Increasing the Efficiency of Cytochrome P450 3A4 Electrocatalysis Using Electrode Modification with Spatially Ordered Anodic Aluminum Oxide-Based Nanostructures for Investigation of Metabolic Transformations of Drugs.

A new approach for modifying electrodes using porous membranes based on anodic aluminum oxide with pore diameters of 0.1 and 0.2 µm and a membrane-like substance didodecyldimethylammonium bromide (DDAB) was proposed to study the electrocatalytic efficiency of the system. This approach makes it possible to increase the catalytic efficiency of the cytochrome P450 3A4-dependent N-demethylation of erythromycin by 132% when using a membrane with pore diameter of 0.1 µm and by 32% when using a membrane with 0.2 µm pore size. Electrode modification using porous membranes shifted the potential of electrochemical reduction and catalysis of cytochrome P450 3A4 in positive direction by 0.070-0.050 V, which indicates a thermodynamically more favorable process of electron transfer and enzymatic electrocatalysis.

[Screening of potential non-azole inhibitors of lanosterol14-alpha demethylase (CYP51) of Candida fungi]. / Skrining potentsial'nykh neazol'nykh ingibitorov 14-al'fademetilazy (CYP51) gribov roda Candida.

Currently, opportunistic fungi of the genus Candida are the main causative agents of mycoses, which are especially severe upon condition of acquired immunodeficiency. The main target for the development of new antimycotics is the cytochrome P450 51 (CYP51) of the pathogenic fungus. Due to the widespread distribution of Candida strains resistancy to inhibitors of the azole class, the screening for CYP51 inhibitors both among non-azole compounds and among clinically used drugs repurposing as antimycotics is becoming urgent. To identify potential inhibitors from the non-azole group, an integrated approach was applied, including bioinformatics analysis, computer molecular modeling, and a surface plasmon resonance (SPR) technology. Using in silico modeling, the binding sites for acetylsalicylic acid, ibuprofen, chlorpromazine and haloperidol (this compounds, according to the literature, showed antimycotic activity) were predicted in the active site of CYP51 of Candida albicans and Candida glabrata. The Kd values of molecular complexes of acetylsalicylic acid, ibuprofen and haloperidol with CYP51, determined by SPR analysis, ranged from 18 µM to 126 µM. It was also shown that structural derivatives of haloperidol, containing various substituents, could be positioned in the active site of CYP51 of Candida albicans with the possible formation of coordination bonds between the hydroxyl groups of the derivatives and the iron atom in the heme of CYP51. Thus, the potential basic structures of non-azole compounds have been proposed, which can be used for the design of new CYP51 inhibitors of Candida fungi.

Substrate-induced modulation of protein-protein interactions within human mitochondrial cytochrome P450-dependent system.

Steroidogenesis is strictly regulated at multiple levels, as produced steroid hormones are crucial to maintain physiological functions. Cytochrome P450 enzymes are key players in adrenal steroid hormone biosynthesis and function within short redox-chains in mitochondria and endoplasmic reticulum. However, mechanisms regulating supply of reducing equivalents in the mitochondrial CYP-dependent system are not fully understood. In the present work, we aimed to estimate how the specific steroids, substrates, intermediates and products of multistep reactions modulate protein-protein interactions between adrenodoxin (Adx) and mitochondrial CYP11 s. Using the SPR technology we determined that steroid substrates affect affinity and stability of CYP11s-Adx complexes in an isoform-specific mode. In particular, cholesterol induces a 4-fold increase in the rate of CYP11A1 - Adx complex formation without significant effect on dissociation (koff decreased ∼1.5-fold), overall increasing complex affinity. At the same time steroid substrates decrease the affinity of both CYP11B1 - Adx and CYP11B2 - Adx complexes, predominantly reducing their stability (4-7 fold). This finding reveals differentiation of protein-protein interactions within the mitochondrial pool of CYPs, which have the same electron donor. The regulation of electron supply by the substrates might affect the overall steroid hormones production. Our experimental data provide further insight into protein-protein interactions within CYP-dependent redox chains involved in steroidogenesis.

[Modeling of drug-drug interactions between omeprazole and erythromycin with cytochrome P450 3A4 in vitro assay]. / Modelirovanie mezhlekarstvennogo vzaimodeistviia omeprazola i éritromitsina s ispol'zovaniem tsitokhrom R450-zavisimoi sistemy in vitro.

In the present study the electrochemical system based on recombinant cytochrome P450 3A4 (CYP3A4) was used for the investigation of potential drug-drug interaction between medicinal preparations employed for Helicobacter pylori eradication therapy. Drug interactions were demonstrated in association of omeprazole as a proton pump inhibitor (PPI) and macrolide antibiotic erythromycin during cytochrome P450 3A4-mediated metabolism. It was shown that in the presence of omeprazole the rate of N-demethylase activity of CYP3A4 to erythromycin measured by means of product (formaldehyde) formation decreased. Mass-spectrometry analysis of omeprazole sulfone as a CYP3A4-mediated metabolite demonstrated the absence of erythromycin influence on CYP3A4-dependent omeprazole metabolism. This phenomenon may be explained by lower spectral dissociation constant of CYP3A4-omeprazole complex (Kd = 18±2 µM) than that of CYP3A4-erythromycin complex (Kd = 52 µM). Using the electrochemical model of electrochemically-driven drug metabolism it is possible to register CYP3A4-mediated catalytic conversion of certain drugs. In vitro experiments of potential CYP3A4-mediated drug-drug interactions are in accordance with in silico modeling with program PASS and PoSMNA descriptors in the case of omeprazole/erythromycin combinations.

[Electroanalytical and electrocatalytical characteristics of cytochrome P450 3A4 using electrodes modified with nanocomposite carbon nanomaterials]. / Tsitokhrom R450 3A4 kak ferment biotransformatsii lekarstv: rol' modifikatsii sensornykh sistem v élektrokatalize i élektroanalize.

The electroanalytical characteristics of recombinant cytochrome P450 3A4 (P450 3A4) immobilized on the surface of screen-printed graphite electrodes modified with multi-walled carbon nanotubes have been studied. The role and the influence of graphite working electrode modification with carbon nanotubes on electroanalytical characteristics of cytochrome P450 3A4 have been demonstrated. The conditions for the immobilization of cytochrome P450 3A4 on the obtained screen-printed graphite electrodes modified with carbon multi-walled nanotubes have been optimized. The electrochemical parameters of the oxidation and reduction of the heme iron of the enzyme have been estimated. The midpoint potential E0' was -0.35±0.01 V vs Ag/AgCl; the calculated heterogeneous electron transfer rate constant ks, was 0.57±0.04 s-1; the amount of electroactive cytochrome P450 3A4 on the electrode Г0, was determined as (2.6±0.6)⋅10-10 mol/cm2. The functioning mechanism of P450 3A4-based electrochemical sensor followed the "protein film voltammetry". In order to develop electrochemical analysis of drugs being substrates of that hemoprotein and respective medical biosensors the voltammetric study of catalytic activity of immobilized cytochrome P450 3A4 was carried out. Electrocatalytic properties of cytochrome P450 3A4, immobilized on modified screen-printed graphite electrodes, has been investigated using erythromycin (macrolide antibiotics). It has been shown that the modification of electrodes plays a decisive role for the study of the properties of cytochromes P450 in electrochemical investigations. Smart electrodes can serve as sensors for analytical purposes, as well as electrocatalysts for the study of biotransformation processes and metabolic processes. Electrodes modified with carbon nanomaterials are applicable for analytical purposes in the registration of hemoproteins. Electrodes modified with synthetic membrane-like compounds (e.g. didodecyldimethylammonium bromide) are effective in enzyme-dependent electrocatalysis.

[Interaction of prostacyclin synthase with cytochromes P450]. / Vzaimodeistvie prostatsiklinsintazy s tsitokhromami P450.

Biosensor experiments on investigation of interaction between prostacyclin synthase (PGIS) and different proteins of the cytochrome P450 monooxygenase systems were perfomed. Interaction of PGIS with microsomal (CYP21A2, CYP2E1) and mitochondrial (CYP27A1, CYP11B1, CYP11B2, CYP11A1) cytochrome P450s was detected. Kinetic and equilibrium parameters of protein complexes formation were determined. Data obtained suggest an essential role of these hemoproteins interaction in regulation of prostacyclin and thromboxane A2 biosynthesis.

[Study specificity of isatin interactions with P450 cytochromes]. / Spetsifichnost' vzaimodeistviia izatina s tsitokhromami R450.

Cytochrome P450-dependent monooxygenase systems exist basically in all living organisms, where they perform various important functions. The coordinated functioning of these systems involves many proteins participating in different protein-protein interactions (PPI). Previously, we have found that the endogenous non-peptide bioregulator isatin (indoledione-2,3), synthesized from indole by means of certain cytochromes P450 (e.g. P450 2E1, P450 2C19, P450 2A6) regulates affinity of some PPI. In this work, an attempt has been undertaken to register a direct interaction of isatin with a set of different proteins related to the functioning of cytochrome P450-dependent monooxygenase: five isoforms of cytochromes P450, two isoforms of cytochrome b5, cytochrome P450 reductase, adrenodoxin, adrenodoxin reductase and ferrochelatase. The study has shown that isatin binds specifically only to cytochromes P450 with high affinity (the equilibrium dissociation constant (Kd) is about 10-8 M).

[The effect of isatin on protein-protein interactions between cytochrome b5 and cytochromes P450]. / Vliianie izatina na vzaimodeistvie tsitokhroma b5 s tsitokhromami R450.

Cytochromes P450 (CYP) are involved in numerous biochemical processes including metabolism of xenobiotics, biosynthesis of cholesterol, steroid hormones etc. Since some CYP catalyze indol oxidation to isatin, we have hypothesized that isatin can regulate protein-protein interactions (PPI) between components of the CYP system thus representing a (negative?) feedback mechanism. The aim of this study was to investigate a possible effect of isatin on interaction of human CYP with cytochrome b5 (CYB5A). Using the optical biosensor test system employing surface plasmon resonance (SPR) we have investigated interaction of immobilized CYB5A with various CYP in the absence and in the presence of isatin. The SPR-based experiments have shown that a high concentration of isatin (270 mM) increases Kd values for complexes CYB5A/CYP3А5 and CYB5A/CYP3A4 (twofold and threefold, respectively), but has no influence on complex formation between CYB5A and other CYP (including indol-metabolizing CYP2C19 and CYP2E1). Isatin injection to the optical biosensor chip with the preformed molecular complex CYB5A/CYP3A4 caused a 30%-increase in its dissociation rate. Molecular docking manipulations have shown that isatin can influence interaction of CYP3А5 or CYP3A4 with CYB5A acting at the contact region of CYB5A/CYP.

[Proteomic analysis of contaminants in recombinant membrane hemeproteins expressed in E. coli and isolated by metal affinity chromatography].

Contaminating proteins have been identified by "shotgun" proteomic analysis in 14 recombinant preparations of human membrane heme- and flavoproteins expressed in Escherichia coli and purified by immobilized metal ion affinity chromatography. Immobilized metal ion affinity chromatography of ten proteins was performed on Ni2+-NTA-sepharose 6B, and the remaining four proteins were purified by ligand affinity chromatography on 2',5'-ADP-sepharose 4B. Proteomic analysis allowed to detect 50 protein impurities from E. coli. The most common contaminant was Elongation factor Tu2. It is characterized by a large dipole moment and a cluster arrangement of acidic amino acid residues that mediate the specific interaction with the sorbent. Peptidyl prolyl-cis-trans isomerase SlyD, glutamine-fructose-6-phosphate aminotransferase, and catalase HPII that contained repeating HxH, QxQ, and RxR fragments capable of specific interaction with the sorbent were identified among the protein contaminants as well. GroL/GroS chaperonins were probably copurified due to the formation of complexes with the target proteins. The Ni2+ cations leakage from the sorbent during lead to formation of free carboxyl groups that is the reason of cation exchanger properties of the sorbent. This was the putative reason for the copurification of basic proteins, such as the ribosomal proteins of E. coli and the widely occurring uncharacterized protein YqjD. The results of the analysis revealed variation in the contaminant composition related to the type of protein expressed. This is probably related to the reaction of E. coli cell proteome to the expression of a foreign protein. We concluded that the nature of the protein contaminants in a preparation of a recombinant protein purified by immobilized metal ion affinity chromatography on a certain sorbent could be predicted if information on the host cell proteome were available.

Direct Molecular Fishing of New Protein Partners for Human Thromboxane Synthase.

Thromboxane synthase (TBXAS1) catalyzes the isomerization reaction of prostaglandin H2 producing thromboxane A2, the autocrine and paracrine factor in many cell types. A high activity and metastability by these arachidonic acid derivatives suggests the existence of supramolecular structures that are involved in the regulation of the biosynthesis and directed translocation of thromboxane to the receptor. The objective of this study was to identify TBXAS1 protein partners from human liver tissue lysate using a complex approach based on the direct molecular fishing technique, LC-MS/MS protein identification, and protein-protein interaction validation by surface plasmon resonance (SPR). As a result, 12 potential TBXAS1 protein partners were identified, including the components regulating cytoskeleton organization (BBIP1 and ANKMY1), components of the coagulation cascade of human blood (SERPINA1, SERPINA3, APOH, FGA, and FN1), and the enzyme involved in the metabolism of xenobiotics and endogenous bioregulators (CYP2E1). SPR validation on the Biacore 3000 biosensor confirmed the effectiveness of the interaction between CYP2E1 (the enzyme that converts prostaglandin H2 to 12-HHT/thromboxane A2 proantagonist) and TBXAS1 (Kd = (4.3 ± 0.4) × 10-7 M). Importantly, the TBXAS1•CYP2E1 complex formation increases fivefold in the presence of isatin (indole-2,3-dione, a low-molecular nonpeptide endogenous bioregulator, a product of CYP2E1). These results suggest that the interaction between these hemoproteins is important in the regulation of the biosynthesis of eicosanoids.

Identification of Mycobacterium tuberculosis enzyme involved in vitamin D and 7-dehydrocholesterol metabolism.

Problems arising during treatment of tuberculosis are well known, therefore studies of Mycobacterium drug molecular targets are an area of particular importance. Members of the cytochrome P450 family (CYP) may belong to potential candidates for drug targets being involved in metabolism of biologically important molecules in the host organism. CYP124 of Mycobacterium tuberculosis (MTCYP124) catalyzes ω-hydroxylation of methyl-branched lipids. The data obtained in the present study indicate that this enzyme can also oxidize provitamin D3 (7-dehydrocholesterol) and vitamin D3. We found that the final product is different from 1α- and 25-hydroxyvitamin D3, so we propose that MTCYP124 is involved in alternative pathway for metabolism of vitamin D3.

[The screening of the inhibitors of the human cytochrome P450(51) (CYP51A1): the plant and animal structural lanosterol's analogs].

The cholesterol biosynthesis regulation is the important part of the hypercholesterolemia diseases therapy. The inhibition of the post-squalene cholesterol biosynthesis steps provide the alternative to classic statin therapy. Sterol-14a-demethylase (CYP51) is one of the hypothetical targets for it. In this work the screening of the ability to interact with human CYP51 (CYP51A1) for the nature low-weight compounds with steroid-like scaffold were performed by integration of the surface plasmon resonance biosensor and spectral titration methods. The results of the selection were 4 compounds (betulafolientriol, holothurin A, teasaponin, capsicoside A) witch had high affinity to the CYP51A1 active site. These data extend the range of compounds which may be used as specific inhibitors of CYP51 and give the permission to suggest the dynamic of the enzyme.

The role of cytochrome b5 structural domains in interaction with cytochromes P450.

To understand the role of the structural elements of cytochrome b5 in its interaction with cytochrome P450 and the catalysis performed by this heme protein, we carried out comparative structural and functional analysis of the two major mammalian forms of membrane-bound cytochrome b5 - microsomal and mitochondrial, designed chimeric forms of the heme proteins in which the hydrophilic domain of one heme protein is replaced by the hydrophilic domain of another one, and investigated the effect of the highly purified native and chimeric heme proteins on the enzymatic activity of recombinant cytochromes P4503A4 and P45017A1 (CYP3A4 and CYP17A1). We show that the presence of a hydrophobic domain in the structure of cytochrome b5 is necessary for its effective interaction with its redox partners, while the nature of the hydrophobic domain has no significant effect on the ability of cytochrome b5 to stimulate the activity of cytochrome P450-catalyzed reactions. Thus, the functional properties of cytochrome b5 are mainly determined by the structure of the heme-binding domain.

[An effective scheme to produce recombinant uracil-DNA glycosylase of Escherichia coli for PCR diagnostics].

An effective scheme has been developed to produce recombinant uracil-DNA glycosylase of Escherichia coli K12 intended to be used for PCR diagnostics, making it possible to achieve a high yield of the end product using a two-stage purification. The gene encoding this enzyme was cloned into the pCWori vector within the same reading frame with six residues of histidine in the C-erminal sequence. Using this vector and the E. coli DH5alpha, a host-vector expression system has been developed and conditions for protein synthesis have been optimized. To purify the protein, metal affinity chromatography with further dialysis was used to remove imidazole. The enzyme yield was no less than 60 mg of the end protein per 1 L of the culture medium. The concordance between amino acid sequences of the recombinant and native enzymes was proved by peptide mass fingerprinting and mass spectrometry. A rapid test to determine the activity of the enzyme preparation was suggested. It was found that the activity of 1.0 mg of the recombinant protein is no less than 3 x 10(3) units. The recombinant enzyme was most stable at pH 8.0 and an ionic strength of the solution equal to 200 mM; it lost its activity completely for 10 min at 60 degrees C. Storage during 1 h at 20 degrees C resulted in the loss of no more than 30% of activity. In the enzyme preparation, the activity of DNase was absent. The free energy of the unfolding of the protein globule of the recombinant uracil-DNA glycosylase is 23.1 +/- 0.2 kJ/mol. The data obtained indicate that the recombinant enzyme may be recommended for use in PCR diagnostics to prevent the appearance of false positive results caused by pollution of the reaction mixture by products of the preceding reactions.

Role of microsomal steroid hydroxylases in Δ7-steroid biosynthesis.

CYP17 (steroid 17α-hydroxylase/17,20-lyase) is a key enzyme in steroid hormone biosynthesis. It catalyzes two independent reactions at the same active center and has a unique ability to differentiate Δ(4)-steroids and Δ(5)-steroids in the 17,20-lyase reaction. The present work presents a complex experimental analysis of the role of CYP17 in the metabolism of 7-dehydrosteroids. The data indicate the existence of a possible alternative pathway of steroid hormone biosynthesis using 7-dehydrosteroids. The major reaction products of CYP17 catalyzed hydroxylation of 7-dehydropregnenolone have been identified. Catalytic activity of CYP17 from different species with 7-dehydropregnenolone has been estimated. It is shown that CYP21 cannot use Δ(5)-Δ(7) steroids as a substrate.

[SPR-biosensor assay for analysis of small compounds interaction with human cytochrome P450 51A1 (CYP51A1)].

The SPR assay for human cytochrome P450 51A1's (CYP51A1) ligand screening was developed. Assay has been validated with known azole inhibitors of cytochrome P450s. The studied azoles selectively interacted with human cytochrome P450 51A1, which showed the highest affinity towards ketoconazole. The efficiency of the SPR assay was showed with 19 steroid and triterpene compounds, which were not investigated as potential ligands of CYP51A1.

Mechanism of intermolecular interactions of microsomal cytochrome P450s CYP17 and CYP21 involved in steroid hormone biosynthesis.

Protein-protein interactions play a significant role in regulation of functional activity of cytochrome P450s. The aim of the present study was to elucidate the molecular interactions between steroidogenic enzymes CYP17 and CYP21 localized in endoplasmic reticulum membranes of adrenal cortex and involved in biosynthesis of corticosteroid hormones. In the present work, we demonstrate for the first time the direct interaction at molecular level between highly purified human recombinant cytochrome P450s in a mixed reconstituted system. The dependence of the interaction between CYP17 and CYP21 on concentration of the redox-partner - NADPH-cytochrome P450 reductase - is demonstrated, and it is shown that electrostatic interactions play a crucial role in the interaction between CYP17 and CYP21.

Molecular modeling of human lanosterol 14α-demethylase complexes with substrates and their derivatives.

Lanosterol 14α-demethylase (CYP51A1) is a key enzyme in sterol biosynthesis. In humans, this enzyme is involved in the cholesterol biosynthesis pathway. The majority of antifungal drugs are aimed at the inhibition of CYP51 in fungi. To elucidate the molecular mechanisms of highly specific protein-ligand recognition, we have developed a full-atomic model of human CYP51A1 and performed docking of natural substrates and their derivatives to the active site of the enzyme. The parameters of the binding enthalpy of substrates, intermediates, and final products of the reaction of 14α-demethylation were estimated using the MMPB(GB)SA algorithm. Dynamic properties and conformational changes of the protein globule upon binding of the ligand near the active site have been investigated by the molecular dynamics method. Our studies reveal that hydroxylated intermediate reaction products have a greater affinity than the initial substrates, which facilitates the multistage reaction without accumulation of intermediate products. The contribution to the free energy of steroid ligand binding of 30 amino acids forming the substrate-binding region of CYP51A1, as well as the influence of their substitutions to alanine on the stability of the protein molecule, has been clarified using alanine scanning modeling. We demonstrate that the most serious weakening of the binding is observed in the case of substitutions Y137A, F145A, V149A, I383A, and R388A. The results of molecular modeling are in agreement with the data obtained by analysis of primary sequences of representatives of the CYP51 family.

Electron transfer properties and catalytic competence of cytochrome b5 in the fusion protein Hmwb5-EGFP in reactions catalyzed by cytochrome P450 3A4.

In the present paper we describe studies on molecular mechanisms of protein-protein interactions between cytochrome P450 3A4 (CYP3A4) and cytochrome b(5), the latter being incorporated into the artificial recombinant protein Hmwb(5)-EGFP containing full-length cytochrome b(5) (functional module) and a mutant form of the green fluorescent protein EGFP (signal module) fused into a single polypeptide chain. It is shown that cytochrome b(5) within the fusion protein Hmwb(5)-EGFP can be reduced by NADPH-cytochrome P450 reductase in the presence of NADPH, the rate of reduction being dependent on solution ionic strength, indicating that the signal module does not prevent the interaction of the flavo- and hemeproteins. Interaction of cytochrome P450 3A4 and Hmwb(5)-EGFP was estimated based on spin equilibrium shift of cytochrome P450 3A4 to high-spin state in the presence of Hmwb(5)-EGFP, as well as based on steady-state fluorescence anisotropy of the EGFP component of the fusion protein in the presence of CYP3A4. The engineering of chimeric protein Hmwb(5)-EGFP gives an independent method to determine dissociation constant for the complex of cytochrome P450 and cytochrome b(5) that is less sensitive to environmental factors compared to spectrophotometric titration used before. Reconstitution of catalytic activity of cytochrome P450 3A4 in the reaction of testosterone 6beta-hydroxylation in the presence of Hmwb(5)-EGFP indicates that cytochrome b(5) in the fusion protein is able to stimulate the hydroxylation reaction. Using other fusion proteins containing either cytochrome b(5) or its hydrophilic domain to reconstitute catalytic activity of cytochrome P450 3A4 showed that the hydrophobic domain of cytochrome b(5) participates not only in hemeprotein interaction, but also in electron transfer from cytochrome b(5) to cytochrome P450.

Conformational stability of cytochrome b5, enhanced green fluorescent protein, and their fusion protein Hmwb5-EGFP.

The conformational stabilities of chimeric protein Hmwb(5)-EGFP and its constituents (cytochrome b(5) and enhanced green fluorescent protein) in guanidine hydrochloride solutions are reported in this paper. Intensity of fluorescence of tryptophan residues, intensity of EGFP fluorescence in the visible region, absorbance of cytochrome b(5) heme and EGFP fluorophore, and fluorescence anisotropy were used to follow the unfolding process. Thermodynamic parameters of protein unfolding were obtained using different approaches. The data were analyzed using a two-stage model and a linear extrapolation method. Unfolding of protein molecules was additionally monitored by measuring Stern-Volmer constants for tryptophan fluorescence quenching by acrylamide, cesium, and iodide. The accessibility of tryptophan residues of both components in the fusion molecule is lower than in the separate molecules. The thermodynamic stability of the protein globules in the fusion protein is much lower than in the individual protein molecules in solution, the difference in free energy of unfolding being more considerable for cytochrome b(5) (29 +/- 4 and 13 +/- 2 kJ/mol) than for EGFP (26 +/- 0.9 and 20 +/- 2.7 kJ/mol). The data indicate that artificial protein fusion can greatly affect total structural stability, and in the case of cytochrome b(5) and EGFP it results in decrease in free energy of transition from native to denatured unfolded form and consequently to decrease in thermodynamic stability of protein globules compared to the separate proteins.