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Proceedings of the National Academy of... Aug 2013P-glycoprotein (P-gp) is one of the best-known mediators of drug efflux-based multidrug resistance in many cancers. This validated therapeutic target is a prototypic,...
P-glycoprotein (P-gp) is one of the best-known mediators of drug efflux-based multidrug resistance in many cancers. This validated therapeutic target is a prototypic, plasma membrane resident ATP-Binding Cassette transporter that pumps xenobiotic compounds out of cells. The large, polyspecific drug-binding pocket of P-gp recognizes a variety of structurally unrelated compounds. The transport of these drugs across the membrane is coincident with changes in the size and shape of this pocket during the course of the transport cycle. Here, we present the crystal structures of three inward-facing conformations of mouse P-gp derived from two different crystal forms. One structure has a nanobody bound to the C-terminal side of the first nucleotide-binding domain. This nanobody strongly inhibits the ATP hydrolysis activity of mouse P-gp by hindering the formation of a dimeric complex between the ATP-binding domains, which is essential for nucleotide hydrolysis. Together, these inward-facing conformational snapshots of P-gp demonstrate a range of flexibility exhibited by this transporter, which is likely an essential feature for the binding and transport of large, diverse substrates. The nanobody-bound structure also reveals a unique epitope on P-gp.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Animals; Drug Delivery Systems; Epitope Mapping; Mice; Models, Molecular; Protein Conformation; Single-Domain Antibodies
PubMed: 23901103
DOI: 10.1073/pnas.1309275110 -
Biochimica Et Biophysica Acta Dec 1999The P-glycoprotein multidrug transporter is a 170-kDa efflux pump which exports a diverse group of natural products, chemotherapeutic drugs, and hydrophobic peptides... (Review)
Review
The P-glycoprotein multidrug transporter is a 170-kDa efflux pump which exports a diverse group of natural products, chemotherapeutic drugs, and hydrophobic peptides across the plasma membrane, driven by ATP hydrolysis. The transporter has been proposed to interact with its drug substrates within the membrane environment; however, much remains to be learned about the nature and number of the drug binding site(s). The two nucleotide binding domains are responsible for ATP binding and hydrolysis, which is coupled to drug movement across the membrane. In recent years, P-glycoprotein has been purified and functionally reconstituted in amounts large enough to allow biophysical studies. The use of spectroscopic techniques has led to insights into both its secondary and tertiary structure, and its interaction with nucleotides and drugs. In this review, we will summarise what has been learned by application to purified P-glycoprotein of fluorescence spectroscopy, circular dichroism spectroscopy and infra-red spectroscopy.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Adenosine Triphosphate; Anilino Naphthalenesulfonates; Binding Sites; Carrier Proteins; Cell Membrane; Circular Dichroism; Intracellular Signaling Peptides and Proteins; Lipid Bilayers; Pharmaceutical Preparations; Protein Conformation; Solubility; Spectrometry, Fluorescence; Spectroscopy, Fourier Transform Infrared; Substrate Specificity
PubMed: 10581365
DOI: 10.1016/s0005-2736(99)00166-2 -
Biophysical Journal Mar 2012P-glycoprotein-ATPase is an efflux transporter of broad specificity that counteracts passive allocrit influx. Understanding the rate of allocrit transport therefore...
P-glycoprotein-ATPase is an efflux transporter of broad specificity that counteracts passive allocrit influx. Understanding the rate of allocrit transport therefore matters. Generally, the rates of allocrit transport and ATP hydrolysis decrease exponentially with increasing allocrit affinity to the transporter. Here we report unexpectedly strong down-modulation of the P-glycoprotein-ATPase by certain detergents. To elucidate the underlying mechanism, we chose 34 electrically neutral and cationic detergents with different hydrophobic and hydrophilic characteristics. Measurement of the P-glycoprotein-ATPase activity as a function of concentration showed that seven detergents activated the ATPase as expected, whereas 27 closely related detergents reduced it significantly. Assessment of the free energy of detergent partitioning into the lipid membrane and the free energy of detergent binding from the membrane to the transporter revealed that the ratio, q, of the two free energies of binding determined the rate of ATP hydrolysis. Neutral (cationic) detergents with a ratio of q = 2.7 ± 0.2 (q > 3) followed the aforementioned exponential dependence. Small deviations from the optimal ratio strongly reduced the rates of ATP hydrolysis and flopping, respectively, whereas larger deviations led to an absence of interaction with the transporter. P-glycoprotein-ATPase inhibition due to membrane disordering by detergents could be fully excluded using (2)H-NMR-spectroscopy. Similar principles apply to modulating drugs.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Adenosine Triphosphatases; Air; Animals; Cell Membrane; Detergents; Kinetics; Lipids; Magnetic Resonance Spectroscopy; Membrane Transport Proteins; Mice; Models, Molecular; Thermodynamics; Unilamellar Liposomes
PubMed: 22455921
DOI: 10.1016/j.bpj.2012.02.018 -
The Journal of Veterinary Medical... Feb 2007Cellular drug resistance to antineoplastic drugs is often due to the presence of a drug efflux pump that reduces intracellular drug accumulation and chemosensitivity....
Cellular drug resistance to antineoplastic drugs is often due to the presence of a drug efflux pump that reduces intracellular drug accumulation and chemosensitivity. P-glycoprotein (P-gp), which is encoded by the MDR1 gene, is considered to function as an ATP-driven membrane drug efflux pump and appears to play an important role in tumor cell resistance. In the present report, we assessed the expression of MDR1 by RT-PCR in three canine mast cell tumor cell lines, TiMC, CoMS and LuMC, originating from a cutaneous tumor, an oral-mucosal tumor and a gastrointestinal tumor, respectively. P-gp expression was also examined by Western blot analysis, while the functional activity of P-gp was assessed by flowcytometric analysis of intracellular rhodamine-123 (Rhd-123) uptake. The results revealed that MDR1 gene and P-gp were both expressed in CoMS and LuMC cells, whereas neither was present in TiMC cells. In CoMS and LuMC cells, intracellular uptake of Rhd-123 increased in the presence of verapamil, a functional modulator of P-gp. In contrast, TiMC cells did not show any changes in the intracellular accumulation of Rhd-123 after the verapamil addition. These findings suggest that the expressions of MDR1 gene and P-gp probably contribute to cellular drug resistance in canine mast cell tumors.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Animals; Blotting, Western; Cell Line, Tumor; Dog Diseases; Dogs; Flow Cytometry; Fluorescent Dyes; Gene Expression; Genes, MDR; Mastocytoma; Reverse Transcriptase Polymerase Chain Reaction; Rhodamine 123; Verapamil
PubMed: 17339753
DOI: 10.1292/jvms.69.111 -
Journal of Medicinal Food Apr 2014Glyceollins are phytoalexins produced in soybeans from their isoflavone precursor daidzein. Their impressive anticancer and glucose normalization effects in rodents have...
Glyceollins are phytoalexins produced in soybeans from their isoflavone precursor daidzein. Their impressive anticancer and glucose normalization effects in rodents have generated interest in their therapeutic potential. The aim of the present studies was to begin to understand glyceollin intestinal transport and metabolism, and their potential effects on P-glycoprotein (Pgp) in Caco-2 cells. At 10 and 25 μM, glyceollin permeability was 2.4±0.16×10(-4) cm/sec and 2.1±0.15×10(-4) cm/sec, respectively, in the absorptive direction. Basolateral to apical permeability at 25 μM was 1.6±0.10×10(-4) cm/sec. Results suggest high absorption potential of glyceollin by a passive-diffusion-dominated mechanism. A sulfate conjugate at the phenolic hydroxyl position was observed following exposure to Caco-2 cells. In contrast to verapamil inhibition of the net secretory permeability of rhodamine 123 (R123) and its enhancement of calcein AM uptake into Caco-2 cells, neither glyceollin nor genistein inhibited Pgp (MDR1; ABCB1) up to 300 μM. There was no significant change in MDR1 mRNA expression, Pgp protein expression, or R123 transport in cells exposed to glyceollin or genistein for 24 h up to 100 μM. Collectively, these results suggest that glyceollin has the potential to be well absorbed, but that, similar to the isoflavone genistein, its absorption may be reduced substantially by intestinal metabolism; further, they indicate that glyceollin does not appear to alter Pgp function in Caco-2 cells.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Caco-2 Cells; Humans; Intestinal Mucosa; Intestines; Plant Extracts; Protein Transport; Pterocarpans; Glycine max
PubMed: 24476214
DOI: 10.1089/jmf.2013.0115 -
British Journal of Haematology Jun 2003Concurrent resistance mechanisms, such as P-glycoprotein (PGP) and bcl-2, may contribute to a worse outcome in adult acute lymphoblastic leukaemia (ALL). Between 1990...
Concurrent resistance mechanisms, such as P-glycoprotein (PGP) and bcl-2, may contribute to a worse outcome in adult acute lymphoblastic leukaemia (ALL). Between 1990 and 2000, we analysed PGP and bcl-2 by flow cytometry, using two anti-PGP (C219 and JSB-1) monoclonal antibodies (mAbs) and an anti-bcl-2 mAb in 115 de novo adult ALL patients. Both a longer overall survival (OS) and longer disease-free survival (DFS) were observed in PGP-negative patients (23%vs 0% at 3 years, P = 0.011 and 29%vs 0% at 2 years, P = 0.006 for C219 respectively; 42%vs 0% at 1.5 years, P = 0.004 and 53%vs 0% at 8.5 months, P = 0.00006 for JSB-1 respectively). Bcl-2 positivity was associated with a significantly higher complete remission rate (90%vs 66%, P = 0.01). Moreover, in 69 patients not presenting with either t(9;22) or B-mature immunophenotype, PGP negativity (JSB-1) maintained its significant favourable prognostic impact with regard to OS (41%vs 0% at 1.5 years, P = 0.009) and DFS (83%vs 0% at 6 months, P = 0.0005). Importantly, within a subset of 62 patients with normal (n = 31) or unknown (n = 31) karyotype, PGP (JSB-1)-negative patients showed both a significantly longer OS and DFS (63%vs 0% at 1.4 years, P = 0.018 and 84%vs 0% at 6 months, P = 0.001 respectively). In multivariate analysis, JSB-1 (P = 0.008) and cytogenetics (P = 0.02) were found to be independent prognostic factors with regard to DFS. Therefore, in adult ALL, PGP and bcl-2 represent sensitive indicators of clinical outcome, and potential targets of novel molecules aimed at overcoming chemoresistance and recurrent relapses.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Adolescent; Adult; Aged; Disease-Free Survival; Female; Humans; Male; Middle Aged; Phenotype; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Prognosis; Proto-Oncogene Proteins c-bcl-2
PubMed: 12780787
DOI: 10.1046/j.1365-2141.2003.04343.x -
Planta Medica Sep 2012The use of herbal/botanical products, also referred to as complementary and alternative medicines (CAM), worldwide enjoys increasing popularity. It appears in particular... (Review)
Review
The use of herbal/botanical products, also referred to as complementary and alternative medicines (CAM), worldwide enjoys increasing popularity. It appears in particular highly prevalent in patient populations already exposed to complex treatment algorithms and polypharmacotherapy, frequently involving narrow therapeutic index drugs. Accordingly, the potential clinical dimension and relevance of herb-drug interactions has received considerable attention over the last years. However, review of pertinent literature indicates that the available clinical evidence in this regard is still limited and sometimes inconclusive. Also, communication of herb-drug interaction data in the biopharmaceutical/medical literature is often complex and confusing, not always unbiased, and in many cases appears not to strive for clear-cut and useful guidance in terms of the clinical relevance of such findings.This systematic review summarizes and interprets the published evidence on clinical herb-drug interaction studies which examined the potential of six popular herbal drugs (Echinacea, garlic, gingko, ginseng, goldenseal, and milk thistle) as perpetrators of pharmacokinetic (PK) drug interactions. Reported effect sizes were systematically categorized according to FDA drug interaction guideline criteria. A total of 66 clinical PK interaction studies, meeting the scope of the present review, were identified. The clinical evidence was found to be most robust and informative for Gingko biloba (GB; 21 studies) and milk thistle/silymarin (MT; 13), and appears still limited for ginseng (9), goldenseal/berberine (GS; 8), garlic (8), and Echinacea (7). Collectively, the available evidence indicates that, at commonly recommended doses, none of these herbs act as potent or moderate inhibitors or inducers of cytochrome P450 (CYP) enzymes or P-glycoprotein (ABCB1). Weak effects in terms of either induction or inhibition were found for GB (presystemic/hepatic CYP3A4 induction/inhibition, CYP2C19 induction at high doses), milk thistle/silymarin (CYP2C9 inhibition), GS/berberine (CYP3A4 and CYP2D6 inhibition), Echinacea (presystemic/hepatic CYP3A4 inhibition/induction, CYP1A2 and CYP2C9 inhibition at high doses). Information was found not always complete for the major drug metabolizing CYP enzymes in the less well-studied herbs and is largely limited to P-glycoprotein (ABCB1) when effects on drug transporters have been investigated.
Topics: ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cytochrome P-450 Enzyme System; Echinacea; Garlic; Ginkgo biloba; Herb-Drug Interactions; Humans; Hydrastis; Silybum marianum; Panax; Pharmacokinetics; Plant Preparations
PubMed: 22855269
DOI: 10.1055/s-0032-1315117 -
Cancer Imaging : the Official... Mar 2011Primary intrinsic and/or acquired multidrug resistance (MDR) is the main obstacle to successful cancer treatment. Functional molecular imaging of MDR in cancer using... (Review)
Review
Primary intrinsic and/or acquired multidrug resistance (MDR) is the main obstacle to successful cancer treatment. Functional molecular imaging of MDR in cancer using single photon or positron emitters may be helpful to identify multidrug-resistant tumours and predict not only those patients who are resistant to treatment, with a clinically unfavourable prognosis, but also those who are susceptible to the development of drug toxicity or even certain tumours . Variations in the mdr1 gene product may directly affect the therapeutic effectiveness, and single nucleotide polymorphisms for the mdr1 gene may be associated with altered oral bioavailability of MDR1 substrates, drug resistance, and a susceptibility to some human diseases. The challenge of translating the concept of MDR modulation in vivo involves a complex cellular interplay between both malignant and normal cells. Integration and correlation of functional single photon emission tomography or positron emission tomography imaging findings with mdr1 genotype and clinical data may contribute to efficient management by selecting cancer patients with the appropriate molecular phenotype for maximal individual therapeutic benefit, as well as those who are non-responders. This review describes a role for functional imaging of classical mechanisms of MDR with an emphasis on readily available [(99m)Tc]MIBI scintigraphy. MIBI scintigraphy has been shown to be a non-invasive cost-effective in vivo assay of ATP-binding cassette transporters associated with MDR in cancer, including P-glycoprotein, multidrug-resistant protein 1 and breast cancer resistant protein. New imaging agents for molecular targets such as vascular endothelial growth factor and HER2 receptors, may potentially be combined with MDR imaging substrates to more accurately predict the therapeutic response to anticancer drugs, guiding individualised treatment while minimising the economic health costs of ineffective therapy in an era of personalised medicine.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP-Binding Cassette Transporters; Drug Resistance, Neoplasm; Genes, MDR; Genotype; Humans; Neoplasms; Radionuclide Imaging; Technetium Tc 99m Sestamibi
PubMed: 21362586
DOI: 10.1102/1470-7330.2011.0001 -
The Journal of Biological Chemistry Jan 2014The P-glycoprotein (P-gp) drug pump (ABCB1) has two transmembrane domains and two nucleotide-binding domains (NBDs). Coupling of the drug-binding sites in the...
The P-glycoprotein (P-gp) drug pump (ABCB1) has two transmembrane domains and two nucleotide-binding domains (NBDs). Coupling of the drug-binding sites in the transmembrane domains to the NBDs occurs through interaction of the intracellular helices (IHs) with residues in the NBDs (IH1/IH4/NBD1 and IH2/IH3/NBD2). We showed previously that cross-linking of cysteines in IH3 and IH1 with a short cross-linker mimicked drug binding as it activated P-gp ATPase activity. Here we show that residue A259C(IH2) could be directly cross-linked to W803C(IH3). Cross-linking was inhibited by the presence of ATP and adenosine 5'-(β,γ-imino)triphosphate but not by ADP. Cross-linking of mutant A259C/W803C inhibited its verapamil-stimulated ATPase activity mutant, but activity was restored after addition of dithiothreitol. Because these residues are close to the ball-and-socket joint A266C(IH2)/Phe(1086)(NBD2), we mutated the adjacent Tyr(1087)(NBD2) close to IH3. Mutants Y1087A and Y1087L, but not Y1087F, were misprocessed, and all inhibited ATPase activity. Mutation of hydrophobic residues (F793A, L797A, L814A, and L818A) flanking IH3 also inhibited maturation. The results suggest that these residues, together with Trp(803) and Phe(804), form a large hydrophobic pocket. The results show that there is an important hydrophobic network at the IH2/IH3/NBD2 transmission interface that is critical for folding and activity of P-gp.
Topics: ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; Adenosine Triphosphate; Amino Acid Substitution; Cross-Linking Reagents; HEK293 Cells; Humans; Mutation, Missense; Protein Folding; Protein Structure, Secondary; Protein Structure, Tertiary
PubMed: 24275649
DOI: 10.1074/jbc.M113.527804 -
The AAPS Journal Nov 2017The blood-brain barrier (BBB) is essential for proper neuronal function, homeostasis, and protection of the central nervous system (CNS) microenvironment from... (Review)
Review
The blood-brain barrier (BBB) is essential for proper neuronal function, homeostasis, and protection of the central nervous system (CNS) microenvironment from blood-borne pathogens and neurotoxins. The BBB is also an impediment for CNS penetration of drugs. In some neurologic conditions, such as epilepsy and brain tumors, overexpression of P-glycoprotein, an efflux transporter whose physiological function is to expel catabolites and xenobiotics from the CNS into the blood stream, has been reported. Recent studies reported that overexpression of P-glycoprotein and increase in its activity at the BBB drives a progressive resistance to CNS penetration and persistence of riluzole, the only drug approved thus far for treatment of amyotrophic lateral sclerosis (ALS), rapidly progressive and mostly fatal neurologic disease. This review will discuss the impact of transporter-mediated pharmacoresistance for ALS drug therapy and the potential therapeutic strategies to improve the outcome of ALS clinical trials and efficacy of current and future drug treatments.
Topics: ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; Amyotrophic Lateral Sclerosis; Blood-Brain Barrier; Brain; Clinical Trials as Topic; Drug Resistance; Humans
PubMed: 28779378
DOI: 10.1208/s12248-017-0120-6