lead to pharmacokinetic drug-drug interactions (DDIs) and to altered plasma and tissue concentrations of the target drug.P-glycoprotein (ABCB1/MDR1) (10, 11), breast cancer resistance protein (ABCG2/ BCRP) (12), and multidrug resistance-associated protein 2 (ABCC2/MRP2) (13) are well-described members of ATP-binding cassette (ABC) drug transporters that are functionally expressed in the small intestine, blood-tissue barriers, and excretory organs (14-17). Together with organic cation transporters (OCTs; SLC22A) of the SLC super-

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“…Complex competitive and mixed-type inhibition were also observed in drug-stimulated ATPase assays suggesting positional overlap of classical transport substrates with ligands localized in the structures 39 . The structures have motivated a flurry of site directed mutagenesis studies to probe contact residues for binding of cancer drugs and classical substrates 32,[40][41][42] (reviewed in 3 ) as well as other inhibitors for which structures are not yet available, supported by molecular dynamics and docking simulations [43][44][45][46] . The emerging picture is that of spatially distinct binding sites that can simultaneously bind multiple drugs, as well as spatially overlapping binding sites for certain drugs that are linked through cooperative and non-cooperative interactions 3,41,42,45 .…”

mentioning

confidence: 99%

Replacing the eleven native tryptophans by directed evolution produces an active P-glycoprotein with site-specific, non-conservative substitutions

Swartz

Singh

Sok

et al. 2020

Sci Rep

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P-glycoprotein (Pgp) pumps an array of hydrophobic compounds out of cells, and has major roles in drug pharmacokinetics and cancer multidrug resistance. Yet, polyspecific drug binding and ATP hydrolysisdriven drug export in pgp are poorly understood. fluorescence spectroscopy using tryptophans (trp) inserted at strategic positions is an important tool to study ligand binding. In Pgp, this method will require removal of 11 endogenous Trps, including highly conserved Trps that may be important for function, protein-lipid interactions, and/or protein stability. Here, we developed a directed evolutionary approach to first replace all eight transmembrane Trps and select for transport-active mutants in Saccharomyces cerevisiae. Surprisingly, many Trp positions contained non-conservative substitutions that supported in vivo activity, and were preferred over aromatic amino acids. The most active construct, W(3Cyto), served for directed evolution of the three cytoplasmic Trps, where two positions revealed strong functional bias towards tyrosine. W(3Cyto) and Trp-less Pgp retained wild-type-like protein expression, localization and transport function, and purified proteins retained drug stimulation of ATP hydrolysis and drug binding affinities. The data indicate preferred Trp substitutions specific to the local context, often dictated by protein structural requirements and/or membrane lipid interactions, and these new insights will offer guidance for membrane protein engineering.

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Location of contact residues in pharmacologically distinct drug binding sites on P-glycoprotein

Cited by 29 publications

References 42 publications

Overcoming P-Glycoprotein–Mediated Drug Resistance with Noscapine Derivatives

Overcoming P-Glycoprotein–Mediated Drug Resistance with Noscapine Derivatives

MDR1 and BCRP Transporter-Mediated Drug-Drug Interaction between Rilpivirine and Abacavir and Effect on Intestinal Absorption

Replacing the eleven native tryptophans by directed evolution produces an active P-glycoprotein with site-specific, non-conservative substitutions

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