Equine cytochrome P450 2C92: cDNA cloning, expression and initial characterization

Substantial gaps exist in our knowledge of the metabolic clearance of therapeutic agents in horses. Accordingly, a cytochrome P450 monooxygenase in the 2C family was cloned from an equine liver, sequenced and expressed in a baculovirus expression system. Catalytic activities of the recombinant protein were measured with a number of substrates. The protein, assigned CYP2C92, displayed optimal catalytic activity with diclofenac using molar ratios of CYP2C92 to NADPH CYP450 reductase of 1:18. Addition of cytochrome b₅ to diclofenac incubations had no significant effect on metabolic turnover. CYP2C92 catalyzed diclofenac metabolism was 20-fold slower than the human counterpart, CYP2C9. CYP2C92 demonstrated comparable tolbutamide and (S)-warfarin hydroxylase activity compared to CYP2C9, upon addition of b₅ to the reactions. The results of this study demonstrate substantial interspecies differences in metabolism of substrates by CYP2C orthologues in the horse and human and support the need to fully characterize this enzyme system in equids.     

Inhibition of human drug metabolizing cytochrome P450 enzymes by plant isoquinoline alkaloids

The human cytochrome P450 (CYP) enzymes play a major role in the metabolism of endobiotics and numerous xenobiotics including drugs. Therefore it is the standard procedure to test new drug candidates for interactions with CYP enzymes during the preclinical development phase. The purpose of this study was to determine in vitro CYP inhibition potencies of a set of isoquinoline alkaloids to gain insight into interactions of novel chemical structures with CYP enzymes. These alkaloids (n = 36) consist of compounds isolated from the Papaveraceae family (n = 20), synthetic analogs (n = 15), and one commercial compound. Their inhibitory activity was determined towards all principal human drug metabolizing CYP enzymes: 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4. All alkaloids were assayed in vitro in a 96-well plate format using pro-fluorescent probe substrates and recombinant human CYP enzymes. Many of these alkaloids inhibited the CYP3A4 form, with 30/36 alkaloids inhibiting CYP3A4 with at least moderate potency (IC₅₀ < 10 μM) and 15/36 inhibiting CYP3A4 potently (IC₅₀ < 1 μM). Among them corydine, parfumine and 8-methyl-2,3,10,11-tetraethoxyberbine were potent and selective inhibitors for CYP3A4. CYP2D6 was inhibited with at least moderate potency by 26/34 alkaloids. CYP2C19 was inhibited by 15/36 alkaloids at least moderate potently, whereas CYP1A2, CYP2B6, CYP2C8, and CYP2C9 were inhibited to a lesser degree. CYP2A6 was not significantly inhibited by any of the alkaloids. The results provide initial structure-activity information about the interaction of isoquinoline alkaloids with major human xenobiotic-metabolizing CYP enzymes, and illustrate potential novel structures as CYP form-selective inhibitors.     

Substrate selectivity of human cytochrome P450 2C9: importance of residues 476, 365, and 114 in recognition of diclofenac and sulfaphenazole and in mechanism-based inactivation by tienilic acid

A series of six site-directed mutants of CYP 2C9 were constructed with the aim to better define the amino acid residues that play a critical role in substrate selectivity of CYP 2C9, particularly in three distinctive properties of this enzyme: (i) its selective mechanism-based inactivation by tienilic acid (TA), (ii) its high affinity and hydroxylation regioselectivity toward diclofenac, and (iii) its high affinity for the competitive inhibitor sulfaphenazole (SPA). The S365A mutant exhibited kinetic characteristics for the 5-hydroxylation of TA very similar to those of CYP 2C9; however, this mutant did not undergo any detectable mechanism-based inactivation by TA, which indicates that the OH group of Ser 365 could be the nucleophile forming a covalent bond with an electrophilic metabolite of TA in TA-dependent inactivation of CYP 2C9. The F114I mutant was inactive toward the hydroxylation of diclofenac; moreover, detailed analyses of its interaction with a series of SPA derivatives by difference visible spectroscopy showed that the high affinity of SPA to CYP 2C9 (K(s)=0.4 microM) was completely lost when the phenyl substituent of Phe 114 was replaced with the alkyl group of Ile (K(s)=190+/-20 microM), or when the phenyl substituent of SPA was replaced with a cyclohexyl group (K(s)=120+/-30 microM). However, this cyclohexyl derivative of SPA interacted well with the F114I mutant (K(s)=1.6+/-0.5 microM). At the opposite end, the F94L and F110I mutants showed properties very similar to those of CYP 2C9 toward TA and diclofenac. Finally, the F476I mutant exhibited at least three main differences compared to CYP 2C9: (i) big changes in the k(cat) and K(m) values for TA and diclofenac hydroxylation, (ii) a 37-fold increase of the K(i) value found for the inhibition of CYP 2C9 by SPA, and (iii) a great change in the regioselectivity of diclofenac hydroxylation, the 5-hydroxylation of this substrate by CYP 2C9 F476I exhibiting a k(cat) of 28min(-1). These data indicate that Phe 114 plays an important role in recognition of aromatic substrates of CYP 2C9, presumably via Pi-stacking interactions. They also provide the first experimental evidence showing that Phe 476 plays a crucial role in substrate recognition and hydroxylation by CYP 2C9.     

Modification of the heme active site to increase the peroxidase activity of thermophilic cytochrome P450: A rational approach

The site specific mutants of the thermophilic P450 (P450 175A1 or CYP175A1) were designed to introduce residues that could act as acid-base catalysts near the active site to enhance the peroxidases activity. The Leu80 in the distal heme pocket of CYP175A1 was located at a position almost equivalent to the Glu183 that is involved in stabilization of the ferryl heme intermediate in chloroperoxidase (CPO). The Leu80 residue of CYP175A1 was mutated with histidine (L80H) and glutamine (L80Q) that could potentially form hydrogen bond with hydrogen peroxide and facilitate formation and stabilization of the putative redox intermediate of the peroxidase cycle. The mutants L80H and L80Q of CYP175A1 showed higher peroxidase activity compared to that of the wild type (WT) CYP175A1 enzyme at 25 °C. The activity constants (k_cat) for the L80H and L80Q mutants of CYP175A1 were higher than those of myoglobin and wild type cytochrome b562 at 25 °C. The optimum temperature for the peroxidase activity of the WT and mutants of CYP175A1 was ~ 70 °C. The rate of catalysis at temperatures above ~ 70 °C was higher for L80Q mutant of CYP175A1 compared to that of the well known natural peroxidase, horseradish peroxidase (HRP) that denatures at such high temperature. The peroxidase activities of the mutants of CYP175A1 were maximum at pH 9, unlike that of HRP which is at pH ~ 5. The results have been discussed in the light of understanding the structure-function relationship of the peroxidase properties of these thermostable heme proteins.     

Characterization of inhibitory effects of perfluorooctane sulfonate on human hepatic cytochrome P450 isoenzymes: focusing on CYP2A6

Perfluorooctane sulfonate (PFOS) is a chemically stable compound extensively used as oil and water repellent, surface active agents in our daily life. Accumulative research evidence gradually appears the toxicity of PFOS against mammals, but the whole figure remains to be elucidated. The present study was conducted to know the effects of PFOS on human hepatic drug metabolizing-type cytochrome P450 (CYP) isoenzymes such as CYP1A2 (7-ethoxyresorufin as a substrate), CYP2A6 (coumarin), CYP2B6 (7-ethoxy-4-trifluoromethylcoumarin), CYP2C8 (paclitaxel), CYP2C9 (diclofenac), CYP2C19 (S-mephenytoin), CYP2D6 (bufuralol), CYP2E1 (chlorzoxazone) and CYP3A4 (testosterone) in human livers employing their typical substrates. Although all of the oxidation reactions tested were more or less inhibited by PFOS, diclofenac 4'-hydroxylation mediated mainly by CYP2C9 was most strongly inhibited (K(i) value of 40 nM), followed by paclitaxel 6α-hydroxylation mediated mainly by CYP2C8 (K(i) value of 4 μM). The substrate oxidation reactions catalyzed by CYP2A6, CYP2B6, CYP2C19 and CYP3A4 were moderately (K(i) values of 35 to 45 μM), and those by CYP1A2, CYP2D6 and CYP2E1 were weakly inhibited by PFOS (K(i) values of 190-300 μM). The inhibition by PFOS for coumarin 7-hydroxylation mainly catalyzed by human liver microsomal CYP2A6 as well as by the recombinant enzyme was found to be enhanced by the preincubation of PFOS with human liver microsomes and NADPH as compared to the case without preincubation. The inhibition of the human liver microsomal cumarin 7-hydroxylation was PFOS concentration-dependent, and exhibited pseudo-first-order kinetics with respect to preincubation time, yielding K(inact) and K(I) values of 0.06 min(-1) and 23 μM, respectively. These results suggest that the metabolism of medicines which are substrates for CYP2C9 may be altered by PFOS in human bodies, and that PFOS is a mechanism-based inhibitor of CYP2A6.     

Important amino acid residues that confer CYP2C19 selective activity to CYP2C9

Although CYP2C9 and CYP2C19 display 91% sequence identity at the amino acid level, the two enzymes have distinct substrate specificities for compounds such as diclofenac, progesterone and (S)-mephenytoin. Amino acid substitutions in CYP2C9 were made based on an alignment of CYP2C9, CYP2C19 and monkey CYP2C43 sequences. Mutants of CYP2C9 were expressed in Escherichia coli. Sixteen amino acids, which are common to both CYP2C19 and CYP2C43 but different between CYP2C9 and CYP2C19, were substituted in CYP2C9 (CYP2C9-16aa). Next, the mutated amino acids in CYP2C9-16aa were individually reverted to those of CYP2C9 to examine the effect of each substitution on the enzymatic activity for CYP2C marker substrates. In addition, the role of the F-G loop in CYP2C9 and CYP2C19 was examined for substrate specificity and enzymatic activity. Our results showed: (i) CYP2C9-16aa displays 11% (S)-mephenytoin 4'-hydroxylase and full omeprazole 5-hydroxylase activity compared with that of CYP2C19; (ii) residue 286 is important for conferring CYP2C9-like enzyme activity on CYP2C9-16aa and residue 442 in CYP2C19 may be involved in the interaction with NADPH-P450 reductase; (iii) substitution of the F-G loop in CYP2C9 to that of CYP2C19 enhances tolbutamide p-methyhydroxylase and diclofenac 4'-hydroxylase activities and confers partial (S)-mephenytoin 4'-hydroxylase and omeprazole 5-hydroxylase activities, which are attributed to CYP2C19.     

Effect of ethanol on spectral binding, inhibition, and activity of CYP3A4 with an antiretroviral drug nelfinavir

Cytochrome P450 3A4 (CYP3A4) is the most abundant CYP enzyme in the liver and metabolizes approximately 50% of the drugs, including antiretrovirals. Although CYP3A4 induction by ethanol and impact of CYP3A4 on drug metabolism and toxicity is known, CYP3A4-ethanol physical interaction and its impact on drug binding, inhibition, or metabolism is not known. Therefore, we studied the effect of ethanol on binding and inhibition of CYP3A4 with a representative protease inhibitor, nelfinavir, followed by the effect of alcohol on nelfinavir metabolism. Our initial results showed that methanol, ethanol, isopropanol, isobutanol, and isoamyl alcohol bind in the active site of CYP3A4 and exhibit type I spectra. Among these alcohol compounds, ethanol showed the lowest K_D (5.9 ± 0.34 mM), suggesting its strong binding affinity with CYP3A4. Ethanol (20 mM) decreased the K_D of nelfinavir by >5-fold (0.041 ± 0.007 vs. 0.227 ± 0.038 μM). Similarly, 20 mM ethanol decreased the IC₅₀ of nelfinavir by >3-fold (2.6 ± 0.5 vs. 8.3 ± 3.1 μM). These results suggest that ethanol facilitates binding of nelfinavir with CYP3A4. Furthermore, we performed nelfinavir metabolism using LCMS. Although ethanol did not alter k_cat, it decreased the K_m of nelfinavir, suggesting a decrease in catalytic efficiency (k_cat/K_m). This is an important finding because alcoholism is prevalent in HIV-1-infected persons and alcohol is shown to decrease the response to antiretroviral therapy.     

Development of pH-sensitive tamarind seed polysaccharide-alginate composite beads for controlled diclofenac sodium delivery using response surface methodology

The present study deals with the development of novel pH-sensitive tamarind seed polysaccharide (TSP)-alginate composite beads for controlled diclofenac sodium delivery using response surface methodology by full 3² factorial design. The effect of polymer-blend ratio (sodium alginate:TSP) and cross-linker (CaCl₂) concentration on the drug encapsulation efficiency (DEE, %) and drug release from diclofenac sodium loaded TSP-alginate composite beads prepared by ionotropic gelation was optimized. The observed responses were coincided well with the predicted values by the experimental design. The DEE (%) of these beads containing diclofenac sodium was within the range between 72.23 ± 2.14 and 97.32 ± 4.03% with sustained in vitro drug release (69.08 ± 2.36-96.07 ± 3.54% in 10 h). The in vitro drug release from TSP-alginate composite beads containing diclofenac sodium was followed by controlled-release pattern (zero-order kinetics) with case-II transport mechanism. Particle size range of these beads was 0.71 ± 0.03-1.33 ± 0.04 mm. The swelling and degradation of the developed beads were influenced by different pH of the test medium. The FTIR and NMR analyses confirmed the compatibility of the diclofenac sodium with TSP and sodium alginate used to prepare the diclofenac sodium loaded TSP-alginate composite beads. The newly developed TSP-alginate composite beads are suitable for controlled delivery of diclofenac sodium for prolonged period.     

Substrate stiffness regulates apoptosis and the mRNA expression of extracellular matrix regulatory genes in the rat annular cells

Cells are subjected to static tension of different magnitudes when cultured on substrates with different stiffnesses. It has long been recognized that mechanical stress is an important modulator of the intervertebral disc degeneration. Here we studied the influence of substrate stiffness on cell morphology, apoptosis and extracellular matrix (ECM) metabolism of the rat annulus fibrosus (AF) cells which are known to be mechanosensitive cells. Polyacrylamide gel substrates with three different stiffnesses were prepared by varying the concentration of acrylamide and bisacrylamide, and the elastic modulus of the different gel substrates were measured with atomic force microscopy (AFM). First-passage rat annular cells were cultured on soft, intermediate, rigid substrates or plastics for 24 or 48 h. The percentages of apoptotic cells were detected by flow cytometry and caspase-3 activity, and morphologic changes were visualized by Hoechst 33258 staining and F-actin staining. In addition, the expression of ECM genes (Col1α1, Col2α1, aggrecan, MMP-3, MMP-13 and ADAMTS-5) were analyzed by RT-PCR. The three different substrates had elastic moduli varying between 1 ± 0.23 kPa (soft, 5% gel with 0.06% bis), 32 ± 2.89 kPa (intermediate, 10% gel with 0.13% bis) and 63 ± 3.45 kPa (rigid, 10% gel with 0.26% bis) with a thickness about 60-70 μm. Most of the rat AF cells appeared small and rounded, and lost most of their stress fibers when cultured on soft substrate. There was a significant increase in the percentage of apoptotic cells in the rat AF cells cultured on soft and intermediate substrates relative to those on plastic surface, with a parallel decrease in the area of cell spreading and nucleus. The AF cells grown on intermediate or rigid substrate had reduced expression of Col1α1, Col2α1 and aggrecan and enhanced expression of MMP-3, MMP-13, and ADAMTS-5 at 24 h or 48 h, respectively, relative to those cultured on plastic surface. Conversely, we observed an up-regulation of Col2α1 and aggrecan and no change in the gene expression of MMP-3, MMP-13, and ADAMTS-5 in AF cells on soft substrates. Rat AF cells are sensitive to substrate stiffness which can regulate the morphology, growth, apoptosis and ECM metabolism of rat AF cells, thus indicating the importance of substrate choice for cell transplantation and regeneration for the treatment of disc degeneration using tissue-engineering technique.     

CYP2C9 genotype and association with bone mineral density: A pilot study

Background

In recent years reduced bone mineral density (BMD) and osteoporosis have become major public health problems. Single nucleotide polymorphisms (SNPs) in the cytochrome P450 2C9 (CYP2C9) gene influence the response to oral anticoagulant drugs, which are positively associated with the risk to develop osteoporosis. The aim of the present investigation was to clarify a potential role of CYP2C9 sequence variations and susceptibility to develop osteoporosis.

Subjects and methods

Ninety two consecutive angiologic outpatients, mean age: 60.3 ± 14.4, without secondary causes of bone loss were genotyped and classified as patients with normal BMD, osteopenia and osteoporosis according to WHO criteria by dual-energy X-ray absorptiometry at the lumbar spine and/or the femoral neck. Potential association between the CYP2C9 genotype and BMD was tested.

Results

59% of the patients (n = 54) presented with reduced BMD and were compared to 38 age-matched persons with normal BMD. The genotype distribution showed 15% heterozygous for CYP2C9*2 p.Arg144Cys, 14% for CYP2C9*3 p.IIe359Leu, 2% for both polymorphisms, and 69% had wildtype genotypes. Patients with CYP2C9 mutations had significantly lower BMD values at the femoral neck and displayed a four-fold higher adjusted risk to suffer from reduced BMD than individuals with wildtype genotypes (p = 0.02).

Discussion

Oral anticoagulant treatment is common in angiologic outpatients. The gene variants CYP2C9*2 and CYP2C9*3 have been shown to require lower maintenance doses of oral anticoagulant drugs. An association between oral anticoagulant drugs and the susceptibility to develop osteoporosis in relation to sequence variations in the CYP2C9 gene is suggested to be mediated via the glucocorticoid synthesis pathway.

Conclusion

The CYP2C9*2/CYP2C9*3 variants were significantly associated with femoral BMD in a selected elderly Austrian population. These variants could contribute to the complex risk to develop osteoporosis.     

Analysis of human cytochrome P450 2C8 substrate specificity using a substrate pharmacophore and site-directed mutants

The structural determinants of substrate specificity of human liver cytochrome P450 2C8 (CYP2C8) were investigated using site-directed mutants chosen on the basis of a preliminary substrate pharmacophore and a three-dimensional (3D) model. Analysis of the structural features common to CYP2C8 substrates exhibiting a micromolar K(m) led to a substrate pharmacophore in which the site of oxidation by CYP2C8 is 12.9, 8.6, 4.4, and 3.9 A from features that could establish ionic or hydrogen bonds, and hydrophobic interactions with protein amino acid residues. Comparison of this pharmacophore with a 3D model of CYP2C8 constructed using the X-ray structure of CYP2C5 suggested potential CYP2C8 amino acid residues that could be involved in substrate recognition. Twenty CYP2C8 site-directed mutants were constructed and expressed in yeast to compare their catalytic activities using five CYP2C8 substrates that exhibit different structures and sizes [paclitaxel, fluvastatin, retinoic acid, a sulfaphenazole derivative (DMZ), and diclofenac]. Mutation of arginine 241 had marked effects on the hydroxylation of anionic substrates of CYP2C8 such as retinoic acid and fluvastatin. Serine 100 appears to be involved in hydrogen bonding interactions with a polar site of the CYP2C8 substrate pharmacophore, as shown by the 3-4-fold increase in the K(m) of paclitaxel and DMZ hydroxylation after the S100A mutation. Residues 114, 201, and 205 are predicted to be in close contact with substrates, and their mutations lead either to favorable hydrophobic interactions or to steric clashes with substrates. For instance, the S114F mutant was unable to catalyze the 6alpha-hydroxylation of paclitaxel. The S114F and F205A mutants were the best catalysts for retinoic acid and paclitaxel (or fluvastatin) hydroxylation, respectively, with k(cat)/K(m) values 5 and 2.1 (or 2.4) times higher, respectively, than those found for CYP2C8. Preliminary experiments of docking of the substrate into the experimentally determined X-ray structure of substrate-free CYP2C8, which became available quite recently [Schoch, G. A., et al. (2004) J. Biol. Chem. 279, 9497], were consistent with key roles for S100, S114, and F205 residues in substrate binding. The results suggest that the effects of mutation of arginine 241 on anionic substrate hydroxylation could be indirect and result from alterations of the packing of helix G with helix B'.     

On the human CYP2C9*13 variant activity reduction: a molecular dynamics simulation and docking study

Cytochrome P450 2C9 (CYP2C9) plays a key role in the metabolism of clinical drugs. CYP2C9 is a genetically polymorphic enzyme and some of its allelic variants have less activity compared to the wild-type form. Drugs with a narrow therapeutic index may cause serious toxicity to the individuals who carry such allele. CYP2C9*13, firstly identified by some of the present authors in a Chinese poor metabolizer of lornoxicam, is characterized by mutation encoding Leu90Pro substitution. Kinetic experiments show that CYP2C9*13 has less catalytic activity in elimination of diclofenac and lornoxicam in vitro. In order to explore the structure-activity relationship of CYP2C9*13, the three-dimensional structure models of the substrate-free CYP2C9*1 and its variant CYP2C9*13 are constructed on the basis of the X-ray crystal structure of human CYP2C9*1 (PDB code 1R9O) by molecular dynamics simulations. The structure change caused by Leu90Pro replacement is revealed and used to explain the dramatic decrease of the enzymatic activity in clearance of the two CYP2C9 substrates: diclofenac and lornoxicam. The trans configuration of the bond between Pro90 and Asp89 in CYP2C9*13 is firstly identified. The backbone of residues 106-108 in CYP2C9*13 turns over and their side chains block the entrance for substrates accessing so that the entrance of *13 shrinks greatly than that in the wild-type, which is believed to be the dominant mechanism of the catalytic activity reduction. Consequent docking study which is consistent with the results of the kinetic experiments by Guo et al. identifies the most important residues for enzyme-substrate complexes.     

Arginines 97 and 108 in CYP2C9 are important determinants of the catalytic function

Human cytochrome P450 2C9 (CYP2C9) is one of the major drug metabolising enzymes which exhibits a broad substrate specificity. The B-C loop is located in the active-site but has been difficult to model, owing to its diverse and flexible structure. To elucidate the function of the B-C loop we used homology modelling based on the Cyp102 structure in combination with functional studies of mutants using diclofenac as a model substrate for CYP2C9. The study shows the importance of the conserved arginine in position 97 and the arginine in position 108 for the catalytic function. The R97A mutant had a 13-fold higher K(m) value while the V(max) was in the same order as the wild type. The R108 mutant had a 100-fold lower activity with diclofenac compared to the wild-type enzyme. The other six mutants (S95A, F100A, L102A, E104A, R105A, and N107A) had kinetic parameters similar to the CYP2C9 wild-type. Our homology model based on the CYP102 structure as template indicates that R97, L102, and R105 are directed into the active site, whereas R108 is not. The change in catalytic function when arginine 97 was replaced with alanine and the orientation of this amino acid in our homology model indicates its importance for substrate interaction.     

Cynomolgus monkey cytochrome P450 2C43: cDNA cloning, heterologous expression, purification and characterization

The cDNA of cytochrome P450 (CYP) 2C43 was cloned from cynomolgus monkey liver by RT-PCR. The deduced amino acid sequence showed 93% and 91% identity to human CYP2C9 and CYP2C19, respectively. The cDNA was expressed in Escherichia coli and purified by a series of chromatography steps, yielding a specific content of 11.5 nmol P450/mg protein. The substrate specificity of the purified CYP2C43 was examined in a reconstitution system comprising NADPH-P450 reductase, lipid, cytochrome b(5) and CYP2C marker substrates. The purified CYP2C43 showed high activity for testosterone 17-oxidation and progesterone 21-hydroxylation, which were also observed for CYP2C19 but not CYP2C9. In addition, CYP2C43 showed activity for (S)-mephenytoin 4'-hydroxylation, a marker reaction for CYP2C19. With CYP2C9 marker substrates, CYP2C43 exhibited low activity for diclofenac 4'-hydroxylation and no activity for tolbutamide p-methylhydroxylation. Therefore, in terms of substrate specificity, our results indicate that CYP2C43 is similar to CYP2C19, rather than CYP2C9.     

Reduction potential of yeast cytochrome c peroxidase and three distal histidine mutants: dependence on pH

The pH dependence of the Fe(III) reduction potential, E⁰′, for yeast cytochrome c peroxidase (yCcP) and three distal pocket mutants, CcP(H52L), CcP(H52Q), and CcP(R48L/W51L/H52L), has been determined between pH 4 and 8. E⁰′ values at pH 7.0 for the yCcP, CcP(H52L), CcP(H52Q), and CcP(R48L/W51L/H52L) are − 189, − 170, − 224, and − 146 mV, respectively. A heme-linked ionization in the reduced enzyme affects the reduction potential for yCcP and all three mutants. Apparent pK_A values for the heme-linked ionization are 7.5 ± 0.2, 6.5 ± 0.3, 6.4 ± 0.2, and 7.0 ± 0.3 for yCcP and the H52L, H52Q, and R48L/W51L/H52L mutants, respectively. A cooperative, two-proton ionization causing a spectroscopically-detectable transition was observed in the ferrous states of yCcP, CcP(H52L) and CcP(H52Q), with apparent pK_A values of 7.7 ± 0.2, 7.4 ± 0.1 and 7.8 ± 0.1, respectively. These data indicate that: (1) the distal histidine in CcP is not the site of proton binding upon reduction of the ferric CcP, (2) the distal histidine is not one of the two groups involved in the cooperative, two-proton ionization observed in ferrous CcP, and (3) the proton-binding site is not involved in the cooperative, two-proton ionization observed in the reduced enzyme.     

Analysis of diclofenac and its metabolites by high-performance liquid chromatography: relevance of CYP2C9 genotypes in diclofenac urinary metabolic ratios

In humans, diclofenac is metabolised to 4'-hydroxy (OH), 3'-OH and 5-OH metabolites. The polymorphic CYP2C9 is involved in the metabolism of diclofenac to 4'-OH diclofenac and 3'-OH diclofenac. The aim of the present study was to develop a high-performance liquid chromatographic method to simultaneously measure diclofenac and its metabolites in urine, suitable for metabolic studies. After liquid-liquid extraction the compounds were separated in a reversed-phase column and measured by ultraviolet absorption at 282 nm. For all compounds intra-day and inter-day variations were less than 7%, and the limits of quantitation were 0.25 mg/l. No analytical interference with endogenous compounds was found. The relationship between diclofenac metabolic ratios among different CYP2C9 genotypes is reported. The CYP2C9*3/*3 subject had the highest diclofenac/4'-OH ratios. However no difference was found between CYP2C9*2/*2 and *1/*1 genotypes. The chromatographic method developed was sensitive and reliable for the measurement of diclofenac and its metabolites simultaneously in human urine, and is suitable for use in diclofenac metabolism studies.     

Giardia duodenalis: adhesion-deficient clones have reduced ability to establish infection in Mongolian gerbils

The role of Giardia duodenalis surface molecules in the attachment of trophozoites to epithelial cells has been established through the dual strategies of characterizing G. duodenalis clones with deficient adhesion and blocking experiments with surface-specific monoclonal antibodies. Also, the infectivity of the analyzed clones was tested using Mongolian gerbils as experimental model. Two adhesion-deficient G. duodenalis clones, C6 and C7, were isolated from the wild type C5 clone which in turn was obtained from the WB strain. The adhesion efficiencies of C6 and C7 clones (48.2 ± 4.9 and 32.6 ± 2.4, respectively) were significantly lower as compared with WB strain or C5 clone (82.8 ± 6.4 and 79.9 ± 7.9). Analysis of radiolabel surface proteins by 1D and 2D SDS-PAGE revealed prominently labelled 28 and 88 kDa components in C6 and C7 clones and a major 200 kDa protein in the C5 clone and the WB strain. The 88 and 200 kDa components are acidic proteins by two-dimensional electrophoretic analyses. The most striking difference between wild-type and adhesion-deficient Giardia trophozoites was the reduced expression of a 200 kDa surface protein in the latter. Significantly, a mAb (IG3) specific for the 200 kDa protein that reacted with more than 99% of WB and C5 trophozoites and less than 1% of C6 and C7 trophozoites as determined by indirect immunofluorescence inhibited the adhesion of trophozoites from WB and C5 clone to Madin Darby Canine Kidney cells by 52% and 40.9%, respectively, suggesting a participation of this antigen in adherence. Finally, the functional relevance of trophozoite adhesion to epithelial cells was indicated by the reduced capacity of the adhesion-deficient clones to establish the infection in Mongolian gerbils.     

Comparison of "high throughput" micromethods for determination of cytochrome P450 activities with classical methods using HPLC for product identification

Enzyme activities of the CYP enzymes (CYP3A4, CYP2C9 and CYP2A6) were determined using classical substrates (testosterone, diclofenac and coumarin, respectively) as well as with luminogenic or fluorogenic substrates in micromethod arrangement. The luciferin-based luminogenic substrates for CYP3A4 and CYP2C9 as well as coumarin in micromethod for assay of CYP2A6 activity gave results well comparable with the classical methods with determination of reaction products by the HPLC.     

Reduced catalytic activity of human CYP2C9 natural alleles for gliclazide: molecular dynamics simulation and docking studies

Amongst sulfonylureas, gliclazide is one of the mostly prescribed drugs to diabetic patients and is metabolized extensively by P450 CYP2C9. Among 24-CYP2C9 alleles, the *2/*2 and *3/*3 genotypes showed significantly lower gliclazide clearances with reductions of 25 and 57%, respectively. However, the reason for the change in drug-metabolizing activity induced by these natural alleles is unknown. In the present study, we used molecular dynamics simulation and autodocking studies to provide models for gliclazide-bound complexes of CYP2C9*2, *3 and *2/*3 mutants, which give insight into CYP2C9-gliclazide interactions and explain the reduced enzymatic activity seen in these variants. Our data shows that the size of the substrate-access entry site is significantly reduced in mutants, which limits the access of gliclazide to heme and the active site. The distance from the substrate oxidation site and heme is >5 Å in *3 and *2/*3. Therefore, the addition of an active oxygen molecule by heme-Fe is hindered. The absence of F100, F114 and F476 in the interacting amino acid pocket in *3 reduces catalytic efficiency toward gliclazide. In *1, gliclazide is stabilized by the formation of two hydrogen bonds with R108 while it is absent in mutants. Further in *3 and *2/*3, the key heme-stabilizing residue, R97 stabilization is greatly reduced. Therefore, the decreased catalytic activity of these variants can be explained from the reduced access of the gliclazide to heme, and the interaction between heme and substrate is affected due to their instability in the active site.