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)

K02 (morpholine-urea-Phe-Hphe-vinylsulfone), a newly developed peptidomimetic, acts as a potent cysteine protease inhibitor, especially of cathepsins B and L (which are associated with cancer progression) and cruzain (a cysteine protease of Trypanosoma cruzi, which is responsible for Chagas' disease). Here we investigated features of the disposition of K02 using in vitro systems, characterizing the interaction of the drug with human cytochrome P450 (CYP) 3A and P-glycoprotein (P-gp), a mediator of multidrug resistance (MDR) to cancer chemotherapy and a countertransporter in the intestine that limits oral drug bioavailability. P-gp functions as an ATP-dependent drug efflux pump to reduce intracellular cytotoxic concentrations. An HPLC assay was developed to analyze K02 and its metabolites formed in human liver microsomes. Three major primary metabolites were determined by LC/MS/MS to be hydroxylated products of the parent compound. A rabbit anti-CYP3A polyclonal antibody (200 microl antibody/mg microsomal protein) produced 75-94% inhibition of the formation of these three hydroxylated metabolites. Ketoconazole (5 microM), a selective CYP3A inhibitor, produced up to 75% inhibition, whereas other CYP-specific inhibitors, i.e. quinidine (CYP2D6), 7,8-benzoflavone (CYP1A2), and sulfaphenazole (CYP2C9), showed no significant effects. An identical metabolite formation profile for K02 was observed with cDNA-expressed human CYP3A4 (Gentest). These data demonstrate that K02 is a substrate for CYP3A. Formation of 1'-hydroxymidazolam, the primary human midazolam metabolite, was markedly inhibited by K02 via competitive processes, which suggests the potential for drug-drug interactions of K02 with other CYP3A substrates. K02 significantly inhibited the photoaffinity labeling of P-gp with azidopine and LU-49888, a photoaffinity analogue of verapamil. Transport studies with [14C]K02, using MDR1-transfected Madin-Darby canine kidney cell monolayers in the Transwell system, demonstrated that the basolateral-to-apical flux of K02 across MDR1-transfected Madin-Darby canine kidney cells was markedly greater than the apical-to-basolateral flux (ratio of 63 with 10 microM [14C]K02). This suggests that K02 is also a P-gp substrate. These studies are important for formulating strategies to increase the absorption and/or decrease the elimination of K02 and to optimize its delivery to malignant cells and parasite-infected host cells.
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PMID:Overlapping substrate specificities of cytochrome P450 3A and P-glycoprotein for a novel cysteine protease inhibitor. 953 25

We investigated the effects of ketoconazole on the oral bioavailability of morpholine-urea-phenylalanine-homophenylalanine-vinylsulfone-phenyl (K02), a vinylsulfone peptidomimetic cysteine protease inhibitor, and a P450 3A (CYP3A) and P-glycoprotein dual substrate, in male Sprague-Dawley rats, so as to evaluate the roles of CYP3A and P-gp in K02 disposition. Male Sprague-Dawley rats (8-10 wk old, n = 3-6) were administered a single dose of K02 (10 mg/kg) i.v. or (30 mg/kg) p.o. with or without a concomitant oral dose of ketoconazole (20 mg/kg). Blood samples were collected from 2 min to 8 h after administration through a implanted jugular vein cannula. K02 plasma concentrations were determined by liquid chromatography/mass spectrometer/mass spectrometer analysis. Ketoconazole markedly raised the area under the curve of orally administered K02 from 9.4 +/- 4.4 to 102 +/- 24 mg . min/liter and decreased K02 oral plasma clearance from 3810 +/- 1620 to 306 +/- 60 ml/min/kg. With concomitant ketoconazole dosing, the changes of AUC of i.v. administered K02 (from 94 +/- 17 to 107 +/- 14 mg . min/liter) and clearance (from 110 +/- 22 to 95 +/- 13 ml/min/kg) were not significant, although K02 oral bioavailability increased from 2.9 +/- 1.4 to 31.0 +/- 7.5% (P < .001). In summary, ketoconazole, a dual inhibitor of CYP3A and P-glycoprotein, can effectively increase K02 oral bioavailability by inhibiting the CYP3A/P-gp absorption barrier in the small intestine.
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PMID:Effects of ketoconazole on the intestinal metabolism, transport and oral bioavailability of K02, a novel vinylsulfone peptidomimetic cysteine protease inhibitor and a P450 3A, P-glycoprotein dual substrate, in male Sprague-Dawley rats. 976 44

Cytochrome P450 3A4 (CYP3A4), the major phase I drug metabolizing enzyme in humans, and the MDR1 gene product P-glycoprotein (P-gp) are present at high concentrations in villus tip enterocytes of the small intestine and share a significant overlap in substrate specificity. A large body of research both in vitro and in vivo has established metabolism by intestinal CYP3A4 as a major determinant of the systemic bioavailability of orally administered drugs. More recently it has been recognized that drug extrusion by intestinal P-gp can both reduce drug absorption and modulate the effects of inhibitors and inducers of CYP3A-mediated metabolism. There is relatively little data regarding the effects of CYP3A and P-gp on peptide drugs; however, studies with the cyclic peptide immunosuppresant cyclosporine as well as peptidomimetics such as the HIV-protease inhibitor saquinavir (Invirase) and a new cysteine protease inhibitor K02 (Morpholine-Urea-Phe-Hphe-Vinyl sulfone; Axys Pharmaceuticals) provide some insight into the impact of these systems on the oral absorption of peptides.
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PMID:Role of P-glycoprotein and cytochrome P450 3A in limiting oral absorption of peptides and peptidomimetics. 981 84

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

The objective of this work was to synthesize the cyclic prodrugs 1 and 2 of [Leu5]-enkephalin (Tyr-Gly-Gly-Phe-Leu-OH) and DADLE (Tyr-D-Ala-Gly-Phe-D-Leu-OH), respectively, using an (acyloxy)alkoxy linker. The cyclic prodrugs 1 and 2 were synthesized via a convergent method using the (acyloxy)alkoxy promoiety that connected the C- and N-terminus of the peptides. The key intermediates were compounds 6a and 9a for cyclic prodrug 1 and compounds 6b and 9b for cyclic prodrug 2. The key intermediates 6a and 9a (or 6b and 9b) were coupled to give compound 10a (or 10b). The N- and C-terminus protecting groups were removed from 10a and 10b to give compounds 11a and 11b, respectively, which were then treated with HBTU to give 1 and 2 in 40% and 53% yields, respectively. The cyclic prodrugs 1 and 2 exhibited Stokes-Einstein molecular radii similar to those of [Leu5]-enkephalin and DADLE; however, the cyclic prodrugs were shown to be significantly more lipophilic than the corresponding opioid peptides, as determined by partitioning experiments using immobilized artificial membrane (IAM) column chromatography. In addition, the cyclic prodrugs exhibit stable solution conformations, which reduce their hydrogen bonding potentials. Based on these physicochemical characteristics, the cyclic prodrugs 1 and 2 should have exhibited better transcellular flux across the Caco-2 cell monolayer than [Leu5]-enkephalin and DADLE, respectively. However, the cyclic prodrugs 1 and 2 were shown in separate studies to be substrates for P-glycoprotein, which significantly reduced their ability to permeate across Caco-2 cell monolayers. When P-glycoprotein was inhibited, the permeability characteristics of prodrugs 1 and 2 were consistent with their physicochemical properties.
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PMID:Synthesis and evaluation of the physicochemical properties of esterase-sensitive cyclic prodrugs of opioid peptides using an (acyloxy)alkoxy linker. 1040 17

In an earlier study using Caco-2 cells, an in vitro cell culture model of the intestinal mucosa, we have shown that the acyloxyalkoxy-based cyclic prodrugs 3 and 4 of the opioid peptides [Leu5]-enkephalin(1, H-Tyr-GLY-Gly-Phe-Leu-OH) and DADLE(2, H-Tyr-D-Ala-Gly-Phe-D-Leu-OH), respectively, were substrates for apically polarized efflux systems and therefore less able to permeate the cell monolayers than were the opioid peptides themselves. In an attempt to explain how structure may influence the recognition of these cyclic prodrugs as substrates by the apically polarized efflux systems, we have determined the possible solution conformations of 3 and 4 using spectroscopic techniques (2D-NMR, CD) and molecular dynamics simulations. Spectroscopic as well as computational studies indicate that cyclic prodrug 4 exhibits a major and a minor conformer in a ratio of 3:2 where both conformers exhibit gamma and beta-turn structures. Spectroscopic, as well as molecular dynamics, studies indicate that the difference between the two conformers involves a cis/trans inversion occurring at the amide bond between the promoiety and Tyr1. The major conformer has a trans amide bond between the promoiety and Tyr1, whereas the minor conformer has a cis amide bond. The spectroscopic data indicate that cyclic prodrug 3 has a structure similar to that of the major conformer in cyclic prodrug 4. It has recently been reported that a particular arrangement of polar groups and spatial separation distances is required for substrate recognition by P-glycoprotein. When the conformation of the acyloxyalkoxy linker was investigated in the major and minor conformers of cyclic prodrug 4, with respect to distances between the polar functional groups, this ideal fixed spatial orientation was observed. Interestingly this same spatial orientation of polar functional groups was not observed for other cyclic prodrugs prepared by our laboratory using different chemical linkers (coumarinic acid and phenylpropionic acid) but the same opioid peptides that had previously been shown not to be substrates for the apically polarized efflux systems. Therefore, we hypothesize that the structure and/or the flexibility of the acyloxyalkoxy linker itself allows cyclic prodrugs 3 and 4 to adopt conformations that permit ideal arrangement of polar groups in the linker and their fixed spatial orientation. This possibly induces the substrate activity of cyclic prodrugs 3 and 4 for the apically polarized efflux systems.
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PMID:The effect of conformation of the acyloxyalkoxy-based cyclic prodrugs of opioid peptides on their membrane permeability. 1040 18

This paper describes some successful examples of a tissue selective drug delivery by utilizing specialized transporter(s) expressed in the targeted tissue cells. These are as follows: (1) oral delivery via H(+)/oligopeptide transporter, rat or human Pept1, in the intestine for beta-lactam antibiotics and a newly synthesized dipeptide, L-dopa-L-phenylalanine; (2) tumor cell specific delivery via the newly discovered H(+)/oligopeptide transporter(s) expressed in human fibrosarcoma cell line HT-1080 for model oligopeptides, glycylsarcosine and carnosine; (3) oral and hepatic delivery via an H(+)/monocarboxylate transporter in the intestine and an organic anion transporter in the liver for HMG-CoA reductase inhibitor, pravastatin; and (4) lung selective delivery via some type of transporter and avoidance of transfer into the brain via P-glycoprotein at the blood-brain barrier for a new quinolone antibacterial, HSR-903.
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PMID:Tissue selective drug delivery utilizing carrier-mediated transport systems. 1051 56

The drug-binding domain of the human multidrug resistance P-glycoprotein (P-gp) probably consists of residues from multiple transmembrane (TM) segments. In this study, we tested whether the amino acids in TM11 participate in binding drug substrates. Each residue in TM11 was initially altered by site-directed mutagenesis and assayed for drug-stimulated ATPase activity in the presence of verapamil, vinblastine, or colchicine. Mutants G939V, F942A, T945A, Q946A, A947L, Y953A, A954L, and G955V had altered drug-stimulated ATPase activities. Direct evidence for binding of drug substrate was then determined by cysteine-scanning mutagenesis of the residues in TM11 and inhibition of drug-stimulated ATPase activity by dibromobimane, a thiol-reactive substrate. Dibromobimane inhibited the drug-stimulated ATPase activities of two mutants, F942C and T945C, by more than 75%. These results suggest that residues Phe(942) and Thr(945) in TM11, together with residues previously identified in TM6 (Leu(339) and Ala(342)) and TM12 (Leu(975), Val(982), and Ala(985)) (Loo, T. W., and Clarke, D. M. (1997) J. Biol. Chem. 272, 31945-31948) form part of the drug-binding domain of P-gp.
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PMID:Identification of residues in the drug-binding domain of human P-glycoprotein. Analysis of transmembrane segment 11 by cysteine-scanning mutagenesis and inhibition by dibromobimane. 1058 7

In this study, we further characterize a mutant P-glycoprotein (P-gp) that has a deletion of Phe(335) and is resistant to inhibition by cyclosporins. Photoaffinity labeling with [(3)H]cyclosporine and [(3)H]azidopine revealed markedly decreased binding to the mutant P-gp compared with wild-type P-gp. Expression of the mutant P-gp in multidrug-resistant variant cell line MES-SA/DxP (DxP) cells was associated with a 2-fold higher basal ATPase activity relative to multidrug-resistant cell line MES-SA/Dx5 (Dx5) cells with wild-type P-gp. Cyclosporine inhibited ATPase activity in both cell types, whereas the cyclosporin D analog valspodar (PSC 833), vinblastine, and dactinomycin stimulated ATPase activity in Dx5 but not in mutant DxP cells. Moreover, the cell lines differed in their responses to verapamil, which produced greater stimulation of ATPase in Dx5 than DxP cells. Verapamil significantly reversed the [(3)H]daunorubicin accumulation defect in wild-type Dx5 cells, but it had no significant effect on [(3)H]daunorubicin accumulation in the mutant DxP cells. Verapamil was not transported by cells expressing either mutant or wild-type P-gp. Vanadate trapping of azido-ATP was markedly impaired in mutant P-gp. In conclusion, our data demonstrate that Phe(335) of transmembrane 6 is an important amino acid residue for the formation of cyclosporine and azidopine drug-binding site(s). Phe(335) also plays a role in the coupling of verapamil binding and modulation of daunorubicin intracellular accumulation in wild-type P-gp. In addition, Phe(335) in transmembrane 6 may play a role in coupling drug binding to ATPase activity. The deletion of Phe(335) results in a significant increase in the basal ATPase activity with a concomitant decrease in its ability to trap ATP and transport some P-gp substrates.
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PMID:Loss of cyclosporin and azidopine binding are associated with altered ATPase activity by a mutant P-glycoprotein with deleted phe(335). 1072 24

The presence in orange juice of compounds that specifically inhibit the P-glycoprotein (P-gp) drug efflux transporter, but not the cytochrome P450 (CYP) isozyme CYP3A4, was investigated. The uptake of [(3)H]vinblastine, a substrate of P-gp, by Caco-2 cells was measured. An ethyl acetate extract of orange juice did not affect the initial uptake rate of [(3)H]vinblastine but significantly increased the steady-state uptake, as did cyclosporin A (20 microM), an inhibitor of P-gp. No significant effect on the uptake of 3-O-[(3)H]methylglucose or [(14)C]phenylalanine by Caco-2 cells was found, compared with the control. When the extract was separated on a Cosmosil column, the eluate with 70% methanol showed the most potent ability to increase [(3)H]vinblastine uptake. Additional separation of the 70% methanol eluate on a silica gel column with hexane-acetone (3:1) gave 3,3',4',5,6,7,8-heptamethoxyflavone (HMF) and 4',5,6,7,8-pentamethoxyflavone (tangeretin). HMF, tangeretin, and 3',4',5,6,7,8-hexamethoxyflavone (nobiletin), another methoxyflavone contained in orange juice, all increased the steady-state uptake of [(3)H]vinblastine by Caco-2 cells in a concentration-dependent manner. The order of potency of these compounds at the concentration of 50 microM was tangeretin > HMF > nobiletin. None of these methoxyflavones inhibited 6beta-hydroxylation of testosterone catalyzed by CYP3A4. The ethyl acetate extract of orange juice and these methoxyflavones also increased steady-state [(3)H]vinblastine uptake by LLC-GA5-COL300 cells (a cell line transfected with human MDR1 cDNA). We conclude that these methoxyflavones enhanced vinblastine uptake by specifically inhibiting drug efflux via P-gp. They may have potential as agents for reversing multidrug resistance or for recovering the bioavailability of certain drugs.
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PMID:Polymethoxylated flavones in orange juice are inhibitors of P-glycoprotein but not cytochrome P450 3A4. 1073 74


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