High affinity of ergopeptides for cytochromes <i>P</i>450 3A

Peyronneau, Marie-Anne; Delaforge, Marcel; Riviere, René; Renaud, Jean‐Paul; Mansuy, Daniel

doi:10.1111/j.1432-1033.1994.tb19072.x

Cited by 51 publications

(50 citation statements)

References 44 publications

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“…Bromocriptine, the most bulky of the substrates studied, had the highest apparent affinity (K s ϭ 0.4 M), consistent with a previously reported dissociation constant (16). The titration plot obtained for flavone binding to P450 3A4 displayed apparent sigmoidicity (supplementary data).…”

Section: Resultssupporting

confidence: 87%

Kinetics and Thermodynamics of Ligand Binding by Cytochrome P450 3A4

Isin

Guengerich

2006

Journal of Biological Chemistry

134

195

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Cytochrome P450 (P450) 3A4, the major catalyst involved in human drug oxidation, displays substrate-and reaction-dependent homotropic and heterotropic cooperative behavior. Although several models have been proposed, these mainly rely on steadystate kinetics and do not provide information on the contribution of the individual steps of P450 catalytic cycle to the observed cooperativity. In this work, we focused on the kinetics of substrate binding, and the fluorescent properties of bromocriptine and ␣-naphthoflavone allowed analysis of an initial ligand-P450 3A4 interaction that does not cause a perturbation of the heme spectrum. The binding stoichiometry for bromocriptine was determined to be unity using isothermal titration calorimetry and equilibrium dialysis methods, suggesting that the ligand bound to the peripheral site during the initial encounter dissociates subsequently. A three-step substrate binding model is proposed, based on absorbance and fluorescence stopped-flow kinetic data and equilibrium binding data obtained with bromocriptine, and evaluated using kinetic modeling. The results are consistent with the substrate molecule binding at a site peripheral to the active site and subsequently moving toward the active site to bind to the heme and resulting in a low to high spin iron shift. The last step is attributed to a conformational change in the enzyme active site. The later steps of binding were shown to have rate constants comparable with the subsequent steps of the catalytic cycle. The P450 3A4 binding process is more complex than a two-state system, and the overlap of rates of some of the events with subsequent steps is proposed to underlie the observed cooperativity.Cytochrome P450 (P450) 2 enzymes are found throughout nature, from bacteria to humans. These enzymes generally catalyze mixed function oxidation reactions that have similar chemistry or else utilize parts of the general catalytic mechanism for reductions and rearrangements (2, 3). The wide diversity of substrates of these enzymes and the basis of catalytic selectivity is a topic of considerable interest in the context of both basic biochemistry and practical applications (4). P450 3A4 is one of the most widely studied of the 57 human P450s (5), mainly due to its role in the metabolism of more than one-half of the drugs on the market as well as various endogenous and exogenous molecules (6, 7). In addition, P450 3A4 is the major P450 expressed in liver (8) and in the intestine (9). Recently solved P450 3A4 crystal structures (10 -12) demonstrate the presence of a large active site, consistent with the broad range of substrates that P450 3A4 can accommodate (7, 13), including (in order of increasing size) acetaminophen (14) (M r 151), testosterone (15) (M r 288), bromocriptine (16) (M r 655), and cyclosporin (17) (M r 1201). Despite its seemingly flexible substrate selectivity, P450 3A4 displays a high degree of regio-and stereoselectivity in many substrate oxidations (18,19).One of the important features of P450 3A4 is its cooperativ...

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Section: Resultssupporting

confidence: 87%

Kinetics and Thermodynamics of Ligand Binding by Cytochrome P450 3A4

Isin

Guengerich

2006

Journal of Biological Chemistry

134

195

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“…Single-use 20 mM aliquots in dimethylsulfoxide were stored at 220°C. Enzymatic synthesis of 8'-OH-dihydroergotamine (8'-OH-DHE) from DHE was performed according to published methods (Maurer and Frick, 1984a;Peyronneau et al, 1994). Test incubations were conducted with dexamethasone pretreated rat and human liver microsomes.…”

Section: Methodsmentioning

confidence: 99%

Kinetics of 5-HT_2BReceptor Signaling: Profound Agonist-Dependent Effects on Signaling Onset and Duration

Unett

Gatlin

Anthony

et al. 2013

J Pharmacol Exp Ther

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The kinetics of drug-receptor interactions can profoundly influence in vivo and in vitro pharmacology. In vitro, the potencies of slowly associating agonists may be underestimated in assays capturing transient signaling events. When divergent receptormediated signaling pathways are evaluated using combinations of equilibrium and transient assays, potency differences driven by kinetics may be erroneously interpreted as biased signaling. In vivo, drugs with slow dissociation rates may display prolonged physiologic effects inconsistent with their pharmacokinetic profiles. We evaluated a panel of 5-hydroxytryptamine 2B (5-HT 2B ) receptor agonists in kinetic radioligand binding assays and in transient, calcium flux assays, and inositol phosphate accumulation assays; two functional readouts emanating from Ga qmediated activation of phospholipase C. In binding studies, ergot derivatives demonstrated slow receptor association and dissociation rates, resulting in significantly reduced potency in calcium assays relative to inositol phosphate accumulation assays. Ergot potencies for activation of extracellular signalregulated kinases 1 and 2 were also highly time-dependent. A number of ergots produced wash-resistant 5-HT 2B signaling that persisted for many hours without appreciable loss of potency, which was not explained simply by slow receptordissociation kinetics. Mechanistic studies indicated that persistent signaling originated from internalized or sequestered receptors. This study provides a mechanistic basis for the long durations of action in vivo and wash-resistant effects in ex vivo tissue models often observed for ergots. The 5-HT 2B agonist activity of a number of ergot-derived therapeutics has been implicated in development of cardiac valvulopathy in man. The novel, sustained nature of ergot signaling reported here may represent an additional mechanism contributing to the valvulopathic potential of these compounds.

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“…Miconazole is an azole drug that is a known inhibitor of CYP3A4. Bromocriptine, an ergot alkaloid used as a dopamine receptor agonist, is also a substrate (28) and potent inhibitor of CYP3A4 (29). …”

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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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High affinity of ergopeptides for cytochromes P450 3A

Cited by 51 publications

References 44 publications

Kinetics and Thermodynamics of Ligand Binding by Cytochrome P450 3A4

Kinetics and Thermodynamics of Ligand Binding by Cytochrome P450 3A4

Kinetics of 5-HT_2BReceptor Signaling: Profound Agonist-Dependent Effects on Signaling Onset and Duration

Ligand Binding to Cytochrome P450 3A4 in Phospholipid Bilayer Nanodiscs

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High affinity of ergopeptides for cytochromes P450 3A

Cited by 51 publications

References 44 publications

Kinetics and Thermodynamics of Ligand Binding by Cytochrome P450 3A4

Kinetics and Thermodynamics of Ligand Binding by Cytochrome P450 3A4

Kinetics of 5-HT2BReceptor Signaling: Profound Agonist-Dependent Effects on Signaling Onset and Duration

Ligand Binding to Cytochrome P450 3A4 in Phospholipid Bilayer Nanodiscs

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Kinetics of 5-HT_2BReceptor Signaling: Profound Agonist-Dependent Effects on Signaling Onset and Duration