Verapamil: new insight into the molecular mechanism of drug oxidation in the human heart

Verapamil is a commonly prescribed cardiovascular drug, but surprisingly its metabolism in the target tissue of pharmacotherapy is basically unknown. We therefore investigated its biotransformation in human heart tissue and correlate the production of metabolites with the gene expression of major drug metabolising enzymes. Using electrospray LC–MS–MS and LC–MS³ experiments, a total of nine metabolites were observed in incubation experiments with verapamil and microsomes isolated from the human heart tissue, and this included a carbinolamine-, N-formyl-, ahemiacetale-, and formate-intermediate of N-demethyl- and O-demethylverapamil. We also observed a hydroxylation product at the benzylic position of atom C-7 (M9). Metabolites M5–M9 are novel and were not observed in previous studies with liver or other human tissues. A fine example of the considerable metabolic competence of human heart is the formation of M1–M4, e.g. dealkylverapamil, norverapamil and isomers of O-demethylverapamil, which were believed to be exclusively produced by the liver.     

细胞色素P450的电化学研究进展

细胞色素P450的电化学研究从一个侧面反映了为使细胞色素P450达到工业催化剂的最终目的人们所作的不懈努力。本文从细胞色素P450在电极上的电子转移研究,隧道扫描显微镜的微观成像研究和使用电极作为细胞色素P450的电子给体从而实现细胞色素P450底物转化三方面,评述了近年来细胞色素P450的电化学研究进展。     

The stereochemistry of fatty acid hydroxylation by cytochrome P450BM3

The stereochemical preference for the cytochrome P450_BM3-catalysed hydroxylation of tetradecanoic and pentadecanoic acids has been determined via comparison with authentic non-racemic standards utilising enantioselective HPLC. The sub-terminal hydroxylation of these fatty acids by P450_BM3 is highly selective for the formation of the R-alcohols. This is the same enantioselectivity as is seen for hexadecanoic acid oxidation but contrasts with a previous report of S-hydroxylation of pentadecanoic acid by P450_BM3.     

Concomitant detection of CYP1A1 enzymatic activity and CYP1A1 protein in individual cells of a human urothelial cell line using a bilayer microfluidic device

To understand molecular networking at the cellular level, analyses of processes and effects at the single-cell level are most appropriate. Usual biochemical or molecular biological analyses are based on integrated signals of numerous cells which differ, however, in their expression and activity profiles. Here we show that it is possible to determine different types of properties of individual cells by means of a specifically designed microfluidic device. As part of investigations to characterize the human urothelial cell line 5637 as a potential model system for studies of toxic and carcinogenic effects on urothelial cells, we use this cell line to assign cytochrome P450 activity, and expression of the enzymes involved, to individual cells. It is shown that the cell population is very heterogeneous with respect to the extent and kinetics of CYP1A1-dependent ethoxyresorufin O-deethylase (EROD). This is also true for the cells’ CYP1A1 protein content. With some exceptions, the EROD activity largely coincides with the presence of CYP1A1 protein in the cells. The results obtained with the microfluidic device are promising and open up new perspectives with regard to multi-property determinations in individual cells and to studies focusing on the biochemical and molecular heterogeneity of cells. Figure Formation of fluorescent resorufin from ethoxyresorufin by cytochrome P450 activity in urothelial cells attached within the chamber of a microfluidic device     

Direct electrochemistry of human and rat NADPH cytochrome P450 reductase

The diflavo-protein NADPH cytochrome P450 reductase (CPR) is the key electron transfer partner for all drug metabolizing cytochrome P450 enzymes in humans. The protein delivers, consecutively, two electrons to the heme active site of the P450 in a carefully orchestrated process which ultimately leads to the generation of a high valent oxo-heme moiety. Despite its central role in P450 function, no direct electrochemical investigation of the purified protein has been reported. Here we report the first voltammetric study of purified human CPR where responses from both the FMN and FAD cofactors have been identified using both cyclic and square wave voltammetry. For human CPR redox responses at −2 and −278 mV (with a ratio of 1e⁻:3e⁻) vs NHE were seen at pH 7.9 while the potentials for rat CPR at pH 8.0 were −20 and −254 mV. All redox responses exhibit a pH dependence of approximately −59 mV/pH unit consistent with proton coupled electron transfer reactions of equal stoichiometry.     

Studies on metabolites and metabolic pathways of bulleyaconitine A in rat liver microsomes using LC‐MSn combined with specific inhibitors

Bulleyaconitine A (BLA) from Aconitum bulleyanum plants is usually used as anti‐inflammatory drug in some Asian countries. It has a variety of bioactivities, and at the same time some toxicities. Since the bioactivities and toxicities of BLA are closely related to its metabolism, the metabolites and the metabolic pathways of BLA in rat liver microsomes were investigated by HPLC–MSⁿ. In this research, the 12 metabolites of BLA were identified according to the results of HPLC‐MSⁿ data and the relevant literature. The results showed that there are multiple metabolites of BLA in rat liver microsomes, including demethylation, deacetylation, dehydrogenation deacetylation and hydroxylation. The major metabolic pathways of BLA in rat liver microsomes were clarified by HPLC‐MS combined with specific inhibitors of CYP450 isoforms. As a result, CYP3A and 2C were found to be the principal CYP isoforms contributing to the metabolism of BLA. Moreover, CYP2D6 and 2E1 are also more important CYP isoforms for the metabolism of BLA. While CYP1A2 only affected the formation rate of M11, its effect on the metabolism of BLA is very small. Copyright © 2014 John Wiley & Sons, Ltd.     

Faradic Peaks Enhanced by Carbon Nanotubes in Microsomal Cytochrome P450 Electrodes

In this work we present an investigation on the behavior of microsomes containing human cytochrome P450 in cyclic voltammetry for drug detection. The microsomes are adsorbed on the surface of multi‐walled carbon nanotubes by drop‐casting. We demonstrate that the hydrophobic and highly electroactive surface of multi‐walled carbon nanotubes enables to distinguish more clearly the contributions in reduction peak current attributed to the enzymatic components of microsomes. Voltammetric measurements were performed under several experimental conditions with two cytochrome P450‐isoforms, 1A2 and 3A4. We show that the reduction current for the component of cytochrome P450‐microsome linearly increases in the presence of a substrate.     

Engineering and assaying of cytochrome P450 biocatalysts

Cytochrome P450s constitute a highly fascinating superfamily of enzymes which catalyze a broad range of reactions. They are essential for drug metabolism and promise industrial applications in biotechnology and biosensing. The constant search for cytochrome P450 enzymes with enhanced catalytic performances has generated a large body of research. This review will concentrate on two key aspects related to the identification and improvement of cytochrome P450 biocatalysts, namely the engineering and assaying of these enzymes. To this end, recent advances in cytochrome P450 development are reported and commonly used screening methods are surveyed.     

尿中咖啡因代谢产物的测定及其在肝功能评价中的应用

建立了高效液相色谱分析尿中咖啡因及其7种代谢产物的方法。测定了饮用咖啡后尿样中13X／CA和17U／CA的比率,以此评价乙肝表面抗原呈阳性患者的P450酶活性。     

Molecular characterization,modeling and docking of CYP107CB2 from Bacillus lehensis G1, an alkaliphile

Cytochrome P450s are a superfamily of heme monooxygenases which catalyze a wide range of biochemical reactions. The reactions involve the introduction of an oxygen atom into an inactivated carbon of a compound which is essential to produce an intermediate of a hydroxylated product. The diversity of chemical reactions catalyzed by cytochrome P450s has led to their increased demand in numerous industrial and biotechnology applications. A recent study showed that a gene sequence encoding a CYP was found in the genome of Bacillus lehensis G1, and this gene shared structural similarity with the bacterial vitamin D hydroxylase (Vdh) from Pseudonocardia autotrophica. The objectives of present study was to mine, for a novel CYP from a new isolate B. lehensis G1 alkaliphile and determine the biological properties and functionalities of CYP in this bacterium. Our study employed the usage of computational methods to search for the novel CYP from CYP structural databases to identify the conserved pattern, functional domain and sequence properties of the uncharacterized CYP from B. lehensis G1. A computational homology model of the protein’s structure was generated and a docking analysis was performed to provide useful structural knowledge on the enzyme’s possible substrate and their interaction. Sequence analysis indicated that the newly identified CYP, termed CYP107CB2, contained the fingerprint heme binding sequence motif FxxGxxxCxG at position 336-345 as well as other highly conserved motifs characteristic of cytochrome P450 proteins. Using docking studies, we identified Ser-79, Leu-81, Val-231, Val-279, Val-383, Ala-232, Thr-236 and Thr-283 as important active site residues capable of stabilizing interactions with several potential substrates, including vitamin D₃, 25-hydroxyvitamin D₃ and 1α-hydroxyvitamin D₃, in which all substrates docked proximally to the enzyme’s heme center. Biochemical analysis indicated that CYP107CB2 is a biologically active protein to produce 1α,25-dihydroxyvitamin D₃ from 1α-hydroxyvitamin D₃. Based on these results, we conclude that the novel CYP107CB2 identified from B. lehensis G1 is a putative vitamin D hydroxylase which is possibly capable of catalyzing the bioconversion of parental vitamin D₃ to calcitriol, or related metabolic products.     

Study of recombinant cytochrome P450 2C9 activity with diclofenac by MEKC

Cytochrome P450 2C9 (CYP2C9) is one of the most important isoforms in human liver involved in the metabolism of a large number of therapeutic agents. The aim of this paper is to demonstrate the applicability of CE for the determination of the enzymatic activity of CYP2C9 with diclofenac as a probe substrate. MEKC with SDS as a pseudostationary phase was used for this purpose. Compared to other assays, the MEKC-based method is rapid, can be automated and requires only a small quantity of enzymes and substrate. Moreover, the enzymatic reaction can be monitored with high sensitivity and repeatability even when the reaction mixture is used for the analysis without any pretreatment. The kinetic study on the given enzymatic reaction was also performed since the basic characterization of drug biotransformation generally begins with the enzyme kinetic analysis of metabolite formation. As a result, the Michaelis constant and maximum reaction velocity were evaluated, the values 3.44 +/- 0.45 microM and 19.78 +/- 0.76 nmol min(-1) nmol(-1), respectively, were in agreement with the literature data. On the other hand, a slight deviation from typical Michaelis-Menten kinetics with a weak positive cooperativity was found at diclofenac concentrations below 2 microM. The same atypical kinetic behavior of CYP2C9 was also observed by other authors.     

Monitoring Caffeine in Human Saliva Using a Newly Developed ELISA

Caffeine is a useful indicator to quickly assess liver function. High-throughput tests are needed for single-point caffeine measurements, with low cross-reactivity toward its major metabolite, paraxanthine. A newly developed ELISA was compared with an LC-MS/MS reference method, using 60 saliva samples from 10 individuals, before and after caffeine intake. Bland-Altman plot, Student t-test and F-test were used to compare the two methods. Proteins were precipitated using organic solvent and the caffeine recoveries compared with those obtained using sample microfiltration. The antibody, with a low cross-reactivity toward paraxanthine (0.08%), allows quantification of caffeine in saliva samples from 2.5 µg/L to 125 µg/L with high precision and the ELISA shows comparable results to those obtained by LC-MS/MS. A one-step protein precipitation using an organic solvent provides comparable results to a more costly and time-consuming microfiltration pre-treatment of samples. The new ELISA is a fit-for-purpose method to accurately and precisely determine caffeine in saliva samples.     

Analysis of CYP1A1 induction in single cells of urothelial cell populations by flow cytometry

As carcinogenesis is a process starting at the single-cell level it is desirable to study carcinogen-mediated effects in individual cells. A primary step in chemically induced carcinogenesis is the formation of reactive DNA-binding metabolites by cytochromes P450 (CYP). We applied indirect immunofluorescence to stain CYP1A1 in urothelial cells for quantification by flow cytometry. Our studies were carried out with metabolically competent primary porcine urinary bladder epithelial cells (PUBECs) and the human urothelial cell line 5637 for which we have previously demonstrated CYP1A1 mRNA induction by the polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (B[a]P) applying real-time RT-PCR. Flow cytometric analysis revealed that for PUBEC and 5637 cells the fraction of CYP1A1-induced cells increased with B[a]P concentration. Furthermore, in 5637 cells this effect was time-dependent, being more pronounced after 48 h than after 24 h. However, CYP1A1 induction could not be detected in all analyzed PUBEC and 5637 cells after treatment with up to 50 μM B[a]P. The reason for this remains unknown at the moment. Overall, B[a]P-treated cells could be divided into fractions of clearly CYP1A1-induced and clearly uninduced cells. Another fraction of “unclear” CYP1A1-induced cells and one of unclassifiable cells remained, as quantification of CYP1A1 induction by flow cytometry was hampered by B[a]P-related fluorescence. This is ascribed to phenolic B[a]P metabolites formed by CYP1A1 and which are known to fluoresce at wavelengths above 500 nm, whereas B[a]P does not. Overall, the method permits the detection of CYP1A1 protein level in large numbers of individual cells, thereby providing an adequate basis for statistical analyses. Flow cytometric detection of CYP1A1 induction in individual cells allows further insight into the metabolic competence of single cells and therefore could be a valuable tool for toxicological studies. Figure Flow cytometric analysis of immunostained PUBEC cells reveals that CYP1A1 is induced by B[a]P in a concentration-dependent manner Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cytochrome P450 BM-3 Evolved by Random and Saturation Mutagenesis as an Effective Indole-Hydroxylating Catalyst

Cytochrome P450 BM-3 with the mutations A74G, F87V, and L188Q could catalyze indole to produce indigo and indirubin. To further enhance this capability, site-directed and random mutageneses on the monooxygenase domain of P450 BM-3 mutant (A74G/F87V/L188Q; 3X) were performed. The mutant libraries created by error-prone polymerase chain reaction were screened using a colorimetric colony-based method on agar plates followed by a spectroscopic assay involving in absorption of indigo at 670 nm and NADPH at 340 nm in microtiter plate. Three mutants (K434R/3X, E435D/3X, and D168N/A225V/K440N/3X) exhibited higher hydroxylation activity toward indole in comparison to parent enzyme. Moreover, using saturation site-directed mutagenesis at amino acid positions 168, 225, 434, 435, and 440, two P450 BM-3 variants (D168H/3X, E435T/3X) with an up to sixfold increase in catalytic efficiency (k _cat/K _m) were identified, and the mutant D168H/3X acquired higher regioselectivity resulting in more indigo (dimerized 3-hydroxy-indole) compared to parent mutant (93 vs72%).