575-03-1Relevant articles and documents
Mechanism-based inactivation of cytochromes P450 2B1 and P450 2B6 by n- propylxanthate
Kent, Ute M.,Yanev, Stanislav,Hollenberg, Paul F.
, p. 317 - 322 (1999)
n-Propylxanthate (nPX) inactivated the 7-ethoxy-4- (trifluoromethyl)coumarin (7-EFC) O-deethylation activity of purified, reconstituted rat hepatic P450 2B1 or human P450 2B6 in a mechanism-based manner. The inactivation followed pseudo-first-order kinetics and was entirely dependent on both NADPH and nPX. The maximal rate constant for inactivation of P450 2B1 at 30 °C was 0.2 min-1. The apparent K(I) was 44 μM, and the half-time for inactivation was 4.1 min. Purified, reconstituted human P450 2B6 was also inactivated by nPX with a K(I) of 12 μM. The κ(inactivation) for P450 2B6 was 0.06 min-1, and the t(1/2) was 11 min. Incubations of P450 2B1 with nPX and NADPH for 20 min resulted in a 75% loss in enzymatic activity and a concurrent 25% loss of the enzyme's ability to form a reduced CO complex. Little loss in the absolute spectrum of nPX- inactivated P450 2B1 was observed. With P450 2B6, an 83% loss in enzymatic activity and a 12% loss in the CO-reduced spectra were observed. The extrapolated partition ratio for nPX with P450 2B1 was 32. P450 2B1 could be protected from inactivation by nPX by adding an alternate substrate to the reaction mixture. Removal of unbound nPX by dialysis did not reverse the inactivation. The alternate oxidant iodosobenzene was able to partially restore enzymatic activity to nPX-inactivated P450 2B1 samples. A stoichiometry for labeling of 1.2:1 for binding of radiolabeled nPX metabolite to P450 2B1 was seen. These results indicated that nPX inactivated P450 2B1 and P450 2B6 in a mechanism-based manner. P450 2B1 was inactivated primarily by a nPX reactive intermediate that bound to the apoprotein.
Insight into the Mechanism of the Pechmann Condensation Reaction Using NMR
Tyndall, Stephen,Wong, Koon Fai,Vanalstine-Parris, Melissa A.
, p. 8951 - 8953 (2015)
The mechanism of the Pechmann condensation is still controversial despite the technological and biochemical importance of coumarins. Here, we present NMR evidence for a mechanism featuring the sequence of initial electrophilic aromatic substitution followed by transesterification and a final dehydration. This mechanism has been convincingly defined and supported by the temporal evolution of two key intermediates which could be purified and identified.
Functional expression and comparative characterization of four feline P450 cytochromes using fluorescent substrates
Okamatsu, Gaku,Kawakami, Kei,Komatsu, Tetsuya,Kitazawa, Takio,Uno, Yasuhiro,Teraoka, Hiroki
, p. 951 - 961 (2017)
1.?Cytochrome P450s (CYP) are a major group of metabolizing enzymes for xenobiotics in humans and other mammals. The properties of CYP isoforms in the domestic cat, an obligate carnivore, are largely unknown at present. In this study, we studied relative expression in tissues and enzymatic properties of nine significant feline CYP isoforms. 2.?CYP2E2 transcript was most abundant in the feline liver, followed by CYP2A13 and 2E1. Transcripts of CYP3A131, 1A2 and 1A1 were also present in the liver, while CYP2D6 and 3A132 were only slightly expressed. CYP3A131 was a major transcript in the small intestine. 3.?Four major CYP isoforms in the feline liver and small intestine (CYP1A2, CYP2A13, CYP2E2 and CYP3A131) were heterologously expressed in Escherichia coli to generate functional monooxygenase systems. We carried out screenings of 17 test compounds known to be inhibitors of CYP isoforms in other mammals as well as two anticancer drugs to assess the activity modulation of feline CYP isoforms using fluorogenic substrates. These CYP isoforms showed similar selectivity to counterparts in other mammals against inhibitors as a whole but with many exceptions. 4.?The present study suggests the usefulness of the feline CYP recombinant system to obtain chemical affinity information and possible drug interactions in CYP metabolism of domestic cats.
Metabolism of 7-benzyloxy-4-trifluoromethylcoumarin by human hepatic cytochrome P450 isoforms
Renwick,Surry,Price,Lake,Evans
, p. 955 - 969 (2000)
1. The metabolism of 7-benzyloxy-4-trifluoromethylcoumarin (BFC) to 7-hydroxy-4-trifluoromethylcoumarin (HFC) was studied in human liver microsomal preparations and in cDNA-expressed human cytochrome P450 (CYP) isoforms. 2. Kinetic analysis of the NADPH-dependent metabolism of BFC to HFC in four preparations of pooled human liver microsomes revealed mean (± SEM) K(m) and V(max) of 8.3 ± 1.3 μM and 454 ± 98 pmol/min/mg protein respectively. 3. The metabolism of BFC to HFC was determined in a characterized bank of 24 individual human liver microsomal preparations employing BFC substrate concentrations of 20 and 50 μM (i.e. about two and six times K(m) respectively). With 20 μM BFC the highest correlations were observed between BFC metabolism and markers of CYP1A2 (r2 = 0.784-0.797) and then with CYP3A (r2 = 0.434-0.547) isoforms, whereas with 50 μM BFC the highest correlations were observed between BFC metabolism and markers of CYP3A (r2= 0.679-0.837) and then with CYP1A2 (r2 = 0.421-0.427) isoforms. At both BFC substrate concentrations, lower correlations were observed between BFC metabolism and enzymatic markers for CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP4A9/11. 4. Using human β-lymphoblastoid cell microsomes containing cDNA-expressed CYP isoforms, 20 μM BFC was metabolized by CYP1A2 and CYP3A4, with lower rates of metabolism being observed with CYP2C9 and CYP2C19. Kinetic studies with the CYP1A2 and CYP3A4 preparations demonstrated a lower K(m) with the CYP1A2 preparation, but a higher V(max) with the CYP3A4 preparation. 5. The metabolism of 20 μM BFC in human liver microsomes was inhibited to 37-48 % of control by 5-100 μM of the mechanism-based CYP1A2 inhibitor furafylline and to 64-69 % of control by 5-100 μM of the mechanism-based CYP3A4 inhibitor troleandomycin. While some inhibition of BFC metabolism was observed in the presence of 100 and 200 μM diethyldithiocarbamate, the addition of 2-50 μM sulphaphenazole, 50-500 μM S-mephenytoin and 2-50 μM quinidine had little effect. 6. The metabolism of 20 μM BFC to HFC in human liver microsomes was also inhibited by an antibody to CYP3A4, whereas antibodies to CYP2C8/9 and CYP2D6 had no effect. 7. In summary, by correlation analysis, use of cDNA-expressed CYP isoforms, chemical inhibition and inhibitory antibodies, BFC appears metabolized by a number of CYP isoforms in human liver. BFC metabolism appears to be primarily catalysed by CYP1A2 and CYP3A4, with possibly some contribution by CYP2C9, CYP2C19 and perhaps other CYP isoforms. 8. The results also demonstrate the importance of the selection of an appropriate substrate concentration when conducting reaction phenotyping studies with human hepatic CYP isoforms.
Scalable production and application of Pichia pastoris whole cell catalysts expressing human cytochrome P450 2C9
Garrigós-Martínez, Javier,Weninger, Astrid,Montesinos-Seguí, José Luis,Schmid, Christian,Valero, Francisco,Rinnofner, Claudia,Glieder, Anton,Garcia-Ortega, Xavier
, (2021)
Background: Currently, the numerous and versatile applications in pharmaceutical and chemical industry make the recombinant production of cytochrome P450 enzymes (CYPs) of great biotechnological interest. Accelerating the drug development process by simple, quick and scalable access of human drug metabolites is key for efficient and targeted drug development in response to new and sometimes unexpected medical challenges and needs. However, due its biochemical complexity, scalable human CYP (hCYP) production and their application in preparative biotransformations was still in its infancy. Results: A scalable bioprocess for fine-tuned co-expression of hCYP2C9 and its essential complementary human cytochrome P450 reductase (hCPR) in the yeast Pichia pastoris (Komagataella phaffii) is presented. High-throughput screening (HTS) of a transformant library employing a set of diverse bidirectional expression systems with different regulation patterns and a fluorimetric assay was used in order to fine-tune hCYP2C9 and hCPR co-expression, and to identify best expressing clonal variants. The bioprocess development for scalable and reliable whole cell biocatalyst production in bioreactors was carried out based on rational optimization criteria. Among the different alternatives studied, a glycerol carbon-limiting strategy at high μ showed highest production rates, while methanol co-addition together with a decrease of μ provided the best results in terms of product to biomass yield and whole cell activity. By implementing the mentioned strategies, up to threefold increases in terms of production rates and/or yield could be achieved in comparison with initial tests. Finally, the performance of the whole cell catalysts was demonstrated successfully in biotransformation using ibuprofen as substrate, demonstrating the expected high selectivity of the human enzyme catalyst for 3′hydroxyibuprofen. Conclusions: For the first time a scalable bioprocess for the production of hCYP2C9 whole cell catalysts was successfully designed and implemented in bioreactor cultures, and as well, further tested in a preparative-scale biotransformation of interest. The catalyst engineering procedure demonstrated the efficiency of the employment of a set of differently regulated bidirectional promoters to identify transformants with most effective membrane-bound hCYP/hCPR co-expression ratios and implies to become a model case for the generation of other P. pastoris based catalysts relying on co-expressed enzymes such as other P450 catalysts or enzymes relying on co-expressed enzymes for co-factor regeneration.
Metabolism of 2,5-bis(trifluoromethyl)-7-benzyloxy-4-trifluoromethylcoumarin by human hepatic CYP isoforms: Evidence for selectivity towards CYP3A4
Renwick,Lewis,Fulford,Surry,Williams,Worboys,Cai,Wang,Price,Lake,Evans
, p. 187 - 204 (2001)
1. The metabolism of 2,5-bis(trifluoromethyl)-7-benzyloxy-4-trifluoromethylcoumarin (BFBFC) to 7-hydroxy-4-trifluoromethylcoumarin (HFC) was studied in human liver microsomes and in cDNA-expressed human liver CYP isoforms. For purposes of comparison, some limited studies were also performed with 7-benzyloxyquinoline (7BQ). 2. Initial interactive docking studies with a homology model of human CYP3A4 indicated that BFBFC was likely to be a selective substrate for CYP3A4 with a relatively high binding affinity, due to the presence of several key hydrogen bonds with active site amino acid residues. 3. Kinetic analysis of NADPH-dependent BFBFC metabolism to HFC in three preparations of pooled human liver microsomes revealed mean (±SEM) Km and Vmax = 4.6 ± 0.3 μM and 20.0 ± 3.8 pmol/min/mg protein, respectively. 4. The metabolism of BFBFC to HFC was determined in a characterized bank of 24 individual human liver microsomal preparations employing a BFBFC substrate concentration of 10 μM (i.e. around twice Km). Good correlations (r2 = 0.736-0.904) were observed between BFBFC metabolism and markers of CYP3A isoforms. 5. While 10 μM BFBFC was metabolized to HFC by cDNA-expressed CYP3A4, little or no metabolism was observed with cDNA-expressed CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1. 6. The metabolism of 10 μM BFBFC in human liver microsomes was markedly inhibited by 5-50 μM troleandomycin and 0.2-5 μM ketoconazole, but stimulated by 0.2-10 μM α-naphthoflavone. The metabolism of 10 μM BFBFC in human liver microsomes was also markedly inhibited by an antibody to CYP3A4. 7. Kinetic analysis of NADPH-dependent 7BQ metabolism to 7-hydroxyquinoline (7HQ) in human liver microsomes revealed Km and Vmax = 70 μM and 3.39 nmol/min/mg protein, respectively. 8. While 80 μM 7BQ was metabolized to 7HQ by cDNA-expressed CYP3A4, only low rates of metabolism were observed with cDNA-expressed CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1. 9. In summary, by correlation analysis, the use of cDNA-expressed CYP isoforms, chemical inhibition and inhibitory antibodies, BFBFC metabolism in human liver microsomes appears to be primarily catalysed by CYP3A4. BFBFC may be a useful fluorescent probe substrate for human hepatic CYP3A4, but compared with 7BQ has only a low rate of metabolism in human liver microsomes.
A double-triggered self-immolative spacer for increased selectivity of molecular release
Huvelle, Steve,Le Saux, Thomas,Jullien, Ludovic,Schmidt, Frédéric
, p. 240 - 246 (2021/12/29)
A self-immolative spacer based on dissymmetricalN,N′-bis-carbamate aniline is introduced to liberate a substrate from a precursor after dual activation. The proof of principle of its exclusive selectivity for substrate liberation has been conducted on a profluorophore.
Novel fluorinated 7-hydroxycoumarin derivatives containing an oxime ether moiety: Design, synthesis, crystal structure and biological evaluation
Dai, Peng,Jiao, Jian,Wang, Qing-Qing,Zhang, Shu-Guang,Zhang, Wei-Hua
, (2021/06/28)
A series of fluorinated 7-hydroxycoumarin derivatives containing an oxime ether moiety have been designed, synthesized and evaluated for their antifungal activity. All the target compounds were determined by1H-NMR,13C-NMR, FTIR and HR-MS spectra. The single-crystal structures of compounds 4e, 4h, 5h and 6c were further confirmed using X-ray diffraction. The antifungal activities against Botrytis cinerea (B. cinerea), Alternaria solani (A. solani), Gibberella zeae (G. zeae), Rhizoctorzia solani (R. solani), Colletotrichum orbiculare (C. orbiculare) and Alternaria alternata (A. alternata) were evaluated in vitro. The preliminary bioassays showed that some of the designed compounds displayed the promising antifungal activities against the above tested fungi. Strikingly, the target compounds 5f and 6h exhibited outstanding antifungal activity against B. cinerea at 100 μg/mL, with the corresponding inhibition rates reached 90.1 and 85.0%, which were better than the positive control Osthole (83.6%) and Azoxystrobin (46.5%). The compound 5f was identified as the promising fungicide candidate against B. cinerea with the EC50 values of 5.75 μg/mL, which was obviously better than Osthole (33.20 μg/mL) and Azoxystrobin (64.95 μg/mL). Meanwhile, the compound 5f showed remarkable antifungal activities against R. solani with the EC50 values of 28.96 μg/mL, which was better than Osthole (67.18 μg/mL) and equivalent to Azoxystrobin (21.34 μg/mL). The results provide a significant foundation for the search of novel fluorinated 7-hydroxycoumarin derivatives with good antifungal activity.
