The catalytic mechanism of steroidogenic cytochromes P450 from all-atom simulations: Entwinement with membrane environment, redox partners, and post-transcriptional regulation

Cytochromes P450 (CYP450s) promote the biosynthesis of steroid hormones with major impact on the onset of diseases such as breast and prostate cancers. By merging distinct functions into the same catalytic scaffold, steroidogenic CYP450s enhance complex chemical transformations with extreme efficiency and selectivity. Mammalian CYP450s and their redox partners are membrane-anchored proteins, dynamically associating to form functional machineries. Mounting evidence signifies that environmental factors are strictly intertwined with CYP450s catalysis. Atomic-level simulations have the potential to provide insights into the catalytic mechanism of steroidogenic CYP450s and on its regulation by environmental factors, furnishing information often inaccessible to experimental means. In this review, after an introduction of computational methods commonly employed to tackle these systems, we report the current knowledge on three steroidogenic CYP450s\u2014CYP11A1, CYP17A1, and CYP19A1\u2014endowed with multiple catalytic functions and critically involved in cancer onset. In particular, besides discussing their catalytic mechanisms, we highlight how the membrane environment contributes to (i) regulate ligand channeling through these enzymes, (ii) modulate their interactions with specific protein partners, (iii) mediate post-transcriptional regulation induced by phosphorylation. The results presented set the basis for developing novel therapeutic strategies aimed at fighting diseases originating from steroid metabolism dysfunction

Regulation of 3β-Hydroxysteroid Dehydrogenase/∆5-∆4 Isomerase: A Review

This review focuses on the expression and regulation of 3β-hydroxysteroi ddehydrogenase/Δ5-Δ4 isomerase (3β-HSD), with emphasis on the porcine version. 3β-HSD is often associated with steroidogenesis, but its function in the metabolism of both steroids and xenobiotics is more obscure. Based on currently available literature covering humans,rodents and pigs, this review provides an overview of the present knowledge concerning the regulatory mechanisms for 3β-HSD at all omic levels. The HSD isoenzymes are essential in steroid hormone metabolism, both in the synthesis and degradation of steroids. They display tissue-specific expression and factors influencing their activity, which therefore indicates their tissue-specific responses. 3β-HSD is involved in the synthesis of a number of natural steroid hormones, including progesterone and testosterone, and the hepatic degradation of the pheromone androstenone. In general, a number of signaling and regulatory pathways have been demonstrated to influence 3β-HSD transcription and activity, e.g., JAK-STAT, LH/hCG, ERα, AR, SF-1 and PPARα. The expression and enzymic activity of 3β-HSD are also influenced by external factors, such as dietary composition. Much of the research conducted on porcine 3β-HSD is motivated by its importance for the occurrence of the boar taint phenomenon that results from high concentrations of steroids such as androstenone. This topic is also examined in this review

An overview on the allelic variant of CYP2D6 genotype

The paper gives an overview on the allelic variant of CYP2D6 genotype. The gene CYP2D6*3 encodes a member of the cytochrome P450 super family of enzymes. The cytochrome P450 proteins are monooxygenases which catalyze many reactions involved in drug metabolism and synthesis ofcholesterol, steroids and other lipids. The protein localizes to the endoplasmic reticulum and is known to metabolize as many as 20% of commonly prescribed drugs. Its substrates include debrisoquine, anadrenergic-blocking drug; sparteine and propafenone, both anti-arrythmic drugs; and amitryptiline, an anti-depressant. The emerging application of pharmacogenomics in the clinical trials requires careful comparison with the traditional genotypic methodologies particularly in the drug metabolism area.Keywords: CYP2D6 gene, PCR, CYP2D6*3, allelic variant

Regulating Retinoic Acid Availability during Development and Regeneration: The Role of the CYP26 Enzymes.

This review focuses on the role of the Cytochrome p450 subfamily 26 (CYP26) retinoic acid (RA) degrading enzymes during development and regeneration. Cyp26 enzymes, along with retinoic acid synthesising enzymes, are absolutely required for RA homeostasis in these processes by regulating availability of RA for receptor binding and signalling. Cyp26 enzymes are necessary to generate RA gradients and to protect specific tissues from RA signalling. Disruption of RA homeostasis leads to a wide variety of embryonic defects affecting many tissues. Here, the function of CYP26 enzymes is discussed in the context of the RA signalling pathway, enzymatic structure and biochemistry, human genetic disease, and function in development and regeneration as elucidated from animal model studies

P450 biochips : development of a protein microarray platform for investigating cytochrome P450 clinical drug metabolism

Includes bibliographical references (leaves 224-243).This thesis describes the development of a novel cytochrome P450 array format, the P450 Biochip that allows quantitative and truly high-throughput measurement of cytochrome P450-mediated turnover reactions in sub-nanolitre volumes

Use of protein immobilization to measure cytochrome P450 conduction and metabolism kinetics

Cytochrome P450s (P450s) are a large family (\u3e11,000) of heme thiolated proteins that are responsible for ~ 75% of the metabolism of pharmaceuticals on the market. Understanding P450 mediated metabolism is crucial for accurate in vitro predictions of drug metabolism. P450 protein-protein interactions have been shown to alter enzyme catalytic activity. Furthermore, these interactions are isoform specific, and can elicit activation, inhibition, or no effect on enzymatic activity. Studies show these effects are also dependent on the protein binding partner cytochrome P450 reductase (CPR), and the order of protein addition to purified reconstituted enzyme systems. In the current work, we use controlled immobilization of P450s to a gold surface to gain a better understanding of P450-P450 interactions between three key drug-metabolizing isoforms (CYP2C9, CYP3A4, and CYP2D6). Molecular modeling was used to assess the favorability of homo/heteromeric P450 complex formation. P450 complex formation in vitro was analyzed in real-time utilizing surface plasmon resonance (SPR). Lastly, the effects of P450 complex formation were investigated utilizing our immobilized platform and reconstituted enzyme systems.;Molecular modeling shows favorable binding of CYP2C9-CPR, CYP2C9-CYP2D6, CYP2C9-CYP2C9, and CYP2C9-CYP3A4 in rank order. KD values obtained via SPR show strong binding, in the nanomolar range, of the above pairs, with CYP2D6 yielding the lowest KD, followed by CYP2C9, CPR, and CYP3A4. Metabolic incubations show immobilized CYP2C9 metabolism was activated by homomeric complex formation. CYP2C9 metabolism was not affected by the presence of CYP3A4 with saturating CPR concentrations. CYP2C9 metabolism was activated by CYP2D6 in solution, but inhibited when CYP2C9 was immobilized, both at saturating and sub-saturating CPR concentrations. Order of addition of proteins (CYP2C9, CYP2D6, CYP3A4, and CPR) influenced magnitude of inhibition for CYP3A4, but not CYP2D6. These results indicate isoform specific P450 interactions and effects on P450 mediated-metabolism. These findings are important in evaluating how in vitro results are obtained for measuring P450 kinetics, and provide a better mechanistic understanding of P450-P450 interactions to allow for better prediction of in vivo metabolism from in vitro data.;We also demonstrate that gold nanopillars, functionalized with an organic self-assembled monolayer, can be used to measure the electrical conductance properties of immobilized P450s without aggregation. Given that transfer of the 1st electron to the P450 heme group acts as the gating step for the catalytic cycle, understanding electron transfer in P450s could shed light on metabolism kinetics. Conductance measurements of nanopillars with immobilized CYP2C9 using conducting probe atomic force microscopy demonstrate that a correlation exists between the energy barrier height between hopping sites and CYP2C9 metabolic activity. Measurements performed as a function of tip force indicate that, when subjected to a large force, the protein is more stable in the presence of a substrate. This agrees with the hypothesis that substrate entry into the active site helps to stabilize the enzyme.;The relative distance between hopping sites also increases with increasing force, possibly because protein functional groups responsible for electron transport depend on the structure of the protein. The inhibitor sulfaphenazole, in addition to the previously studied aniline, increased the barrier height for electron transfer and thereby makes CYP2C9 reduction more difficult and inhibits metabolism. This suggests that P450 Type II ligands may decrease the ease of electron transport processes in the enzyme, in addition to occupying the active site. These findings further our understanding of how P450 metabolism is mediated through substrates, and provides an important technological advancement for studying P450s that avoids complications found in current methodologies. These two studies demonstrate the ability of an immobilized P450 platform to provide information on protein-protein interactions, substrate protein interactions, and atypical enzyme kinetics

MODIFICATION OF THE NUCLEOTIDE COFACTOR-BINDING SITE OF CYTOCHROME P450 REDUCTASE TO ENHANCE TURNOVER WITH NADH IN VIVO

NADPH-cytochrome P450 reductase is the electron transfer partner for the cytochromes P450, heme oxygenase, and squalene monooxygenase, and is a component of the nitric oxide synthases and methionine synthase reductase. P450 reductase shows very high selectivity for NADPH and uses NADH only poorly. Substitution of tryptophan 677 with alanine (W677A) has been shown by others to yield a 3-fold increase in turnover with NADH, but profound inhibition by NADP+ makes the enzyme unsuitable for in vivo applications. In the present study site-directed mutagenesis of amino acids in the 2\u27-phosphate-binding site of the NADPH domain, coupled with the W677A substitution, was used to generate a reductase that was able to use NADH efficiently in vivo without inhibition by NADP+. Of 11 single, double, and triple mutant proteins, two (R597M/W677A and R597M/K602W/W677A) showed up to a 500-fold increase in catalytic efficiency (kcat/Km) with NADH. Inhibition by NADP+ was reduced by up to four orders of magnitude relative to the W677A protein and was equal to or less than that of the wild-type reductase. Both proteins were 2- to 3-fold more active than wild-type reductase with NADH in reconstitution assays with cytochrome P450 1A2 and with squalene monooxygenase. In a recombinant cytochrome P450 2E1 Ames bacterial mutagenicity assay the R597M/W677A protein increased the sensitivity to dimethylnitrosamine by approximately 2-fold, suggesting that the ability to use NADH afforded a significant advantage in this in vivo assay. In addition to providing a valuable tool for understanding the determinants of nucleotide cofactor specificity in this and related enzymes, these mutants might also lend themselves to creation of bioremediation schemes with increased enzymatic activity and robustness in situ, as well as cost-effective reconstitution of enzyme systems in vitro that do not require the use of expensive reducing equivalents from NADPH

Effects of ciprofloxacin on drug P450 metabolic pathways in pigs

Drugs are potentially toxic substances that elicit dosage-dependent therapeutic effects for specific disease conditions. The efficacy of a drug regimen depends on the concentration of the drug at the site of needed activity and the duration of time it is maintained. The selection of dosing regimens for different species requires the establishment of pharmacokinetic equivalency between species; that is, achieving equivalent peak serum and tissue concentrations and duration of drug exposure. Drug metabolism is a direct reflection of the multiple enzyme systems that characterize different species and is often the most important single factor in regulation if drug concentrations in the body. The largest concentration of enzymes catalyzing these reactions is located in the liver; however, significant concentrations also exist in other tissues such as the intestine. The cytochrome P450 system metabolizes the majority of drugs. Although cytochrome P450 enzymes exist in all species examined to date, minor changes in the structure or tissue distribution of the enzymes may lead to great differences in the metabolism and elimination of specific drugs. A lack of consideration of the rates of biotransformation and elimination of drugs in animals particularly those intended for food may result in drug residues, such as quinolones, in consumed meats. Alternatively, ineffective drug concentrations may prevent killing of bacteria, leading to contamination of meats. More important, low level antibiotic concentrations may favor development of resistant bacterial strains. In an effort to determine pharmacokinetic differences that can indirectly affect development of quinolone resistance in bacteria, differences in quinolone disposition will be identified by allometric analysis of pharmacokinetic data from different species will be analyzed. Activities of specific cytochrome P450 enzymes will be examined in swine

Current trends in drug metabolism and pharmacokinetics.

Pharmacokinetics (PK) is the study of the absorption, distribution, metabolism, and excretion (ADME) processes of a drug. Understanding PK properties is essential for drug development and precision medication. In this review we provided an overview of recent research on PK with focus on the following aspects: (1) an update on drug-metabolizing enzymes and transporters in the determination of PK, as well as advances in xenobiotic receptors and noncoding RNAs (ncRNAs) in the modulation of PK, providing new understanding of the transcriptional and posttranscriptional regulatory mechanisms that result in inter-individual variations in pharmacotherapy; (2) current status and trends in assessing drug-drug interactions, especially interactions between drugs and herbs, between drugs and therapeutic biologics, and microbiota-mediated interactions; (3) advances in understanding the effects of diseases on PK, particularly changes in metabolizing enzymes and transporters with disease progression; (4) trends in mathematical modeling including physiologically-based PK modeling and novel animal models such as CRISPR/Cas9-based animal models for DMPK studies; (5) emerging non-classical xenobiotic metabolic pathways and the involvement of novel metabolic enzymes, especially non-P450s. Existing challenges and perspectives on future directions are discussed, and may stimulate the development of new research models, technologies, and strategies towards the development of better drugs and improved clinical practice