Drug-drug interactions: Effect of quinidine on nifedipine binding to human cytochrome P450 3A4

doi:10.1016/S0006-2952(96)00836-2

Biochemical Pharmacology

Volume 53, Issue 4, 21 February 1997, Pages 455-460

https://doi.org/10.1016/S0006-2952(96)00836-2 Get rights and content

Abstract

Quinidine is a known inhibitor of cytochrome P450-mediated nifedipine metabolism. The interactions of nifedipine and quinidine with human cytochrome P450 3A4, which metabolizes these drugs, were examined using the kinetics of CO binding to this P450 as a rapid kinetic probe of protein conformation and dynamics. This approach showed that nifedipine and quinidine bind to different P450 3A4 species, respectively termed species I and II, with distinct conformations. When both drugs were present simultaneously, nifedipine interacted with the quinidine-bound P450 species II, but not species I. These findings indicate that quinidine acts as an allosteric inhibitor by switching nifedipine binding from nifedipine-metabolizing species I to the nonmetabolizing species II.

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Cited by (55)

Electron transfer in the complex of membrane-bound human cytochrome P450 3A4 with the flavin domain of P450BM-3: The effect of oligomerization of the heme protein and intermittent modulation of the spin equilibrium
2010, Biochimica et Biophysica Acta - Bioenergetics
Citation Excerpt :
Homo- and hetero-oligomerization of cytochromes P450 in the membrane may play an important functional role (see [14,16,25] for review). According to our hypothesis [14], the conformational and/or steric consequences of the P450–P450 interactions in the oligomers may represent the primary cause of persistent conformational heterogeneity of the enzyme in the membrane, which is known to be tightly related to the mechanisms of heterotropic cooperativity in CYP3A4 [26–29]. Such oligomerization-promoted heterogeneity may have a prominent impact on the kinetics of electron transfer to cytochromes P450 [12,29,30].
We studied the kinetics of NADPH-dependent reduction of human CYP3A4 incorporated into Nanodiscs (CYP3A4–ND) and proteoliposomes in order to probe the effect of P450 oligomerization on its reduction. The flavin domain of cytochrome P450-BM3 (BMR) was used as a model electron donor partner. Unlike CYP3A4 oligomers, where only 50% of the enzyme was shown to be reducible by BMR, CYP3A4–ND could be reduced almost completely. High reducibility was also observed in proteoliposomes with a high lipid-to-protein ratio (L/P = 910), where the oligomerization equilibrium is displaced towards monomers. In contrast, the reducibililty in proteoliposomes with L/P = 76 did not exceed 55 ± 6%. The effect of the surface density of CYP3A4 in proteoliposomes on the oligomerization equilibrium was confirmed with a FRET-based assay employing a cysteine-depleted mutant labeled on Cys-468 with BODIPY iodoacetamide. These results confirm a pivotal role of CYP3A4 oligomerization in its functional heterogeneity. Furthermore, the investigation of the initial phase of the kinetics of CYP3A4 reduction showed that the addition of NADPH causes a rapid low-to-high-spin transition in the CYP3A4–BMR complex, which is followed by a partial slower reversal. This observation reveals a mechanism whereby the CYP3A4 spin equilibrium is modulated by the redox state of the bound flavoprotein.
Kinetics of electron transfer in the complex of cytochrome P450 3A4 with the flavin domain of cytochrome P450BM-3 as evidence of functional heterogeneity of the heme protein
2008, Archives of Biochemistry and Biophysics
Citation Excerpt :
Therefore, the suggested regulatory mechanism based on incomplete reducibility of P450 in the oligomers may have evolved to maintain the balance between the monooxygenase activity of cytochromes P450 and the production of ROS. In summary, our results provide important evidence that the mechanism of action of heterotropic activators, such as ANF, involves redistribution of the CYP3A4 among several stable conformers and activation of an otherwise inactive subpopulation of the enzyme, as hypothesized by Koley and co-authors [36]. Admittedly, the use of oligomers of CYP3A4 in solution in a pair with BMR as a model electron donor does not allow direct extrapolation of our results to the membranous microsomal system.
We used a rapid scanning stop-flow technique to study the kinetics of reduction of cytochrome P450 3A4 (CYP3A4) by the flavin domain of cytochrome P450-BM3 (BMR), which was shown to form a stoichiometric complex (K_D = 0.48 μM) with CYP3A4. In the absence of substrates only about 50% of CYP3A4 was able to accept electrons from BMR. Whereas the high-spin fraction was completely reducible, the reducibility of the low-spin fraction did not exceed 42%. Among four substrates tested (testosterone, 1-pyrenebutanol, bromocriptine, or α-naphthoflavone (ANF)) only ANF is capable of increasing the reducibility of the low-spin fraction to 75%. Our results demonstrate that the pool of CYP3A4 is heterogeneous, and not all P450 is competent for electron transfer in the complex with reductase. The increase in the reducibility of the enzyme in the presence of ANF may represent an important element of the mechanism of action of this activator.
Role of subunit interactions in P450 oligomers in the loss of homotropic cooperativity in the cytochrome P450 3A4 mutant L211F/D214E/F304W
2007, Archives of Biochemistry and Biophysics
The contribution of conformational heterogeneity to cooperativity in cytochrome P450 3A4 was investigated using the mutant L211F/D214E/F304W. Initial spectral studies revealed a loss of cooperativity of the 1-pyrenebutanol (1-PB) induced spin shift (S₅₀ = 5.4 μM, n = 1.0) but retained cooperativity of α-naphthoflavone binding. Continuous variation (Job’s titration) experiments showed the existence of two pools of enzyme with different 1-PB binding characteristics. Monitoring of 1-PB binding by fluorescence resonance energy transfer from the substrate to the heme confirmed that the high-affinity site (K_D = 0.3 μM) is retained in at least some fraction of the enzyme, although cooperativity is masked. Removal of apoprotein on a second column increased the high-spin content and restored cooperativity of 1-PB binding and of progesterone and testosterone 6β-hydroxylation. The loss of cooperativity in the mutant is, therefore, mediated by the interaction of holo- and apo-P450 in mixed oligomers.
Multiple sequential steps involved in the binding of inhibitors to cytochrome P450 3A4
2007, Journal of Biological Chemistry
Citation Excerpt :
Although it is not possible to exclude binding of multiple ligands at significantly higher concentrations, this outcome suggests a 1:1 binding stoichiometry between clotrimazole and P450 3A4, at least in the concentration range studied. Sequential-mixing Stopped-flow Experiments—One possible model proposed for ligand binding to P450 3A4 involves the existence of multiple enzyme populations in dynamic equilibrium (Scheme 1B) (1, 42-45, 62). In principle, multi-phasic kinetics could be the result of different rates of binding of a ligand to individual enzyme species.
Cytochrome P450 (P450) 3A4 is an extensively studied human enzyme involved in the metabolism of >50% of drugs. The mechanism of the observed homotropic and heterotropic cooperativity in P450 3A4-catalyzed oxidations is not well understood, and together with the cooperative behavior, a detailed understanding of interaction of drug inhibitors with P450 3A4 is important in predicting clinical drug-drug interactions. The interactions of P450 3A4 with several structurally diverse inhibitors were investigated using both kinetic and thermodynamic approaches to resolve the steps involved in binding of these ligands. The results of pre-steady-state absorbance and fluorescence experiments demonstrate that inhibitor binding is clearly a multistep process, even more complex than the binding of substrates. Based on spectrophotometric equilibrium binding titrations as well as isothermal titration calorimetry experiments, the stoichiometry of binding appears to be 1:1 in the concentration ranges studied. Using a sequential-mixing stopped-flow approach, we were also able to show that the observed multiphasic binding kinetics is the result of sequential events as opposed to the existence of multiple enzyme populations in dynamic equilibrium that interact with ligands at different rates. We propose a three-step minimal model for inhibitor binding, developed with kinetic simulations, consistent with our previously reported model for the binding of substrates, although it is possible that even more steps are involved.
Kinetics and thermodynamics of ligand binding by cytochrome P450 3A4
2006, Journal of Biological Chemistry
Citation Excerpt :
In light of the many published papers on the basis of cooperativity of P450 3A4 and other P450s, one question is how the work presented here differs from previous studies and models. Other articles have dealt with models involving either multiple ligands bound to P450 3A4 (34, 37–39, 41), multiple conformational states in equilibrium (66–68), or a combination of both (71). A variant on the second theme is a recent proposal involving equilibrium among multiple oligomeric states (69), although exactly how such a phenomenon relates to ligand cooperativity is unclear.4
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 steady-state 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 α-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.
Resolution of two substrate-binding sites in an engineered cytochrome P450eryF bearing a fluorescent probe
2005, Biophysical Journal
Citation Excerpt :
Studies of mutual effects of different substrates and effectors of P450 3A4 have compelled some authors to suggest three or more ligand binding sites in this enzyme (15–18), although direct physical evidence is lacking. The situation becomes even more complex when various indications of conformational heterogeneity of P450 3A4 are incorporated into mechanisms of cooperativity (11,19–21), leading to a nested allostery model for this enzyme (10). This model, which is based on the presumption of two stable conformers, each possessing the properties of an allosteric enzyme with two binding sites, may provide a viable alternative to a scheme with three or more binding sites.
To elucidate the mechanisms of cooperativity of cytochrome P450eryF an SH-reactive fluorescent probe was introduced close to the substrate-binding site. Cys-154, the only accessible cysteine, was eliminated by site-directed mutagenesis, and a novel cysteine was substituted for Ser-93 in the B′/C loop. S93C, C154A, C154S, S93C/C154A, and S93C/S154C were characterized in terms of affinity for 1-pyrenebutanol (1-PB), cooperativity, and ionic-strength dependence of the 1-PB-induced spin shift. S93C/C154S retains the key functional properties of the wild-type, and modification by three different SH-reactive probes had little effect on the characteristics of the enzyme. The labeled proteins exhibited fluorescence resonance energy transfer from 1-PB to the label, which allowed us to resolve two substrate-binding events, and to determine the corresponding K_D values (K_D1 = 1.2 ± 0.2 μM, K_D2 = 9.4 ± 0.8 μM). Using these values for analysis of the substrate-induced spin transition, we demonstrate that the interactions of P450eryF with 1-PB are consistent with a sequential binding mechanism, where substrate interactions at a higher-affinity site cause a conformational transition crucial for the binding of the second substrate molecule and subsequent spin shift. This transition is apparently associated with an important rearrangement of the system of salt links in the proximity of Cys-154.

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Rapid communicationDrug-drug interactions: Effect of quinidine on nifedipine binding to human cytochrome P450 3A4

Abstract

Toxicology

Br J Anaesth

Biochimie

Anal Biochem

J Biol Chem

Trends Pharmacol Sci

Drug Interactions: A Source Book of Adverse Interactions, Their Mechanisms, Clinical Importance and Management

Mechanisms of Drug Interactions

Differential effects of quinidine on the disposition of nifedipine, sparteine, and mephenytoin in humans

Clin Pharmacol Ther

Rapid communication
Drug-drug interactions: Effect of quinidine on nifedipine binding to human cytochrome P450 3A4