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.
Synthesis of amidoalkyl chromen-2-ones by one pot three component reaction under solvent free conditions
Emmadi, Narender Reddy,Atmakur, Krishnaiah,Chennapuram, Madhu,Nanubolu, Jagadeesh Babu
, p. 14501 - 14506 (2014)
A mild and efficient method for the functionalization of chromen-2-ones with amidoalkyl derivatives have been developed starting from 4-trifluoromethyl substituted chromen-2-ones, aromatic aldehydes and acetamide promoted by stannous chloride dihydrate in a one pot three component reaction under solvent free condition. Simple reaction conditions, high yields and environmentally benign procedure are the advantage of this protocol. This journal is the Partner Organisations 2014.
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.
Delivery of coumarin-containing all-: Trans retinoic acid derivatives via targeted nanoparticles encapsulating indocyanine green for chemo/photothermal/photodynamic therapy of breast cancer
Jiao, Jia,Wu, Hongshuai,Chen, Fanghui,Chen, Renjie,Sun, Baiwang,Wang, Mingliang
, p. 8805 - 8814 (2018)
Development of chemo/photothermal/photodynamic therapy with nanoplatforms offers a promising strategy for effective cancer treatment. Recently, all-trans retinoic acid (ATRA), as a potential antitumor drug, has attracted great attention due to its antitumor activity. In this study, a novel coumarin-containing ATRA (AC) and indocyanine green (ICG) dye-loaded nanoparticles with the targeted ligand cyclic (Arg-Gly-Asp-d-Phe-Lys) (cRGD) peptide were fabricated by self-assembly and used as a new theranostic nanoplatform for chemo/photothermal/photodynamic therapy. The as-formed nanoparticles (AC/ICG-TNPs) had a diameter of around 133 nm with uniform monodispersity. Additionally, AC/ICG-TNPs showed marked stability under normal physiological conditions. However, it could rapidly release drugs in a mild acidic microenvironment. Moreover, confocal microscopic observations confirmed that the uptake of AC/ICG-TNPs increased in the breast cancer cells, particularly in MDA-MB-231 cells, probably mediated by cRGD via specific recognition of the overexpressed integrin αvβ3. Moreover, free AC exhibited stronger cytotoxic effects than free ATRA in the MTT assay, and AC/ICG-TNPs were demonstrated to possess excellent antitumor efficacy when exposed to NIR irradiation through the combination therapy. Hence, the therapeutic method designed in this study is a good candidate for improved bioactivity of ATRA and site-specific combinational therapy against breast cancer.
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.
Novel trifluoromethylcoumarinyl urea derivatives: Synthesis, characterization, fluorescence, and bioactivity
Qiao, Lili,Hao, Shuanghong
, (2018)
A series of novel trifluoromethylcoumarinyl urea derivatives were designed, synthesized, and characterized by 1H-NMR, 13C-NMR, and HR-ESI-MS. The fluorescence spectra of the target compounds were recorded. The spectra show that most of the title compounds glow green with λmaxem of 500-517 nm, while compounds 5r, 5s, 5u, and 5l (compounds named by authors) glow violet with λmaxem of 381-443 nm. Moreover, the herbicidal and antifungal activities of the synthesized compounds were evaluated for their potential use as pesticides. The results indicate that compound 5f against the caulis of Amaranthus retroflexus and compounds 5j and 5l against the taproot of Digitaria sanguinalis are equivalent to the commercial herbicide Acetochlor. Nine of the title compounds are more antifungal than commercial fungicide Carbendazim against Botrytis cinerea.
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.
Development of a human lymphoblastoid cell line constitutively expressing human CYP1B1 cDNA: Substrate specificity with model substrates and promutagens
Crespi, Charles L.,Penman, Bruce W.,Steimel, Dorothy T.,Smith, Theresa,Yang, Chung S.,Sutter, Thomas R.
, p. 83 - 89 (1997)
An AHH-1 TK(+/-) cell derivative was developed that stably expresses human cytochrome P4501B1 (CYP1B1) cDNA in an extrachromosomal vector which confers resistance to 1-histidinol and co-expresses NADPH cytochrome P450 oxidoreductase (OR). The CYP1B1-expressing cell line was designated h1B1/OR. Microsomes prepared from CYP1B1 cDNA expressing cells exhibit elevated levels of 7-ethoxyresorufin deethylase (EROD), 7-ethoxy-4-trifluoromethylcoumarin deethylase (EFCD), benzo(a)pyrene hydroxylase (BPH), bufuralol 1'-hydroxylase, testosterone hydroxylase activities and spectrally quantifiable cytochrome P450. CYP1B1-containing microsomes did not contain detectable coumarin 7-hydroxylase, p-nitrophenol hydroxylase, lauric acid hydroxylase, (S)-mephenytoin 4'-hydroxylase or diclofenac 4'-hydroxylase activities. Kinetic parameters for selected substrates were compared among CYP1B1 and the two additional members of the CYP1 family, CYP1A1 and CYP1A2. For BPH and EFCD, the rank order of rates of substrate metabolism were CYP1A1 > CYP1B1 > CYP1A2. For EROD, the rank order of substrate metabolism was CYP1A1 > CYP1A2 > CYP1B1. For both EROD and EFCD the apparent K(m) values for CYP1B1 were more similar to CYP1A1 than to CYP1A2. In order to begin to characterize the promutagen activating ability of CYP1B1, the mutagenicity of selected chemicals was examined in h1B1/OR cells; there was increased sensitivity (CYP1B1-expressing relative to control cells) to the mutagenicity of benzo(a)pyrene, cyclopenta(c,d)pyrene, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and aflatoxin B1 (AFB). CYP1B1, expressed in this system, appears to be particularly efficient at activating AFB.
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.
Revisiting the photophysical properties and excited singlet-state dipole moments of several coumarin derivatives
Cisse, Lamine,Djande, Abdoulaye,Capo-Chichi, Martine,Delatre, Franois,Saba, Adama,Tine, Alphonse,Aaron, Jean-Jacques
, p. 428 - 436 (2011)
The solvent effects on the electronic absorption and fluorescence emission spectra of several coumarins derivatives, containing amino, N,N-dimethyl-amino, N,N-diethyl-amino, hydroxyl, methyl, carboxyl, or halogen substituents at the positions 7, 4, or 3, were investigated in eight solvents with various polarities. The first excited singlet-state dipole moments of these coumarins were determined by various solvatochromic methods, using the theoretical ground-state dipole moments which were calculated by the AM1 method. The first excited singlet-state dipole moment values were obtained by the Bakhshiev, Kawski-Chamma-Viallet, Lippert-Mataga, and Reichardt-Dimroth equations, and were compared to the ground-state dipole moments. In all cases, the dipole moments were found to be higher in the excited singlet-state than in the ground state because of the different electron densities in both states. The red-shifts of the absorption and fluorescence emission bands, observed for most compounds upon increasing the solvent polarity, indicated that the electronic transitions were of π-π* nature.
