Effects of alcohol consumption and tobacco smoking on the composition of the ensemble of drug metabolizing enzymes and transporters in human liver.

We examined the effect of alcohol consumption and smoking on the abundance of drug-metabolizing enzymes and transporters (DMET) in human liver microsomes (HLM) isolated from liver tissues of 94 donors. Global proteomics analysis was performed and DMET protein levels were analyzed in relation to alcohol consumption levels, smoking history, and sex using non-parametric tests (p-value ≤ 0.05; cutoff of 1.

High-Throughput Assay of Cytochrome P450-Dependent Drug Demethylation Reactions and Its Use to Re-Evaluate the Pathways of Ketamine Metabolism.

In a search for a reliable, inexpensive, and versatile technique for high-throughput kinetic assays of drug metabolism, we elected to rehire an old-school approach based on the determination of formaldehyde (FA) formed in cytochrome P450-dependent demethylation reactions. After evaluating several fluorometric techniques for FA detection, we chose the method based on the Hantzsch reaction with acetoacetanilide as the most sensitive, robust, and adaptable to high-throughput implementation. Here we provide a detailed protocol for using our new technique for automatized assays of cytochrome P450-dependent drug demethylations and discuss its applicability for high-throughput scanning of drug metabolism pathways in the human liver.

Probing functional interactions between cytochromes P450 with principal component analysis of substrate saturation profiles and targeted proteomics.

We investigated the correspondence between drug metabolism routes and the composition of the P450 ensemble in human liver microsomes (HLM). As a probe, we used Coumarin 152 (C152), a fluorogenic substrate metabolized by multiple P450 species. Studying the substrate-saturation profiles (SSP) in seven pooled HLM preparations, we sought to correlate them with the P450 pool's composition characterized by targeted proteomics.

Effects of alcohol-induced increase in CYP2E1 content in human liver microsomes on the activity and cooperativity of CYP3A4.

We investigate the effect of the alcohol-induced increase in the content of CYP2E1 in human liver microsomes (HLM) on the function of CYP3A4. Membrane incorporation of the purified CYP2E1 into HLM considerably increases the rate of metabolism of 7-benzyloxyquinoline (BQ) and attenuates the homotropic cooperativity observed with this CYP3A4-specific substrate. It also eliminates the activating effect of α-naphthoflavone (ANF) seen in some HLM samples.

Nonadditivity in human microsomal drug metabolism revealed in a study with coumarin 152, a polyspecific cytochrome P450 substrate.

We closely characterized 7-Dimethylamino-4-trifluromethylcoumarin (Coumarin 152, C152), a substrate metabolized by multiple P450 species, to establish a new fluorogenic probe for the studies of functional integration in the cytochrome P450 ensemble. Scanning fluorescence spectroscopy and LC/MS-MS were used to characterize the products of N-demethylation of C152 and optimize their fluorometric detection. The metabolism of C152 by the individual P450 species was characterized using the microsomes containing cDNA-expressed enzymes.

Toward a systems approach to cytochrome P450 ensemble: interactions of CYP2E1 with other P450 species and their impact on CYP1A2.

In this study, we investigate the ability of ethanol-inducible CYP2E1 to interact with other cytochrome P450 species and affect the metabolism of their substrates. As a model system, we used CYP2E1-enriched human liver microsomes (HLM) obtained by the incorporation of purified CYP2E1. Using a technique based on homo-FRET in oligomers of CYP2E1 labeled with BODIPY 577/618 maleimide we demonstrated that the interactions of CYP2E1 with HLM result in the formation of its mixed oligomers with other P450 species present in the microsomal membrane.

Kinetic mechanism of time-dependent inhibition of CYP2D6 by 3,4-methylenedioxymethamphetamine (MDMA): Functional heterogeneity of the enzyme and the reversibility of its inactivation.

We investigate the mechanism of time-dependent inhibition (TDI) of human cytochrome P450 2D6 (CYP2D6) by 3,4-methylenedioxymethamphetamine (MDMA, ecstasy), one of the most widespread recreational drugs of abuse. In an effort to unravel the kinetic mechanism of the formation of metabolic inhibitory complex (MIC) of CYP2D6 with MDMA-derived carbene we carried out a series of spectrophotometric studies paralleled with registration of the kinetics of time-dependent inhibition (TDI) in CYP2D6-incorporated proteoliposomes. The high amplitude of spectral signal in this system allowed us to characterize the spectral properties of the formed MIC in details and obtain an accurate spectral signature of MIC formation.

Toward a systems approach to the human cytochrome P450 ensemble: interactions between CYP2D6 and CYP2E1 and their functional consequences.

Functional cross-talk among human drug-metabolizing cytochrome P450 through their association is a topic of emerging importance. Here, we studied the interactions of human CYP2D6, a major metabolizer of psychoactive drugs, with one of the most prevalent human P450 enzymes, ethanol-inducible CYP2E1. Detection of P450-P450 interactions was accomplished through luminescence resonance energy transfer between labeled proteins incorporated into human liver microsomes and the microsomes of insect cells containing NADPH-cytochrome P450 reductase.

Conformational Mobility in Cytochrome P450 3A4 Explored by Pressure-Perturbation EPR Spectroscopy.

We used high hydrostatic pressure as a tool for exploring the conformational landscape of human cytochrome P450 3A4 (CYP3A4) by electron paramagnetic resonance and fluorescence spectroscopy. Site-directed incorporation of a luminescence resonance energy transfer donor-acceptor pair allowed us to identify a pressure-dependent equilibrium between two states of the enzyme, where an increase in pressure increased the spatial separation between the two distantly located fluorophores. This transition is characterized by volume change (ΔV°) and P1/2 values of -36.

Interactions among cytochromes P450 in microsomal membranes: oligomerization of cytochromes P450 3A4, 3A5, and 2E1 and its functional consequences.

The body of evidence of physiologically relevant P450-P450 interactions in microsomal membranes continues to grow. Here we probe oligomerization of human CYP3A4, CYP3A5, and CYP2E1 in microsomal membranes. Using a technique based on luminescence resonance energy transfer, we demonstrate that all three proteins are subject to a concentration-dependent equilibrium between the monomeric and oligomeric states.

Pivotal role of P450-P450 interactions in CYP3A4 allostery: the case of α-naphthoflavone.

We investigated the relationship between oligomerization of CYP3A4 (cytochrome P450 3A4) and its response to ANF (α-naphthoflavone), a prototypical heterotropic activator. The addition of ANF resulted in over a 2-fold increase in the rate of CYP3A4-dependent debenzylation of 7-BFC [7-benzyloxy-4-(trifluoromethyl)coumarin] in HLM (human liver microsomes), but failed to produce activation in BD Supersomes or Baculosomes containing recombinant CYP3A4 and NADPH-CPR (cytochrome P450 reductase). However, incorporation of purified CYP3A4 into Supersomes containing only recombinant CPR reproduced the behaviour observed with HLM.

CYP261 enzymes from deep sea bacteria: a clue to conformational heterogeneity in cytochromes P450.

We have explored the adaptation of the cytochromes P450 (P450) of deep-sea bacteria to high hydrostatic pressures. Strict conservation of the protein fold and functional importance of protein-bound water make P450 a unique subject for the studies of high-pressure adaptation. Earlier, we expressed and purified a fatty-acid binding P450 from the deep-sea bacteria Photobacterium profundum SS9 (CYP261C1).

Peripheral ligand-binding site in cytochrome P450 3A4 located with fluorescence resonance energy transfer (FRET).

The mechanisms of ligand binding and allostery in the major human drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4) were explored with fluorescence resonance energy transfer (FRET) using a laser dye, fluorol-7GA (F7GA), as a model substrate. Incorporation into the enzyme of a thiol-reactive FRET probe, pyrene iodoacetamide, allowed us to monitor the binding by FRET from the pyrene donor to the F7GA acceptor. Cooperativity of the interactions detected by FRET indicates that the enzyme possesses at least two F7GA-binding sites that have different FRET efficiencies and are therefore widely separated.

Multiple substrate-binding sites are retained in cytochrome P450 3A4 mutants with decreased cooperativity.

The basis of decreased cooperativity in substrate binding in the cytochrome P450 3A4 mutants F213W, F304W, and L211F/D214E was studied with fluorescence resonance energy transfer and absorbance spectroscopy. Although in the wild type enzyme, the absorbance changes reflecting the interactions with 1-pyrenebutanol exhibit a Hill coefficient (n(H)) around 1.7 (S(50) = 11.

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.

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.

Allosteric transitions in cytochrome P450eryF explored with pressure-perturbation spectroscopy, lifetime FRET, and a novel fluorescent substrate, Fluorol-7GA.

To establish a direct method for monitoring substrate binding in cytochrome P450eryF applicable at elevated hydrostatic pressures, we introduce a laser dye Fluorol-7GA (F7GA) as a novel fluorescent ligand. The high intensity of fluorescence and the reasonable resolution of the excitation band from the absorbance bands of P450 allowed us to establish highly sensitive binding assays compatible with pressure perturbation. The interactions of F7GA with P450eryF cause an ample spin shift revealing cooperative binding ( S50 = 8.

Effect of glutathione on homo- and heterotropic cooperativity in cytochrome P450 3A4.

Glutathione (GSH) exerted a profound effect on the oxidation of 7-benzyloxy-4-(trifluoromethyl)coumarin (BFC) and 7-benzyloxyquinoline (BQ) by human liver microsomes as well as by CYP3A4-containing insect cell microsomes (Baculosomes). The cooperativity in O-debenzylation of both substrates is eliminated in the presence of 1-4mM GSH. Addition of GSH also increased the amplitude of the 1-PB induced spin shift with purified CYP3A4 and abolished the cooperativity of 1-PB or BFC binding.

Mechanism of interactions of alpha-naphthoflavone with cytochrome P450 3A4 explored with an engineered enzyme bearing a fluorescent probe.

Design of a partially cysteine-depleted C98S/C239S/C377S/C468A cytochrome P450 3A4 mutant designated CYP3A4(C58,C64) allowed site-directed incorporation of thiol-reactive fluorescent probes into alpha-helix A. The site of modification was identified as Cys-64 with the help of CYP3A4(C58) and CYP3A4(C64), each bearing only one accessible cysteine. Changes in the fluorescence of CYP3A4(C58,C64) labeled with 6-(bromoacetyl)-2-(dimethylamino)naphthalene (BADAN), 7-(diethylamino)-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM), or monobromobimane (mBBr) were used to study the interactions with bromocriptine (BCT), 1-pyrenebutanol (1-PB), testosterone (TST), and alpha-naphthoflavone (ANF).

Resolution of two substrate-binding sites in an engineered cytochrome P450eryF bearing a fluorescent probe.

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.