Energetics of heterotropic cooperativity between α-naphthoflavone and testosterone binding to CYP3A4

Roberts, Arthur G.; Atkins, William M.

doi:10.1016/j.abb.2007.03.006

Cited by 43 publications

(57 citation statements)

References 57 publications

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“…5C. When thalidomide docks near the heme iron and/or suitable substrate binding area (Roberts and Atkins, 2007;Skopalík et al, 2008), the proposed substrate pocket of P450 3A5 would be affected, resulting in a large active site. On the other hand, because thalidomide apparently does not dock closely to the heme of the P450 3A4 structure (Fig.…”

Section: Discussionmentioning

confidence: 99%

Drug Interactions of Thalidomide with Midazolam and Cyclosporine A: Heterotropic Cooperativity of Human Cytochrome P450 3A5

Okada

Murayama²,

Yanagida³

et al. 2008

Drug Metab Dispos

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ABSTRACT:There is growing clinical interest of thalidomide because of its immunomodulatory and antiangiogenic properties, despite its teratogenicity. However, little information about thalidomide has been reported regarding its precise effects on drug-metabolizing enzymes. We investigated the effects of thalidomide on cytochrome P450 (P450) enzymes in human liver microsomes to clarify the potential for possible drug interactions. Thalidomide inhibited S-mephenytoin 4-hydroxylation activities of recombinant P450 2C19 and human liver microsomes: the apparent concentration of thalidomide producing 50% inhibition was approximately 270 M for P450 2C19. Midazolam 4-hydroxylation activities were suppressed by thalidomide, but activities of 1-hydroxylation and total midazolam oxidation and testosterone 6␤-hydroxylation were enhanced in the presence of thalidomide. Recombinant P450 3A5 was found to have altered kinetics at clinically relevant concentrations of thalidomide (10-30 M). P450 3A4 was also affected, but only at higher thalidomide concentrations. Enhanced midazolam hydroxylation by thalidomide was also seen in liver microsomal samples harboring the CYP3A5*1 allele. Similarly enhanced rates of cyclosporine A clearance were observed in P450 3A5 and liver microsomes expressing P450 3A5 in the presence of thalidomide. A proposed effector constant for thalidomide corresponded roughly to its clinical plasma levels. Docking studies with a P450 3A5 homology model, based on the published structure of P450 3A4, revealed close interaction between thalidomide and the heme of P450 3A5. The present results suggest that total midazolam metabolism or cyclosporine A clearance may be increased by thalidomide in a dose-dependent manner. Unexpected drug interactions involving thalidomide might occur via heterotropic cooperativity of polymorphic P450 3A5.

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Section: Discussionmentioning

confidence: 99%

Drug Interactions of Thalidomide with Midazolam and Cyclosporine A: Heterotropic Cooperativity of Human Cytochrome P450 3A5

Okada

Murayama²,

Yanagida³

et al. 2008

Drug Metab Dispos

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“…4c). Data from this titration were fit to the model in Scheme 3, and the recovered K 1 Ј value was 5.8 Ϯ 0.4 M. ANF binding to CYP3A4 in solution has been studied extensively (43)(44)(45)(46)(47)(48) and is thought to be cooperative in nature with at least two distinct ANF-binding sites in each CYP3A4 monomer. This interaction has been described frequently (43,46,48) values and cannot be directly compared with K 1 Ј values.…”

Section: Resultsmentioning

confidence: 99%

Ligand Binding to Cytochrome P450 3A4 in Phospholipid Bilayer Nanodiscs

Nath

Grinkova

Sligar

et al. 2007

Journal of Biological Chemistry

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The membrane-bound protein cytochrome P450 3A4 (CYP3A4) is a major drug-metabolizing enzyme. Most studies of ligand binding by CYP3A4 are currently carried out in solution, in the absence of a model membrane. Therefore, there is little information concerning the membrane effects on CYP3A4 ligand binding behavior. Phospholipid bilayer Nanodiscs are a novel model membrane system derived from high density lipoprotein particles, whose stability, monodispersity, and consistency are ensured by their self-assembly. We explore the energetics of four ligands (6-(p-toluidino)-2-naphthalenesulfonic acid (TNS), ␣-naphthoflavone (ANF), miconazole, and bromocriptine) binding to CYP3A4 incorporated into Nanodiscs. Ligand binding to Nanodiscs was monitored by a combination of environment-sensitive ligand fluorescence and ligand-induced shifts in the fluorescence of tryptophan residues present in the scaffold proteins of Nanodiscs; binding to the CYP3A4 active site was monitored by ligand-induced shifts in the heme Soret band absorbance. The dissociation constants for binding to the active site in CYP3A4-Nanodiscs were 4.0 M for TNS, 5.8 M for ANF, 0.45 M for miconazole, and 0.45 M for bromocriptine. These values are for CYP3A4 incorporated into a lipid bilayer and are therefore presumably more biologically relevant that those measured using CYP3A4 in solution. In some cases, affinity measurements using CYP3A4 in Nanodiscs differ significantly from solution values. We also studied the equilibrium between ligand binding to CYP3A4 and to the membrane. TNS showed no marked preference for either environment; ANF preferentially bound to the membrane, and miconazole and bromocriptine preferentially bound to the CYP3A4 active site. Cytochrome P450 3A4 (CYP3A4)2 is a major drug-metabolizing enzyme, and its ligand binding and catalysis are therefore of wide interest. CYP3A4 is an integral membrane protein, interacting with the membrane via an embedded N-terminal helix and other hydrophobic surface regions; however, most investigations of ligand binding are currently carried out in solution using recombinant protein with a partially truncated helical anchor, in the absence of any model membrane. Therefore, there is a critical lack of understanding of how the membrane environment affects ligand binding to CYP3A4. Membrane effects may be especially important when in vitro binding or kinetic data are extrapolated to predictions of in vivo pharmacokinetics.Model membranes can profoundly alter the ligand binding behavior of membrane proteins. For example, the choice of model membranes can shift the apparent affinity of a spider venom toxin for voltage-dependent K ϩ channels more than 4 orders of magnitude (1). Apart from the structural and dynamic effects of incorporation into a model membrane, competition for ligand binding between a model membrane and an incorporated protein can alter the apparent affinity and stoichiometry of ligand binding by the protein in equilibrium and kinetic experiments.Parry et al. (2) presented separate analytical ...

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“…We have previously proposed a two-site sequential model for ANF binding to CYP3A4, with K D s of 1 µM and 55 µM [38]. Global fits (R 2 = 0.97) of both isotherms in Figure 5C The isotherms for the TST competition experiment are not well-fit by the two-site model employed above for ANF.…”

Section: Effectors Compete With the 'Red' Speciesmentioning

confidence: 99%

“…Furthermore, there is potential for communication between this site and the active site via the F-G region, which provides a 'wall' between the surface and active sites, wherein conformational rearrangement of the wall may control access to the active site. In addition to the crystallographic results, spectroscopic studies and energetic analyses [11,38] of the ligand-dependent spin equilibrium suggest that TST and ANF populate a high affinity site that is not tightly coupled to the spin equilibrium, followed by a lower affinity site that induces a larger spin state change. Together, the available data suggest that TST and ANF bind with highest affinity to a site other than the active site, and we have suggested that ANF binds to the TST/progesterone site.…”

Section: Effectors Compete With the 'Red' Speciesmentioning

confidence: 99%

Spectral resolution of a second binding site for Nile Red on cytochrome P4503A4

Nath

Fernández

Lampe

et al. 2008

Archives of Biochemistry and Biophysics

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Nile Red is sequentially metabolized by Cytochrome P4503A4 to the N-monoethyl and N-desethyl products, which typifies the metabolism of many amine-containing drugs. Sequential metabolism of a single substrate results in complex kinetics that confound predictive models of drug clearance. As a fluorescent model for drugs which undergo sequential metabolism, Nile Red provides the opportunity to monitor drug-CYP interactions wherein the fluorescent properties of Nile Red could, in principle, be exploited to determine individual rate and equilibrium constants for the individual reactions. Previously, it was shown that Nile Red binds at the active site and fluoresces (K D = 50 nM) with maximum emission at ~620 nm, but it was unclear whether a red-shifted emission, at ~660 nm, consisted of only free Nile Red or Nile Red bound at a second site on the protein. Here, equilibrium binding studies, including 'reverse titrations' spanning low ratios of CYP3A4/Nile Red, indicate two binding sites for Nile Red with a contribution to the 'red emission' greater than can be accounted for by free Nile Red. Singular value decomposition affords basis spectra for both spectral components and fits well to the experimentally determined concentration dependence of Nile Red emission. In addition, the red spectral component, with an apparent K D = 2.2 µM, is selectively eliminated by titration with the known allosteric effectors of CYP3A4, α-napthoflavone and testosterone. Furthermore, the double mutant L2311F/D214E, previously demonstrated to perturb a peripheral allosteric site, lacks the red-emitting Nile Red binding site, but retains the blue-emitting site. Together these data indicate that a second Nile Red site competes with other effectors of CYP3A4 at a site that results in Nile Red emission at 660 nm. KeywordsCytochrome P450 3A4; drug metabolism; allosterism; homotropic effectors; Nile Red; solvatochromism As detoxification enzymes, Cytochrome P450's (CYPs) 1 catalyze a wide range of oxidative and reductive reactions with an enormous range of structurally distinct substrates [1][2][3][4]. CYPs dominate the metabolism of nearly all prescribed therapeutic drugs, as well as a number of endogenous hormones and xenobiotics, and thus control their steady state plasma and tissue levels [1,5,6]. Their ability to perform complex oxidative metabolism on a structurally diverse set of compounds is often accompanied by complex non-Michaelis-Menten, or "allosteric", kinetics [7][8][9]. The predominant human hepatic drug metabolizing CYP isoform, CYP 3A4, *e-mail: winky@u.washington.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaim...

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Energetics of heterotropic cooperativity between α-naphthoflavone and testosterone binding to CYP3A4

Cited by 43 publications

References 57 publications

Drug Interactions of Thalidomide with Midazolam and Cyclosporine A: Heterotropic Cooperativity of Human Cytochrome P450 3A5

Drug Interactions of Thalidomide with Midazolam and Cyclosporine A: Heterotropic Cooperativity of Human Cytochrome P450 3A5

Ligand Binding to Cytochrome P450 3A4 in Phospholipid Bilayer Nanodiscs

Spectral resolution of a second binding site for Nile Red on cytochrome P4503A4

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