Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.3.44 (P-glycoprotein)
13,344 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

P-glycoprotein (P-gp) is expressed at high levels in a variety of non-cancerous tissues such as the endothelial cells of the blood-brain barrier (BBB) capillaries. These thin capillaries tightly regulate the movement of substrates from the circulating blood into the brain. P-gp may be involved in the exclusion of various drugs from the capillary endothelial cells, blocking their entry into the brain. However, interactions of drugs with P-gp expressed in brain capillaries remain to be characterized. We have performed photoaffinity labeling studies using [125I]arylazidoprazosin (IAAP) to evaluate the inhibitory efficiency of various compounds. Cyclosporin A (CsA) and its derivative PSC 833 (PSC) were the most effective inhibitors of IAAP binding among the drugs tested. The magnitude of inhibition was: PSC > CsA > quinidine > vinblastine > verapamil < actinomycin D > colchicine > reserpine > bilirubin > doxorubicin > progesterone. Cremophor El, the vehicle used to administer CsA and PSC intravenously, was also able to inhibit IAAP photolabeling of P-gp. Labeling experiments were also performed using a photoactivatable [3H]CsA derivative. Photolabeling of P-gp with this compound was abolished almost completely by CsA and PSC. In vivo studies were also performed by treating rats with CsA [10 mg/(kg.day) for 10 days]. Following this treatment, no alteration in the level of P-gp expression in brain capillaries was observed. These results suggest that, at the proper dosage, administration of CsA to cancer patients could help to enhance the response of brain tumors to chemotherapeutic agents without modifying the intrinsic level of P-gp expression in this tissue.
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PMID:Interaction of drugs with P-glycoprotein in brain capillaries. 750 74

Human P-glycoprotein (Pgp) confers multidrug resistance to cancer cells by ATP-dependent extrusion of a great many structurally dissimilar hydrophobic compounds. The manner in which Pgp recognizes these different substrates is unknown. The protein shows internal homology between its N- and C-terminal halves, each comprised of six putative transmembrane helices and a consensus ATP binding/utilization site. Photoactive derivatives of certain Pgp substrates specifically label two regions, one on each half of the protein. In this study, using [125I]iodoarylazidoprazosin ([125I]IAAP), a photoactive analog of prazosin, we have demonstrated the presence of two nonidentical drug-interaction sites within Pgp. Taking advantage of a highly susceptible trypsin cleavage site in the linker region of Pgp, we characterized the [125I]IAAP binding to the N- and C-terminal halves. cis(Z)-Flupentixol, a modulator of Pgp function, preferentially increased the affinity of [125I]IAAP for the C-terminal half of the protein (C-site) by reducing the Kd from 20 to 6 nM without changing the labeling or affinity (Kd = 42-46 nM) of the N-terminal half (N-site). Also, the concentration of vinblastine (Pgp substrate) and cyclosporin A (Pgp modulator) required for 50% inhibition of [125I]IAAP binding to the C-site was increased 5- to 6-fold by cis(Z)-flupentixol without any effect on the N-site. In addition, [125I]IAAP binding to the N-site was less susceptible than to C-site to inhibition by vanadate which blocks ATP hydrolysis and drug transport. These data demonstrate the presence of at least two nonidentical substrate interaction sites in Pgp.
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PMID:Evidence for two nonidentical drug-interaction sites in the human P-glycoprotein. 938 Jun 80

Rats were treated with daily injections of SDZ-PSC 833 (PSC) to study the interaction of this potent modulator of multidrug resistance (MDR) with P-glycoprotein (P-gp) expressed in normal tissues. After 2 days of treatment, the level of P-gp expression, detected by Western blot analysis, was not modified in renal brush border membranes (BBMs) and brain capillaries. However, the amount of P-gp detected with the photoaffinity probe [125I]-arylazidoprazosin (IAAP) was decreased in both tissues, suggesting that the drug binding properties of P-gp were altered by PSC treatment. This effect was further characterized by treating rats with PSC for 10 days. Following these treatments, the amount of immunodetected P-gp was increased in renal BBMs and brain capillaries. However, no increase in P-gp expression was observed in photolabeling experiments, suggesting that induced P-gp was not functional. In vitro experiments performed with renal BBMs showed that the inhibition of P-gp photolabeling by cyclosporin A (CsA), verapamil and vinblastine could be reversed by performing washing steps to remove these drugs before incubating the samples with IAAP. However, the inhibition mediated by PSC was less reversible since photolabeling of P-gp remained inhibited following the washing steps. Pre-incubation of intact CHRC5 cells with PSC, CsA and verapamil also inhibited P-gp photolabeling and increased rhodamine 123 accumulation. For PSC pre-treated samples, these effects were not completely reversed following washing, but were abolished for CsA and Ver pre-treated samples. Our results suggest that PSC could block P-gp function by a different mechanism from that of CsA and verapamil, involving modification of the drug binding sites.
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PMID:Drug binding to P-glycoprotein is inhibited in normal tissues following SDZ-PSC 833 treatment. 961 Jul 33

Both cis and trans isomers of the dopamine receptor antagonist flupentixol inhibit drug transport and reverse drug resistance mediated by the human multidrug transporter P-glycoprotein (Pgp) with a stereoselective potency. The rate of ATP hydrolysis by Pgp and photoaffinity labeling of Pgp with the substrate analogue [125I]iodoarylazidoprazosin ([125I]IAAP) are modulated by each isomer in an opposite manner, suggesting different mechanisms for the inhibitory effect on drug transport. In this study we demonstrate that substitution of a single phenylalanine residue at position 983 (F983) with alanine (F983A) in putative transmembrane (TM) region 12 selectively affects inhibition of Pgp-mediated drug transport by both isomers of flupentixol. In F983A the stimulatory effect of cis(Z)-flupentixol and the inhibitory effect of trans(E)-flupentixol on ATP hydrolysis and [125I]IAAP labeling were significantly altered. This indicates that F983 contributes to inhibition of drug transport by both isomers of flupentixol and plays an important role in stimulation and inhibition of ATP hydrolysis and [125I]IAAP labeling by cis(Z)- and trans(E)-flupentixol, respectively. The near-wild-type level of drug transport by the F983A Pgp mutant dissociates susceptibility to inhibition by flupentixol from drug translocation, indicating the allosteric nature of the flupentixol interaction. The inhibitory effects of cyclosporin A on drug transport, drug-stimulated ATP hydrolysis, and [125I]IAAP labeling as well as the stimulatory effect of verapamil on ATP hydrolysis by Pgp were minimally affected by substitution of F983, suggesting no global alteration in the structural and functional integrity of the mutant. Taken together, our data suggest that distinct mechanisms of inhibition of Pgp-mediated drug transport by the cis and trans isomers of flupentixol are mediated through a common site of interaction.
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PMID:A single amino acid residue contributes to distinct mechanisms of inhibition of the human multidrug transporter by stereoisomers of the dopamine receptor antagonist flupentixol. 1035 Apr 82

Human P-glycoprotein (P-gp) is a cell surface drug efflux pump that contains two nucleotide binding domains (NBDs). Mutations were made in each of the Walker B consensus motifs of the NBDs at positions D555N and D1200N, thought to be involved in Mg(2+) binding. Although the mutant and wild-type P-gps were expressed equivalently at the cell surface and bound the drug analogue [(125)I]iodoarylazidoprazosin ([(125)I]IAAP) comparably, neither of the mutant proteins was able to transport fluorescent substrates nor had detectable basal nor drug-stimulated ATPase activities. The wild-type and D1200N P-gps were labeled comparably with [alpha-(32)P]-8-azido-ATP at a subsaturating concentration of 2.5 microM, whereas labeling of the D555N mutant was severely impaired. Mild trypsin digestion, to cleave the protein into two halves, demonstrated that the N-half of the wild-type and D1200N proteins was labeled preferentially with [alpha-(32)P]-8-azido-ATP. [alpha-(32)P]-8-Azido-ATP labeling at 4 degrees C was inhibited in a concentration-dependent manner by ATP with half-maximal inhibition at approximately 10-20 microM for the P-gp-D1200N mutant and wild-type P-gp. A chimeric protein containing two N-half NBDs was found to be functional for transport and was also asymmetric with respect to [alpha-(32)P]-8-azido-ATP labeling, suggesting that the context of the ATP site rather than its exact sequence is an important determinant for ATP binding. By use of [alpha-(32)P]-8-azido-ATP and vanadate trapping, it was determined that the C-half of wild-type P-gp was labeled preferentially under hydrolysis conditions; however, the N-half was still capable of being labeled with [alpha-(32)P]-8-azido-ATP. Neither mutant was labeled under vanadate trapping conditions, indicating loss of ATP hydrolysis activity in the mutants. In confirmation of the lack of ATP hydrolysis, no inhibition of [(125)I]IAAP labeling was observed in the mutants in the presence of vanadate. Taken together, these data suggest that the two NBDs are asymmetric and intimately linked and that a conformational change in the protein may occur upon ATP hydrolysis. Furthermore, these data are consistent with a model in which binding of ATP to one site affects ATP hydrolysis at the second site.
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PMID:Both ATP sites of human P-glycoprotein are essential but not symmetric. 1052 34

The human P-glycoprotein (Pgp, ABCB1) is an ATP-dependent efflux pump for structurally unrelated hydrophobic compounds, conferring simultaneous resistance to and restricting bioavailability of several anticancer and antimicrobial agents. Drug transport by Pgp requires a coordinated communication between its substrate binding/translocating pathway (substrate site) and the nucleotide binding domains (NBDs or ATP sites). In this study, we demonstrate that certain thioxanthene-based Pgp modulators, such as cis-(Z)-flupentixol and its closely related analogues, effectively disrupt molecular cross talk between the substrate, and the ATP, sites without affecting the basic functional aspects of the two domains, such as substrate recognition, binding, and hydrolysis of ATP and dissociation of ADP following ATP hydrolysis. The allosteric modulator cis-(Z)-flupentixol has no effect on [alpha-(32)P]-8-azido-ATP binding to Pgp under nonhydrolytic conditions or on the K(m) for ATP during ATP hydrolysis. Both hydrolysis of ATP and vanadate-induced [alpha-(32)P]-8-azido-ADP trapping (following [alpha-(32)P]-8-azido-ATP breakdown) by Pgp are stimulated by the modulator. However, the ability of Pgp substrates (such as prazosin) to stimulate ATP hydrolysis and facilitate vanadate-induced trapping of [alpha-(32)P]-8-azido-ADP is substantially affected in the presence of cis-(Z)-flupentixol. Substrate recognition by Pgp as determined by [(125)I]iodoarylazidoprazosin ([(125)I]IAAP) binding both in the presence and in the absence of ATP is facilitated by the modulator, whereas substrate dissociation in response to vanadate trapping is considerably affected in its presence. In the Pgp F983A mutant, which is impaired in modulation by cis-(Z)-flupentixol, the modulator has a minimal effect on substrate-stimulated ATP hydrolysis as well as on substrate dissociation coupled to vanadate trapping. Finally, cis-(Z)-flupentixol has no effect on dissociation of [alpha-(32)P]-8-azido-ADP (or ADP) from vanadate-trapped Pgp, which is essential for subsequent rounds of ATP hydrolysis. Taken together, our results demonstrate a distinct mechanism of Pgp modulation that involves allosteric disruption of molecular cross talk between the substrate, and the ATP, sites without any direct interference with their individual functions.
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PMID:Modulator-induced interference in functional cross talk between the substrate and the ATP sites of human P-glycoprotein. 1648 67

ATP-dependent drug transport by human P-glycoprotein (Pgp, ABCB1) involves a coordinated communication between its drug-binding site (substrate site) and the nucleotide binding/hydrolysis domain (ATP sites). It has been demonstrated that the two ATP sites of Pgp play distinct roles within a single catalytic turnover; whereas ATP binding or/and hydrolysis by one drives substrate translocation and dissociation, the hydrolytic activity of the other resets the transporter for the subsequent cycle (Sauna, Z. E., and Ambudkar, S. V. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 2515-2520; Sauna, Z. E., and Ambudkar, S. V. (2001) J. Biol. Chem. 276, 11653-11661). Trapping of ADP (or 8-azido-ADP) and vanadate (ADP.Vi or 8-azido-ADP.Vi) at the catalytic site, following nucleotide hydrolysis, markedly reduces the affinity of Pgp for its transport substrate [125I]iodoarylazidoprazosin ([125I]IAAP), resulting in dissociation of the latter. Regeneration of the [125I]IAAP site requires an additional round of nucleotide hydrolysis. In this study, we demonstrate that certain thioxanthene-based allosteric modulators, such as cis-(Z)-flupentixol and its closely related analogs, induce regeneration of [125I]IAAP binding to vanadate-trapped (or fluoroaluminate-trapped) Pgp without any further nucleotide hydrolysis. Regeneration was facilitated by dissociation of the trapped nucleotide and vanadate. Once regenerated, the substrate site remains accessible to [125I]IAAP even after removal of the modulator from the medium, suggesting a modulator-induced relaxation of a constrained transition state conformation. Consistent with this, limited trypsin digestion of vanadate-trapped Pgp shows protection by cis-(Z)-flupentixol of two Pgp fragments (approximately 60 kDa) recognizable by a polyclonal antiserum specific for the NH2-terminal half. No regeneration was observed in the Pgp mutant F983A that is impaired in modulation by flupentixols, indicating involvement of the allosteric modulator site in the phenomenon. In summary, the data demonstrate that in the nucleotide-trapped low affinity state of Pgp, the allosteric site remains accessible and responsive to modulation by flupentixol (and its closely related analogs), which can reset the high affinity state for [125I]IAAP binding without any further nucleotide hydrolysis.
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PMID:Allosteric modulation bypasses the requirement for ATP hydrolysis in regenerating low affinity transition state conformation of human P-glycoprotein. 1650 85

The drug-transport function of the human P-glycoprotein (Pgp or ABCB1) is inhibited by a number of structurally unrelated compounds, known as modulators or reversing agents. Among them, the thioxanthene derivative flupentixol inhibits Pgp-mediated drug transport by an allosteric mechanism. Unlike most other Pgp modulators, the cis isomer of flupentixol [cis-(Z)-flupentixol] facilitates interaction of Pgp with its transport-substrate [125I]iodoarylazidoprazosin (or [125I]IAAP), yet inhibits transport. In this study, we show that the flupentixol site acts as a common site of interaction for the tricyclic ring-containing modulators thioxanthenes and phenothiazines. The allosteric stimulation of [125I]IAAP binding to Pgp occurs independent of the phosphorylation status of the transporter. Stimulation is retained in purified Pgp reconstituted into proteoliposomes, suggesting no involvement of any other cellular protein in the phenomenon. However, perturbation of the lipid environment of the reconstituted Pgp by nonionic detergent octylglucoside abolishes stimulation by cis-(Z)-flupentixol of [125I]IAAP binding. Extensive trypsin digestion of the [125I]IAAP-labeled Pgp generates a 5.5 kDa fragment with 80% of the stimulated level of labeling associated with it. Sensitivity to inhibition by transport-substrate vinblastine and competitive modulator cyclosporin A suggests that the elevated level of [125I]IAAP binding to the fragment represents a functionally relevant interaction with the substrate site of Pgp. In summary, we demonstrate that allosteric modulation by cis-(Z)-flupentixol is mediated through its interaction with Pgp at a site specific for tricyclic ring-containing Pgp modulators of thioxanthene and phenothiazine backbone, independent of other cellular components and the phosphorylation status of the protein.
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PMID:Biochemical and pharmacological properties of an allosteric modulator site of the human P-glycoprotein (ABCB1). 1672 76

The majority of chronic phase chronic myeloid leukemia (CML) patients treated with the tyrosine kinase inhibitor (TKI) imatinib mesylate maintain durable responses to the drug. However, most patients relapse after withdrawal of imatinib and advanced stage patients often develop drug resistance. As CML is considered a hematopoietic stem cell cancer, it has been postulated that inherent protective mechanisms lead to relapse in patients. The ATP binding-cassette transporters ABCB1 (MDR-1; P-glycoprotein) and ABCG2 are highly expressed on primitive hematopoietic stem cells (HSCs) and have been shown to interact with TKIs. Herein we demonstrate a dose-dependent, reversible inhibition of ABCG2-mediated Hoechst 33342 dye efflux in primary human and murine HSC by both imatinib and nilotinib (AMN107), a novel aminopyrimidine inhibitor of BCR-ABL. ABCG2-transduced K562 cells were protected from imatinib and nilotinib-mediated cell death and from downregulation of P-CRKL. Moreover, photoaffinity labeling revealed interaction of both TKIs with ABCG2 at the substrate binding sites as they compete with the binding of [(125)I] IAAP and also stimulate the transporter's ATPase activity. Therefore, our evidence suggests for the role of ABC transporters in resistance to TKI on primitive HSCs and CML stem cells and provides a rationale how TKI resistance can be overcome in vivo.
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PMID:Imatinib mesylate and nilotinib (AMN107) exhibit high-affinity interaction with ABCG2 on primitive hematopoietic stem cells. 1751 60

Cellular expression of ATP-binding cassette (ABC) transport proteins, such as P-glycoprotein (Pgp), multidrug resistance-associated protein (MRP1), or ABCG2, is known to confer a drug-resistant phenotype. Thus, the development of effective transporter inhibitors could be of value to cancer treatment. CBT-1 is a bisbenzylisoquinoline plant alkyloid currently in development as a Pgp inhibitor. We characterized its interactions with the three major ABC transporters associated with drug resistance - Pgp, MRP1 and ABCG2 - and compared it to other known inhibitors. CBT-1 completely inhibited rhodamine 123 transport from Pgp-overexpressing cells at a concentration of 1muM. Additionally, 1 microM completely reversed Pgp-mediated resistance to vinblastine, paclitaxel and depsipeptide in SW620 Ad20 cells. CBT-1 was found to compete [(125)I]-IAAP labeling of Pgp with an IC(50) of 0.14 microM, and low concentrations of CBT-1 (<1 microM) stimulated Pgp-mediated ATP hydrolysis. In MRP1-overexpressing cells, 10 microM CBT-1 was found to completely inhibit MRP1-mediated calcein transport. CBT-1 at 25 microM did not have a significant effect on ABCG2-mediated pheophorbide a transport. Serum levels of CBT-1 in samples obtained from eight patients receiving CBT-1 increased intracellular rhodamine 123 levels in CD56+ cells 2.1- to 5.7-fold in an ex vivo assay. CBT-1 is able to inhibit the ABC transporters Pgp and MRP1, making it an attractive candidate for clinical trials in cancers where Pgp and/or MRP1 might be overexpressed. Further clinical studies with CBT-1 are warranted.
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PMID:Inhibition of P-glycoprotein (ABCB1)- and multidrug resistance-associated protein 1 (ABCC1)-mediated transport by the orally administered inhibitor, CBT-1((R)). 1823 54


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