Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.14.13.97 (CYP3A4)
 6,365 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An antipeptide antibody has been produced that recognizes CYP3A4 and exhibits greater than 90-95% inhibition on CYP3A4-mediated reactions [Wang RW and Lu AYH (1997) Drug Metab Dispos 25:762-767]. The inhibitory epitope of the 21-amino acid peptide, corresponding to residues 253 to 273 of CYP3A4, has been identified to reside in a 7-amino acid sequence (LEDTQKH: residues 261-267 of CYP3A4). This conclusion was based on the reversal of antibody inhibition of testosterone 6beta-hydroxylation when peptides with overlapping sequence in this region were preincubated with the antibody. In immunoblotting analysis, this antibody did not recognize CYP3A5 or CYP3A7 in microsomes prepared from baculovirus-infected cells containing these two expressed isoforms. In addition, the antipeptide antibody did not inhibit testosterone 6beta-hydroxylation or midazolam 1'- and 4-hydroxylation in microsomes containing expressed CYP3A5 and CYP3A7. Because the corresponding sequence in CYP3A5 (LNDKQKH) and CYP3A7 (LKETQKH) differs from CYP3A4 by only two amino acids, six peptides with either one or two amino acid changes were used to determine which amino acid is essential for antibody-antigen interaction. Our data indicate that Glu, Asp, and Thr in the 7-amino acid sequence of CYP3A4 are critical determinants of selectivity among CYP3A isoforms.
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PMID:Inhibitory anti-CYP3A4 peptide antibody: mapping of inhibitory epitope and specificity toward other CYP3A isoforms. 992 98

In an E. coli expression system for human cytochrome P450 3A7 (CYP3A7), holo-CYP3A7 was not expressed as judged by CO-difference spectra, although apo-CYP3A7 was clearly detected by Western blot analysis. Unlike CYP3A7, CYP3A4 was expressed efficiently as a hemoprotein in E. coli transformed with a CYP3A4 expression plasmid. To achieve the high yield of the holo-CYP3A7 in E. coli, we examined a causal residue(s) preventing the expression of the holo-CYP3A7 using the chimeric gene of CYP3A4 with CYP3A7. It was found that the region between residues 405 and 503 of CYP3A7 was responsible for the prevention of the holo-CYP3A7 expression in E. coli. Among amino acids examined, substitution of Thr at position 485 in CYP3A7 with Pro, which is at the corresponding position of CYP3A4, resulted in an increase in the amount of holo-CYP3A7. The Thr residue was adjacent to the heme-binding region of CYP3A7. Thus, it appeared that the incorporation of heme into CYP3A7 was possibly affected by this particular amino acid residue. Moreover, holo-CYP3A7 was expressed efficiently when CYP3A7 was co-expressed with molecular chaperone GroEL, known to assist the correct folding of unfolded proteins. Dehydroepiandrosterone 16alpha-hydroxylation was catalyzed by CYP3A7 expressed in the presence of GroEL.
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PMID:Development of bacterial expression system with high yield of CYP3A7, a human fetus-specific form of cytochrome P450. 1070 4

A series of antipeptide antibodies directed against CYP2D6 were produced by immunizing rabbits with peptides that were sterically unrestrained (linear) or conformationally restricted by cyclization. A variety of sites within the region comprising residues 254 to 290 of CYP2D6 were targeted. In immunoblotting studies, each of the antibodies against the linear and cyclic peptides recognized only a single immunoreactive band of 54 kDa in human liver microsomal fraction and bound to recombinant CYP2D6, but not recombinant CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2E1, or CYP3A4. However, the relative intensity of immunoreactive bands was considerably stronger for those antibodies raised against cyclic peptides. Similarly, in an enzyme-linked immunosorbent assay, antibodies raised against cyclic peptides bound 10 to 100 times more strongly to recombinant CYP2D6 than antibodies raised against the corresponding linear peptides. None of the antibodies raised against linear peptides had any effect on debrisoquine 4-hydroxylase activity of human hepatic microsomal fraction; however, anticyclic peptide antibodies targeted against residues 254 to 273, 261 to 272, and 257 to 268 of CYP2D6 inhibited enzyme activity by a maximum of 60, 75, and 91%, respectively. In contrast, despite binding strongly to CYP2D6, an anticyclic peptide antibody directed against residues 278 to 290 did not inhibit enzyme activity. The epitope of the proinhibitory anticyclic peptide antibody directed against residues 257 to 268 of CYP2D6 included Thr-261 and Trp-262, and indicates a role for these residues in enzyme inhibition. In conclusion, immunization with peptides conformationally restricted by cyclization to mimic loop regions of CYP2D6 resulted in strongly binding antibodies that when targeted appropriately were able to inhibit CYP2D6-catalyzed activity.
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PMID:Affinity and potency of proinhibitory antipeptide antibodies against CYP2D6 is enhanced using cyclic peptides as immunogens. 1077 33

Cytochrome P450(eryF) (CYP107A1), which hydroxylates deoxyerythronolide B in erythromycin biosynthesis, lacks the otherwise highly conserved threonine that is thought to promote O-O bond scission. The role of this threonine is satisfied in P450(eryF) by a substrate hydroxyl group, making deoxyerythronolide B the only acceptable substrate. As shown here, replacement of Ala(245) by a threonine enables the oxidation of alternative substrates using either H(2)O(2) or O(2)/spinach ferredoxin/ferredoxin reductase as the source of oxidizing equivalents. Testosterone is oxidized to 1-, 11alpha-, 12-, and 16alpha-hydroxytestosterone. A kinetic solvent isotope effect of 2.2 indicates that the A245T mutation facilitates dioxygen bond cleavage. This gain-of-function evidence confirms the role of the conserved threonine in P450 catalysis. Furthermore, a Hill coefficient of 1.3 and dependence of the product distribution on the testosterone concentration suggest that two testosterone molecules bind in the active site, in accord with a published structure of the P450(eryF)-androstenedione complex. P450(eryF) is thus a structurally defined model for the catalytic turnover of multiply bound substrates proposed to occur with CYP3A4. In view of its large active site and defined structure, catalytically active P450(eryF) mutants are also attractive templates for the engineering of novel P450 activities.
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PMID:An A245T mutation conveys on cytochrome P450eryF the ability to oxidize alternative substrates. 1095 54

Midazolam (MDZ) oxidation by recombinant CYP3A4 purified from Escherichia coli and 30 mutants generated at 15 different substrate recognition site positions has been studied to determine the role of individual residues in regioselectivity and to investigate the possible existence of multiple binding sites. Initial results showed that oxidation of MDZ by CYP3A4 causes time- and concentration-dependent enzyme inactivation with K(I) and k(inact) values of 5.8 microM and 0.15 min(-1), respectively. The different time courses of MDZ hydroxylation by mutants that predominantly formed 1'-OH MDZ as opposed to 4-OH MDZ provided strong evidence that the 1'-OH MDZ pathway leads to CYP3A4 inactivation. Correlational analysis of 1'-OH formation versus 4-OH formation by the mutants supports the inference that the two metabolites result from the binding of MDZ at two separate sites. Thus, substitution of residues Phe-108, Ile-120, Ile-301, Phe-304, and Thr-309 with a larger amino acid caused an increase in the ratio of 1'-OH/4-OH MDZ formation, whereas substitution of residues Ser-119, Ile-120, Leu-210, Phe-304, Ala-305, Tyr-307, and Thr-309 with a smaller amino acid decreased this ratio. Kinetic analyses of nine key mutants revealed that the alteration in regioselectivity is caused by a change in kinetic parameters (V(max) and K(M)) for the formation of both metabolites in most cases. The study revealed the role of various active-site residues in the regioselectivity of MDZ oxidation, identified the metabolic pathway that leads to enzyme inactivation, and provided an indication that the two proposed MDZ binding sites in CYP3A4 may be partially overlapping.
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PMID:Midazolam oxidation by cytochrome P450 3A4 and active-site mutants: an evaluation of multiple binding sites and of the metabolic pathway that leads to enzyme inactivation. 1185 29

The main objective of the present study was to find a fluorescent substrate probe for cytochrome P450eryF (P450eryF). P450eryF is a bacterial P450 that catalyzes the hydroxylation of 6-deoxyerythronolide B at the 6S position, a necessary step in the biosynthesis of erythromycin. The lack of a conserved threonine residue in the I-helix, in contrast to other P450s, makes P450eryF unable to oxidize other substrates. A recent study [Xiang et al. (2000) J. Biol. Chem. 275, 35999-36006] has shown that the substitution of Ala-245 by threonine confers on P450eryF significant testosterone hydroxylase activity. Therefore, we investigated various known fluorescent P450 substrates with P450eryF wild-type as well as two mutants, A245S and A245T. Among the various fluorescent compounds tested, 7-benzyloxyquinoline (7-BQ) was found to be the most suitable probe for P450eryF A245T, with rates of oxidation being lower for A245S and wild-type enzyme. The steady-state kinetics of 7-BQ oxidation by A245T are sigmoidal (V(max) = 0.71 nmol/min/nmol, n = 2.18, and S(50) = 132 microM). alpha-Naphthoflavone (alpha-NF), a well-known activator of CYP3A4, did not stimulate 7-BQ oxidation by A245T, although the S(50) value for alpha-NF binding to wild-type P450eryF was similar to P450 3A4. Interestingly, spectral binding studies of wild-type P450eryF and A245T with ketoconazole and miconazole showed differential binding behaviors. Titration of wild-type with ketoconazole and miconazole and of A245T with miconazole showed the expected type-II binding. However, titration of A245T with ketoconazole produced a spectrum similar to type-I. Inhibition studies showed that both ketoconazole and miconazole are able to inhibit 7-BQ oxidation by A245T, although miconazole showed a slightly higher potency. In brief, the present study reports the discovery of 7-BQ as the first fluorescent and only the second unnatural substrate, and of miconazole as an effective P450eryF inhibitor.
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PMID:7-Benzyloxyquinoline oxidation by P450eryF A245T: finding of a new fluorescent substrate probe. 1206 48

The active site topology of heterologously expressed CYP3A4 purified from an Escherichia coli expression system was examined using phenyldiazene. Incubation of CYP3A4 with phenyldiazene and subsequent oxidation yielded all four potential N-phenylprotoporphyrin IX regioisomers derived from attack on an available nitrogen atom in pyrrole rings B, A, C, or D (N(B):N(A):N(C):N(D) = 6:73:7: 13). Further study using 28 active site mutants showed that substitution of residues closer to the heme, Ala-305, Thr-309, or Ala-370, with a larger residue caused the most drastic changes in regioisomer formation, which reflected the location of each amino acid residue replaced in a CYP3A4 homology model. Previous studies have suggested a conformational change in CYP3A4 upon binding of NADPH-cytochrome P450 reductase (CPR) or cytochrome b(5) (b(5)). Therefore, regioisomer formation was also compared in the absence of redox partners and in the presence of CPR, b(5), or both. Formation of all four regioisomers in CYP3A4 wild type, particularly the minor ones, was reduced in the presence of b(5). CPR also greatly decreased the three minor isomers but increased the major isomer significantly. The presence of b(5) and CPR restored minor isomer formation and suppressed the enhancement of N(A) formation caused by CPR alone. Interestingly, the effects of the redox partners differed among representative active site mutants. In particular, the increase in N(C) upon substitution of Ala-370 with Phe was significantly reversed in the presence of redox partners, strongly suggesting that a conformational change occurs around pyrrole ring C due to protein-protein interactions between CYP3A4 and CPR or b(5).
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PMID:Topological changes in the CYP3A4 active site probed with phenyldiazene: effect of interaction with NADPH-cytochrome P450 reductase and cytochrome b5 and of site-directed mutagenesis. 1470 33

P450cam has long served as a prototype for the cytochrome P450 (CYP) gene family. But, little is known about how substrate enters its active site pocket, and how access is achieved in a way that minimizes exposure of the reactive heme. We hypothesize that P450cam may first bind substrate transiently near the mobile F-G helix that covers the active site pocket. Such a two-step binding process is kinetically required if P450cam rarely populates an open conformation-as suggested by previous literature and the inability to obtain a crystal structure of P450cam in an open conformation. Such a mechanism would minimize exposure of the heme by allowing P450cam to stay in a closed conformation as long as possible, since only brief flexing into an open conformation would be required to allow substrate entry. To test this model, we have attempted to dock a second camphor molecule into the crystal structure of camphor-bound P450cam. The docking identified only one potential entry site pocket, a well-defined cavity on the F-helix side of the F-G flap, 16 A from the heme iron. Location of this entry site pocket is consistent with our NMR T1 relaxation-based measurements of distances for a camphor that binds in fast exchange (active site camphor is known to bind in slow exchange). Presence of a second camphor binding site is also confirmed with [(1)H-(13)C] HSQC titrations of (13)CH3-threonine labeled P450cam. To confirm that camphor can bind outside of the active site pocket, (13)CH3-S-pyridine was bound to the heme iron to physically block the active site, and to serve as an NMR chemical shift probe. Titration of this P450cam-pyridine complex confirms that camphor can bind to a site outside the active site pocket, with an estimated Kd of 43 microM. The two-site binding model that is proposed based on these data is analogous to that recently proposed for CYP3A4, and is consistent with recent crystal structures of P450cam bound to tethered-substrates, which force a partially opened conformation.
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PMID:Structural evidence for a functionally relevant second camphor binding site in P450cam: model for substrate entry into a P450 active site. 1759 43

Cytochromes P450 (P450s) incur phosphorylation. Although the precise role of this post-translational modification is unclear, marking P450s for degradation is plausible. Indeed, we have found that after structural inactivation, CYP3A4, the major human liver P450, and its rat orthologs are phosphorylated during their ubiquitin-dependent proteasomal degradation. Peptide mapping coupled with mass spectrometric analyses of CYP3A4 phosphorylated in vitro by protein kinase C (PKC) previously identified two target sites, Thr(264) and Ser(420). We now document that liver cytosolic kinases additionally target Ser(478) as a major site. To determine whether such phosphorylation is relevant to in vivo CYP3A4 degradation, wild type and CYP3A4 with single, double, or triple Ala mutations of these residues were heterologously expressed in Saccharomyces cerevisiae pep4Delta strains. We found that relative to CYP3A4wt, its S478A mutant was significantly stabilized in these yeast, and this was greatly to markedly enhanced for its S478A/T264A, S478A/S420A, and S478A/T264A/S420A double and triple mutants. Similar relative S478A/T264A/S420A mutant stabilization was also observed in HEK293T cells. To determine whether phosphorylation enhances CYP3A4 degradation by enhancing its ubiquitination, CYP3A4 ubiquitination was examined in an in vitro UBC7/gp78-reconstituted system with and without cAMP-dependent protein kinase A and PKC, two liver cytosolic kinases involved in CYP3A4 phosphorylation. cAMP-dependent protein kinase A/PKC-mediated phosphorylation of CYP3A4wt but not its S478A/T264A/S420A mutant enhanced its ubiquitination in this system. Together, these findings indicate that phosphorylation of CYP3A4 Ser(478), Thr(264), and Ser(420) residues by cytosolic kinases is important both for its ubiquitination and proteasomal degradation and suggest a direct link between P450 phosphorylation, ubiquitination, and degradation.
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PMID:A role for protein phosphorylation in cytochrome P450 3A4 ubiquitin-dependent proteasomal degradation. 1909 58

Sorafenib (Nexavar) is an orally active multikinase inhibitor that is approved in the EU for the treatment of hepatocellular carcinoma. Monotherapy with sorafenib prolongs overall survival and delays the time to progression in patients with advanced hepatocellular carcinoma who are not candidates for potentially curative treatment or transarterial chemoembolization. Sorafenib is generally well tolerated in patients with advanced hepatocellular carcinoma. Thus, sorafenib represents an important advance in the treatment of advanced hepatocellular carcinoma and is the new standard of care for this condition. The bi-aryl urea sorafenib is an oral multikinase inhibitor that inhibits cell surface tyrosine kinase receptors (e.g. vascular endothelial growth factor receptors and platelet-derived growth factor receptor-beta) and downstream intracellular serine/threonine kinases (e.g. Raf-1, wild-type B-Raf and mutant B-Raf); these kinases are involved in tumour cell proliferation and tumour angiogenesis. In vitro, dose-dependent inhibition of cell proliferation and induction of apoptosis was seen with sorafenib in human hepatocellular carcinoma cells lines. Sorafenib demonstrated dose-dependent antitumour activity in a murine xenograft model of human hepatocellular carcinoma. Steady-state plasma concentrations were reached within 7 days in patients with advanced, refractory solid tumours who received twice-daily oral sorafenib. Metabolism of sorafenib occurs primarily in the liver and is mediated via cytochrome P450 (CYP) 3A4 and uridine diphosphate glucuronosyltransferase 1A9. In advanced hepatocellular carcinoma, differences in sorafenib pharmacokinetics between Child-Pugh A and B patients were not considered clinically significant. Sorafenib may be associated with drug interactions. For example, sorafenib exposure was reduced by an average 37% with concomitant administration of the CYP3A4 inducer rifampicin (rifampin); sorafenib concentrations may also be decreased by other CYP3A4 inducers. Monotherapy with oral sorafenib 400 mg twice daily prolonged median overall survival and delayed the median time to progression in patients with advanced hepatocellular carcinoma, according to the results of two randomized, double-blind, placebo-controlled, multicentre, phase III trials (the SHARP trial and the Asia-Pacific trial). There was no significant difference between sorafenib and placebo recipients in the median time to symptomatic progression in either trial. The vast majority of patients included in these trials were Child-Pugh A. Combination therapy with sorafenib plus doxorubicin did not delay the median time to progression to a significant extent compared with doxorubicin alone in patients with advanced hepatocellular carcinoma, according to the results of a randomized, double-blind, phase II trial. However, the median durations of overall survival and progression-free survival were significantly longer in patients receiving sorafenib plus doxorubicin than in those receiving doxorubicin alone. Combination therapy with sorafenib plus tegafur/uracil or mitomycin also showed potential in advanced hepatocellular carcinoma, according to the results of noncomparative trials. Monotherapy with oral sorafenib was generally well tolerated in patients with advanced hepatocellular carcinoma, with a manageable adverse effect profile; diarrhoea and hand-foot skin reaction were consistently the most commonly occurring drug-related adverse events in clinical trials. In the SHARP trial, drug-related adverse events of any grade occurring in significantly more sorafenib than placebo recipients included diarrhoea, hand-foot skin reaction, anorexia, alopecia, weight loss, dry skin, abdominal pain, voice changes and 'other' dermatological events. A similar tolerability profile was seen in the Asia-Pacific trial. As expected given the addition of a chemotherapy agent, the adverse event profile in patients with advanced hepatocellular carcinoma who received combination therapy with sorafenib plus doxorubicin differed somewhat to that seen with sorafenib monotherapy in the SHARP trial. In patients receiving sorafenib plus doxorubicin, the most commonly occurring all-cause adverse events (all grades) included fatigue, neutropenia, diarrhoea, elevated bilirubin levels, abdominal pain, hand-foot skin reaction, left ventricular dysfunction, hypertension and febrile neutropenia.
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PMID:Sorafenib: a review of its use in advanced hepatocellular carcinoma. 1922 77


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