Active Ingredients and Anti-Arthritic Mechanisms of Ba-Wei-Long-Zuan Granule Revealed by 1 H-NMR-Based Metabolomics Combined with Network Pharmacology Analysis.

Ba-Wei-Long-Zuan granule (BWLZ) is a traditional herbal preparation. It has been widely used for the treatment of rheumatoid arthritis (RA). However, its active ingredients and mechanisms of action are still unclear. The present study aims to reveal the active compounds and anti-arthritic mechanisms of BWLZ against collagen-induced arthritis (CIA) by using 1 H-NMR-based metabolomics, molecular docking and network pharmacology methods. After 30 days of administration, BWLZ could effectively improve the metabolic disorders in CIA rats. The anti-arthritic effect of BWLZ was related to its restoration of 16 disturbed serum metabolites. Molecular docking and network analysis showed that 20 compounds present in BWLZ could act on multiple targets. Among them, coclaurine and hesperidin showed the highest hit rates for target proteins related to both metabolic regulation and RA, indicating that these two compounds might be potential active ingredients of BWLZ. Moreover, pathway enrichment analysis suggested that the anti-arthritic mechanisms of BWLZ might be attributed to its network regulation of several biological processes, such as steroid hormone biosynthesis, mTOR signaling pathway, alanine, aspartate and glutamate metabolism, and synthesis and degradation of ketone bodies. These results provide further evidence for the anti-arthritic properties of BWLZ and are beneficial for its quality control and clinical application. The potential targets and biological processes found in this study may provide valuable information for further studying the molecular mechanisms of BWLZ against RA. In addition, our work provides new insights for revealing the active ingredients and regulatory mechanisms of complex herbal preparations.

The citrus flavonone hesperetin attenuates the nuclear translocation of aryl hydrocarbon receptor.

The environmental polycyclic aromatic hydrocarbons (PAH) and dioxins are carcinogens and their adverse effects have been largely attributed to the activation of AhR. Hesperetin is a flavonone found abundantly in citrus fruits and has been shown to be a biologically active agent. In the present study, the effect of hesperetin on the nuclear translocation of AhR and the downstream gene expression was investigated in MCF-7 cells. Confocal microscopy indicated that 7, 12-dimethylbenz[α]anthracene (DMBA) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) -induced nuclear translocation of AhR was deterred by hesperetin treatment. The reduced nuclear translocation could also be observed in Western analysis. Reporter-gene assay further illustrated that the induced XRE transactivation was weakened by the treatment of hesperetin. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay demonstrated that the gene expressions of CYP1A1, 1A2, and 1B1 followed the same pattern of AhR translocation. These results suggested that hesperetin counteracted AhR transactivation and suppressed the downstream gene expression.

The inhibitory effects of ß-caryophyllene, ß-caryophyllene oxide and α-humulene on the activities of the main drug-metabolizing enzymes in rat and human liver in vitro.

Sesquiterpenes, the main components of plant essential oils, are often taken in the form of folk medicines and dietary supplements. Several sesquiterpenes possess interesting biological activities but they could interact with concurrently administered drugs via inhibition of drug-metabolizing enzymes. Therefore, the present study was designed to test the potential inhibitory effect of tree structurally relative sesquiterpenes ß-caryophyllene (CAR), ß-caryophyllene oxide (CAO) and α-humulene (HUM) on the activities of the main drug-metabolizing enzymes. For this purpose, rat and human hepatic subcellular fractions were incubated with CAR, CAO or HUM together with specific substrates for oxidation, reduction and conjugation enzymes and their coenzymes. HPLC, spectrophotometric and spectrofluorimetric analyses of product formations were used. All tested sesquiterpenes significantly inhibited cytochromes P4503A (CYP3A) activities in rats as well as in human hepatic microsomes, with CAO being the strongest inhibitor. A non-competitive type of inhibition was found. On the other hand, none of the tested sesquiterpenes significantly affected the activities of carbonyl-reducing enzymes (CBR1, AKRs, NQO1) or conjugation enzymes (UGTs, GSTs, SULTs, COMT). As CYP3A enzymes metabolize many drugs, their inhibition by CAO, CAR and HUM might affect the pharmacokinetics of concurrently administered drugs. Similar results obtained in rat and human hepatic microsomes indicate that rats could be used for further testing of possible drug-sesquiterpenes interactions in vivo.

Simple chalcones and bis-chalcones ethers as possible pleiotropic agents.

The synthesis, the antioxidative properties and the lipoxygenase (LOX) and acetylcholinesterase (AChE) inhibition of a number of 4-hydroxy-chalcones diversely substituted as well as of a series of bis-chalcones ether derivatives are reported. The chalcones derivatives were readily produced using a Claisen-Schmidt condensation in a ultra sound bath in good yields. The structures of the synthesized compounds were confirmed by spectral and elemental analysis. Their lipophilicity is experimentally determined by reversed-phase thin-layer chromatography method. Most of them are potent in vitro inhibitors of lipid peroxidation and of LOX. Compounds b2 and b3 were found to be the most potent LOX and AChE inhibitors among the tested derivatives with a significant anti-lipid peroxidation profile. The results led us to propose these enone derivatives as new multifunctional compounds against Alzheimer's disease. The results are discussed in terms of structural and physicochemical characteristics of the compounds. Moreover, the pharmacokinetic profile of these compounds was investigated using computational methods.

Cytochrome P450 system proteins reside in different regions of the endoplasmic reticulum.

Cytochrome P450 (P450) function is dependent on the ability of these enzymes to successfully interact with their redox partners, NADPH-cytochrome P450 reductase (CPR) and cytochrome b5, in the endoplasmic reticulum (ER). Because the ER is heterogeneous in lipid composition, membrane microdomains with different characteristics are formed. Ordered microdomains are more tightly packed, and enriched in saturated fatty acids, sphingomyelin and cholesterol, whereas disordered regions contain higher levels of unsaturated fatty acids. The goal of the present study was to determine whether the P450 system proteins localize to different regions of the ER. The localization of CYP1A2, CYP2B4 and CYP2E1 within the ER was determined by partial membrane solubilization with Brij 98, centrifugation on a discontinuous sucrose gradient and immune blotting of the gradient fractions to identify ordered and disordered microdomains. CYP1A2 resided almost entirely in the ordered regions of the ER with CPR also localized predominantly to this region. CYP2B4 was equally distributed between the ordered and disordered domains. In contrast, CYP2E1 localized to the disordered membrane regions. Removal of cholesterol (an important constituent of ordered domains) led to the relocation of CYP1A2, CYP2B4 and CPR to the disordered regions. Interestingly, CYP1A1 and CYP1A2 localized to different membrane microdomains, despite their high degree of sequence similarity. These data demonstrate that P450 system enzymes are organized in specific membrane regions, and their localization can be affected by depletion of membrane cholesterol. The differential localization of different P450 in specific membrane regions may provide a novel mechanism for modulating P450 function.

Inhibition of CYP1 by berberine, palmatine, and jatrorrhizine: selectivity, kinetic characterization, and molecular modeling.

Cytochrome P450 (P450, CYP) 1 family plays a primary role in the detoxification and bioactivation of polycyclic aromatic hydrocarbons. Human CYP1A1, CYP1A2, and CYP1B1 exhibit differential substrate specificity and tissue distribution. Berberine, palmatine, and jatrorrhizine are protoberberine alkaloids present in several medicinal herbs, such as Coptis chinensis (Huang-Lian) and goldenseal. These protoberberines inhibited CYP1A1.1- and CYP1B1.1-catalyzed 7-ethoxyresorufin O-deethylation (EROD) activities, whereas CYP1A2.1 activity was barely affected. Kinetic analysis revealed that berberine noncompetitively inhibited EROD activities of CYP1A1.1 and CYP1B1.1, whereas palmatine and jatrorrhizine caused either competitive or mixed type of inhibition. Among protoberberines, berberine caused the most potent and selective inhibitory effect on CYP1B1.1 with the least Ki value of 44±16 nM. Berberine also potently inhibited CYP1B1.1 activities toward 7-ethoxycoumarin and 7-methoxyresorufin, whereas the inhibition of benzo(a)pyrene hydroxylation activity was less pronounced. Berberine inhibited the polymorphic variants, CYP1B1.3 (V432L) and CYP1B1.4 (N453S), with IC50 values comparable to that for CYP1B1.1 inhibition. Berberine-mediated inhibition was abolished by a mutation of Asn228 to Thr in CYP1B1.1, whereas the inhibition was enhanced by a reversal mutation of Thr223 to Asn in CYP1A2.1. This result in conjugation with the molecular modeling revealed the crucial role of hydrogen-bonding interaction of Asn228 on CYP1B1.1 with the methoxy moiety of berberine. These findings demonstrate that berberine causes a selective CYP1B1-inhibition, in which Asn228 appears to be crucial. The inhibitory effects of berberine on CYP1B1 activities toward structurally diverse substrates can be different.

[Preclinical investigation of pharmaceuticals impact against cytochrome P450 activity and prognosis of substrate affinity as means for providing substrate therapy safety].

Tricyclic antidepressants, not influencing the P450 3A4 activity, are safe in combination with drugs of other groups used in the treatment of comorbid patients. Azaphen is is one of the agents most widely used in the clinical practice. The in vitro electrochemical analysis showed that pipofezin (azaphen) was not a substrate, inductor, and/or inhibitor of cytochrome P450 CYP3A4 isoenzymes. The Guzar programme computer modelling and the literature data demonstrated the substrate affinity of pipifezin to CYP1A2.

Preferred binding orientations of phenacetin in CYP1A1 and CYP1A2 are associated with isoform-selective metabolism.

Human cytochromes P450 1A1 and 1A2 play important roles in drug metabolism and chemical carcinogenesis. Although these two enzymes share high sequence identity, they display different substrate specificities and inhibitor susceptibilities. In the present studies, we investigated the structural basis for these differences with phenacetin as a probe using a number of complementary approaches, such as enzyme kinetics, stoichiometric assays, NMR, and molecular modeling. Kinetic and stoichiometric analyses revealed that substrate specificity (k(cat)/K(m)) of CYP1A2 was approximately 18-fold greater than that of CYP1A1, as expected. Moreover, despite higher H2O2 production, the coupling efficiency of reducing equivalents to acetaminophen formation in CYP1A2 was tighter than that in CYP1A1. CYP1A1, in contrast to CYP1A2, displayed much higher uncoupling, producing more water. The subsequent NMR longitudinal (T1) relaxation studies with the substrate phenacetin and its product acetaminophen showed that both compounds displayed similar binding orientations within the active site of CYP1A1 and CYP1A2. However, the distance between the OCH2 protons of the ethoxy group (site of phenacetin O-deethylation) and the heme iron was 1.5 Å shorter in CYP1A2 than in CYP1A1. The NMR findings are thus consistent with our kinetic and stoichiometric results, providing a likely molecular basis for more efficient metabolism of phenacetin by CYP1A2.

The molecular basis for the inhibition of human cytochrome P450 1A2 by oroxylin and wogonin.

In our previous kinetics studies the natural products oroxylin and wogonin were shown to have strong biological affinity for, and inhibitory effects against, human cytochrome P450 1A2, with IC(50) values of 579 and 248 nM, respectively; this might lead to the occurrence of drug-drug interactions when co-administered clinically. However, their inhibitory mechanisms against 1A2 remain elusive. In this study, molecular docking and molecular dynamics simulations were performed to better understand the molecular basis of their inhibitory mechanisms towards 1A2. Structural analysis revealed that oroxylin has a different binding pattern from wogonin and another very strongly binding inhibitor α-naphthoflavone (ANF, IC(50) = 49 nM). The O(7) atom of oroxylin forms hydrogen bonds with the OD1/OD2 atoms of Asp313, which is not observed in the 1A2-wogonin complex. Because of energetically unfavorable repulsions with the methoxy group at the 6 position of the oroxylin ring, significant conformational changes were observed for the sidechain of Thr118 in the MD simulated model. As a result, the larger and much more open binding-site architecture of the 1A2-oroxylin complex may account for its weaker inhibitory effect relative to the 1A2-ANF complex. Energy analysis indicated that oroxylin has a less negative predicted binding free energy of -19.8 kcal/mol than wogonin (-21.1 kcal/mol), which is consistent with our experimental assays. Additionally, our energy results suggest that van der Waals/hydrophobic and hydrogen-bonding interactions are important in the inhibitory mechanisms of oroxylin whereas the former is the underlying force responsible for strong inhibition by ANF and wogonin.

Computational and in vitro studies on the inhibitory effects of herbal compounds on human cytochrome P450 1A2.

Human CYP1A2 is an important enzyme for drug metabolism and procarcinogen activation. This study aimed to explore the binding mode of ligands with CYP1A2 and to screen potential inhibitors from a library of herbal compounds using computational and in vitro approaches. The heme prosthetic group and six residues (Thr124, Phe125, Phe226, Phe260, Gly316, and Ala317) in the active site of CYP1A2 were identified as important residues for ligand binding using the LIGPLOT program. Ala317 in helix I immediately above heme was highly conserved in most human CYPs with known crystal structures. In molecular docking, 19 of the 56 herbal compounds examined were identified as potential inhibitors of CYP1A2. Up to 21 of the 56 herbal compounds were hit by the pharmacophore model of CYP1A2 inhibitors developed and validated in this study. In the in vitro inhibition study, 8 herbal compounds were identified as moderate to potent inhibitors of CYP1A2. Five of the 8 herbal compounds predicted to be potential inhibitors were confirmed as CYP1A2 inhibitors in the in vitro study. A combination of computational and in vitro approaches, represent a useful tool to identify potential inhibitors for CYP1A2 from herbal compounds.

Effect of physicochemical parameters on enzymatic biodecaffeination during tea fermentation.

We report for the first time the development of a biodecaffeination process for tea synchronised with tea fermentation process using enzymes isolated from Pseudomonas alcaligenes. Cell-free extract was used for biodecaffeination of tea during fermentation of tea and 80% of the caffeine in the tea dhool was degraded within 90 min of incubation. Several factors that tend to effect the biodecaffeination during this stage, like moisture, aeration, intermittent enzyme addition and mixing, were optimized, and inhibitory interactions of proteins with polyphenols, caffeine-polyphenol interactions, which directly influence the biodecaffeination process were prevented by the use of glycine (5% w/w) in the dhool. Tea decaffeinated through the enzymatic route retained the original flavor and aroma, and there was an increase in the total polyphenol content of the tea.

Effects of ephedra water decoction and cough tablets containing ephedra and liquorice on CYP1A2 and the pharmacokinetics of theophylline in rats.

Ephedra water decoction (EWD) and cough tablets containing ephedra and liquorice (maxing cough tablets, MXCT) have been used widely in the treatment of asthma. In the clinic, EWD and MXCT may be prescribed with theophylline, one of the most popular antiasthmatic drugs and a typical substrate of cytochrome P450 (CYP) 1A2. So in the present study the potential effects of EWD and MXCT on CYP1A2 activity and the pharmacokinetics of theophylline in rats were evaluated. In the in vivo CYP1A2 activity research, the rats were given oral caffeine (10 mg/kg) after a 14 day pretreatment with EWD (18 g/kg) and MXCT (0.1, 0.2 or 0.4 g/kg). Then the CYP 1A2 activity was expressed by using the caffeine metabolic ratio (CMR). The results showed that the CMR increased markedly compared with the control groups. In the pharmacokinetics experiment, the rats were given oral theophylline (10 mg/kg) after a 14 day pretreatment with EWD (18 g/kg) and MXCT (0.2 g/kg). The results showed that the AUC(0-24 h) and C(max) of theophylline were reduced markedly compared with the control groups. These results demonstrated that EWD or MXCT pretreatment obviously induced CYP1A2 activity, therefore, speeding up the metabolism of theophylline. The concomitant use of EWD or MXCT may decrease the effect of theophylline in rats.

Theoretical investigation of differences in nitroreduction of aristolochic acid I by cytochromes P450 1A1, 1A2 and 1B1.

OBJECTIVES: The herbal drug aristolochic acid (AA) derived from Aristolochia species has been shown to be the cause of aristolochic acid nephropathy (AAN), Balkan endemic nephropathy (BEN) and their urothelial malignancies. One of the common features of AAN and BEN is that not all individuals exposed to AA suffer from nephropathy and tumor development. One cause for these different responses may be individual differences in the activities of the enzymes catalyzing the biotransformation of AA. Thus, the identification of enzymes principally involved in the metabolism of AAI, the major toxic component of AA, and detailed knowledge of their catalytic specificities is of major importance. Human cytochrome P450 (CYP) 1A1 and 1A2 enzymes were found to be responsible for the AAI reductive activation to form AAI-DNA adducts, while its structurally related analogue, CYP1B1 is almost without such activity. However, knowledge of the differences in mechanistic details of CYP1A1-, 1A2-, and 1B1- mediated reduction is still lacking. Therefore, this feature is the aim of the present study. METHODS: Molecular modeling capable of evaluating interactions of AAI with the active site of human CYP1A1, 1A2 and 1B1 under the reductive conditions was used. In silico docking, employing soft-soft (flexible) docking procedure was used to study the interactions of AAI with the active sites of these human enzymes. RESULTS: The predicted binding free energies and distances between an AAI ligand and a heme cofactor are similar for all CYPs evaluated. AAI also binds to the active sites of CYP1A1, 1A2 and 1B1 in similar orientations. The carboxylic group of AAI is in the binding position situated directly above heme iron. This ligand orientation is in CYP1A1/1A2 further stabilized by two hydrogen bonds; one between an oxygen atom of the AAI nitro-group and the hydroxyl group of Ser122/Thr124; and the second bond between an oxygen atom of dioxolane ring of AAI and the hydroxyl group of Thr497/Thr498. For the CYP1B1:AAI complex, however, any hydrogen bonding of the nitro-group of AAI is prevented as Ser122/Thr124 residues are in CYP1B1 protein replaced by hydrophobic residue Ala133. CONCLUSION: The experimental observations indicate that CYP1B1 is more than 10× less efficient in reductive activation of AAI than CYP1A2. The docking simulation however predicts the binding pose and binding energy of AAI in the CYP1B1 pocket to be analogous to that found in CYP1A1/2. We believe that the hydroxyl group of S122/T124 residue, with its polar hydrogen placed close to the nitro group of the substrate (AAI), is mechanistically important, for example it could provide a proton required for the stepwise reduction process. The absence of a suitable proton donor in the AAI-CYP1B1 binary complex could be the key difference, as the nitro group is in this complex surrounded only by the hydrophobic residues with potential hydrogen donors not closer than 5 Å.

Effects of furanocoumarins from apiaceous vegetables on the catalytic activity of recombinant human cytochrome P-450 1A2.

Inhibition of cytochrome P-450 1A2 (CYP1A2)-mediated activation of procarcinogens may be an important chemopreventive mechanism. Consumption of apiaceous vegetables (rich in furanocoumarins) inhibits CYP1A2 in humans. Because many furanocoumarins are potent inhibitors of several CYPs, we characterized the effects of three furanocoumarins from apiaceous vegetables on human CYP1A2 (hCYP1A2). We assessed hCYP1A2 methoxyresorufin O-demethylase (MROD) activity using microsomes from Saccharomyces cerevisiae expressing hCYP1A2. Isopimpinellin exhibited mechanism-based inactivation (MBI) of hCYP1A2 (K(i) = 1.2 µM, k (inact) = 0.34 min⁻¹, and partition ratio = 8). Imperatorin and trioxsalen were characterized as mixed inhibitors with K(i) values of 0.007 and 0.10 µM, respectively. These results indicate that even if present at low levels in apiaceous vegetables, imperatorin, trioxsalen and isopimpinellin may contribute significantly to CYP1A2 inhibition and potentially decreased procarcinogen activation. Moreover, the in vivo effect of isopimpinellin on CYP1A2 may be longer lasting compared to reversible inhibitors.

Novel natural inhibitors of CYP1A2 identified by in silico and in vitro screening.

Inhibition of cytochrome P450 (CYP) is a major cause of herb-drug interactions. The CYP1A2 enzyme plays a major role in the metabolism of drugs in humans. Its broad substrate specificity, as well as its inhibition by a vast array of structurally diverse herbal active ingredients, has indicated the possibility of metabolic herb-drug interactions. Therefore nowadays searching inhibitors for CYP1A2 from herbal medicines are drawing much more attention by biological, chemical and pharmological scientists. In our work, a pharmacophore model as well as the docking technology is proposed to screen inhibitors from herbal ingredients data. Firstly different pharmaphore models were constructed and then validated and modified by 202 herbal ingredients. Secondly the best pharmaphore model was chosen to virtually screen the herbal data (a curated database of 989 herbal compounds). Then the hits (147 herbal compounds) were continued to be filtered by a docking process, and were tested in vitro successively. Finally, five of eighteen candidate compounds (272, 284, 300, 616 and 817) were found to have inhibition of CYP1A2 activity. The model developed in our study is efficient for in silico screening of large herbal databases in the identification of CYP1A2 inhibitors. It will play an important role to prevent the risk of herb-drug interactions at an early stage of the drug development process.

The effect of infliximab on hepatic cytochrome P450 and pharmacokinetics of verapamil in rats with pre-adjuvant arthritis: a drug-disease and drug-drug interaction.

Inflammatory conditions result in increased concentration but reduced potency of some cardiovascular drugs. This is associated with increased levels of pro-inflammatory mediators. Infliximab reduces pro-inflammatory mediators and reverses the diminishing effect of inflammation on response in the rat. We suggested that infliximab treatment would also reverse the effects of inflammation on drug metabolism and clearance. We examined hepatic cytochrome P450 content and the pharmacokinetics of verapamil in pre-adjuvant arthritic rats treated with infliximab. Pre-adjuvant arthritis was induced in male Sprague-Dawley rats with a tail base injection of Mycobacterium butyricum. Animals were monitored for symptoms of arthritis, serum nitrite and C-reactive protein. On day 6, rats were administered with single s.c. doses of infliximab (10 mg/kg). On day 14, a single i.v. dose of racemic verapamil (2 mg/kg) was administered, and S- and R-verapamil concentrations were determined by high performance liquid chromatography. Hepatic cytochrome P450 content and verapamil protein binding were also measured. Serum nitrite levels were significantly elevated in pre-adjuvant arthritis. Infliximab did not affect mean nitrite concentrations but there was a significant correlation between nitrite and S-verapamil concentrations as well as cytochrome P450, CYP3A, and CYP1A contents. Infliximab increased cytochrome P450 enzymes content that had been diminished by pre-adjuvant arthritis but had no significant effect on verapamil protein binding. Infliximab partially restores hepatic cytochrome P450 enzyme contents. The effect of infliximab on the mean verapamil clearance was not significantly affected due, likely, to the lack of effect on plasma protein binding.

In vitro and in silico identification and characterization of thiabendazole as a mechanism-based inhibitor of CYP1A2 and simulation of possible pharmacokinetic drug-drug interactions.

Thiabendazole (TBZ) and its major metabolite 5-hydroxythiabendazole (5OH-TBZ) were screened for potential time-dependent inhibition (TDI) against CYP1A2. Screen assays were carried out in the absence and presence of NADPH. TDI was observed with both compounds, with k(inact) and K(I) values of 0.08 and 0.02 min(-1) and 1.4 and 63.3 microM for TBZ and 5OH-TBZ, respectively. Enzyme inactivation was time-, concentration-, and NADPH-dependent. Inactivation by TBZ was irreversible by dialysis and oxidation by potassium ferricyanide, and there was no protection by glutathione. 5OH-TBZ was a weak TDI of CYP1A2, and enzyme activity was recovered by dialysis. IC(50) determination of TBZ and 5OH-TBZ showed both compounds to be potent inhibitors, with IC(50) values of 0.83 and 13.05 microM, respectively. IC(50) shift studies also demonstrated that TBZ was a TDI of CYP1A2. In silico methods identified the thiazole group as a TDI fragment and predicted it as the site of metabolism. The observation pointed to epoxidation of the thiazole and the benzyl rings of TBZ as possible routes of metabolism and mechanisms of TDI. Drug-drug interaction (DDI) simulation studies using SimCyp showed good predictions for competitive inhibition. However, predictions for mechanism-based inhibition (MBI)-based DDI were not in agreement with clinical observations. There was no TBZ accumulation upon chronic administration of the drug. The in vitro MBI findings might therefore not be capturing the in vivo situation in which the proposed bioactivation route is minor. This might be the case for TBZ in which, in vivo, UDP glucuronosyltransferases and sulfanotransferase metabolize and eliminate the 5OH-TBZ.

Virtual screening and prediction of site of metabolism for cytochrome P450 1A2 ligands.

With the availability of an increasing number of high resolution 3D structures of human cytochrome P450 enzymes, structure-based modeling tools are more readily used. In this study we explore the possibilities of using docking and scoring experiments on cytochrome P450 1A2. Three different questions have been addressed: 1. Binding orientations and conformations were successfully predicted for various substrates. 2. A virtual screen was performed with satisfying enrichment rates. 3. A classification of individual compounds into active and inactive was performed. It was found that while docking can be used successfully to address the first two questions, it seems to be more difficult to perform the classification. Different scoring functions were included, and the well-characterized water molecule in the active site was included in various ways. Results are compared to experimental data and earlier classification data using machine learning methods. The possibilities and limitations of using structure-based drug design tools for cytochrome P450 1A2 come to light and are discussed.

Mechanism of oxidative DNA damage induced by capsaicin, a principal ingredient of hot chili pepper.

Although capsaicin exhibits antitumor activity, carcinogenic potential has also been reported. To clarify the mechanism for expression of potential carcinogenicity of capsaicin, we examined DNA damage induced by capsaicin in the presence of metal ion and various kinds of cytochrome P450 (CYP) using 32P-5'-end-labeled DNA fragments. Capsaicin induced Cu(II)-mediated DNA damage efficiently in the presence of CYP1A2 and partially in the presence of 2D6. CYP1A2-treated capsaicin caused double-base lesions at 5'-TG-3', 5'-GC-3' and CG of the 5'-ACG-3' sequence complementary to codon 273, a hotspot of p53 gene. DNA damage was inhibited by catalase and bathocuproine, a Cu(I) chelator, suggesting that reactive species derived from the reaction of H2O2 with Cu(I) participate in DNA damage. Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine was significantly increased by CYP1A2-treated capsaicin in the presence of Cu(II). Therefore, we conclude that Cu(II)-mediated oxidative DNA damage by CYP-treated capsaicin seems to be relevant for the expression of its carcinogenicity.

Development of a novel cytochrome p450 bioaffinity detection system coupled online to gradient reversed-phase high-performance liquid chromatography.

A high-resolution screening platform, coupling online affinity detection for mammalian cytochrome P450s (Cyt P450s) to gradient reversed-phase high-performance liquid chromatography (HPLC), is described. To this end, the online Cyt P450 enzyme affinity detection (EAD) system was optimized for enzyme (beta-NF-induced rat liver microsomes), probe substrate (ethoxyresorufine), and organic modifier (methanol or acetonitrile). The optimized Cyt P450 EAD system has first been evaluated in a flow injection analysis (FIA) mode with 7 known ligands of Cyt P450 1A1/1A2 (alpha-naphthoflavone, beta-naphthoflavone, ellipticine, 9-hydroxy-ellipticine, fluvoxamine, caffein, and phenacetin). Subsequently, IC50 values were online in FIA-mode determined and compared with those obtained with standardmicrosomal assay conditions. The IC50 values obtained with the online Cyt P450 EAD system agreed well with the IC50 values obtained in the standard assays. For high affinity ligands of Cyt P450 1A1/1A2, detection limits of 1 to 3 pmol injected (n=3; signal to noise [S/N]=3) were obtained. The individual inhibitory properties of ligands in mixtures of the ligands were subsequently investigated using an optimized Cyt P450 EAD system online coupled to gradient HPLC. Using the integrated online gradient HPLC Cyt P450 EAD platform, detection limits of 10 to 25 pmol injected (n=1; S/N=3) were obtained for high-affinity ligands. It is concluded that this novel screening technology offers new perspectives for rapid and sensitive screening of individual compounds in mixtures exhibiting affinity for liver microsomal Cyt P450s.