7-alkoxyquinolines: New fluorescent substrates for cytochrome p450 monooxygenases

Mayer, Richard T.; Netter, K.J.; Heubel, Friedrich; Hahnemann, B.; Buchheister, Armin; Mayer, Gerhard; Burke, Mary

doi:10.1016/0006-2952(90)90467-y

Cited by 49 publications

(23 citation statements)

References 20 publications

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“…Despite this difference, the K m values reported in these studies are close to that observed in our control group (Table 1). As for resorufin formation from BR via 2B1/2, our two-enzyme kinetic model (Table 1) is different from the previously reported (Mayer et al, 1990) single-enzyme model. This discrepancy is most likely due to a narrower substrate concentration range used by these investigators.…”

Section: Enzyme-selective Effects Of Nitric Oxide On P450contrasting

confidence: 85%

Enzyme-Selective Effects of Nitric Oxide on Affinity and Maximum Velocity of Various Rat Cytochromes P450

Vuppugalla

Mehvar²

2005

Drug Metabolism and Disposition

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ABSTRACT:Nitric oxide (NO) has recently been shown to decrease cytochrome P450 (P450) enzyme activity rapidly (<30 min), concentration dependently, and enzyme-selectively in the rat liver. Interestingly, among all the studied P450 enzymes, only CYP2D1 was not affected by NO donors. However, these studies were conducted using only a single concentration of the substrates, thus lacking information about the possible simultaneous changes in both maximum velocity (V max ) and affinity (K m ) of the enzymes. In the present study, we systematically evaluated the effects of NO on the enzyme kinetic parameters of marker substrates for a range of P450 enzymes, including 2D1. Livers were perfused (1 h) in the absence ( Cytochrome P450 (P450) enzymes are heme-containing proteins critical to the metabolism and subsequent excretion of a large number of drugs and toxic agents as well as endogenous compounds. It is well established today that the ability of the liver to carry out P450-dependent drug biotransformation is compromised in patients with infectious diseases or disease states that are associated with inflammation (Morgan, 1997(Morgan, , 2001Renton, 2004;Riddick et al., 2004). Despite wide acceptance that the stimulation of the immune system can inhibit drug metabolism, the exact mechanisms responsible for this down-regulation are still not well understood. Large quantities of cytokines, produced during inflammation, are believed to be responsible, at least in part, for mediating suppression of P450 enzymes at the transcriptional level (Morgan, 1997). Indeed, several reports (Ghezzi et al., 1986;Warren et al., 1999) have shown that the administration of inflammatory cytokines depresses P450 activities and contents both in vivo and in vitro.In addition to cytokines, inflammatory states induce the expression of inducible nitric-oxide synthase and result in excessive production of nitric oxide (NO) in both hepatocytes and Kupffer cells (Nathan, 1992). Although some reports Takemura et al., 1999) suggest that P450 enzyme down-regulation occurs independently of NO, others (Stadler et al., 1994;Khatsenko and Kikkawa, 1997;Khatsenko, 1998) have shown that NO may also play a role in this event. Besides this indirect evidence for an NO role in inflammation-induced P450 depression, attempts have also been made at delineating the effects of NO directly, using NO donors in the microsomes or purified enzyme systems (Khatsenko et al., 1993;Wink et al., 1993;Minamiyama et al., 1997). Supporting the above findings, very recently (Vuppugalla and Mehvar, 2004a), we showed the inhibitory effects of NO on P450 to be rapid, concentration-dependent, and enzyme-selective in an isolated perfused rat liver (IPRL) model. Furthermore, we also conducted time course studies to assess the mechanisms responsible for the NO-mediated P450 inhibition and demonstrated that the short-term inhibitory effects of NO are time-dependent, consisting of both reversible and irreversible components (Vuppugalla and Mehvar, 2004b).One of the novel findings of our ...

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Section: Enzyme-selective Effects Of Nitric Oxide On P450contrasting

confidence: 85%

Enzyme-Selective Effects of Nitric Oxide on Affinity and Maximum Velocity of Various Rat Cytochromes P450

Vuppugalla

Mehvar²

2005

Drug Metabolism and Disposition

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“…The reaction, however, proceeds with a low efficiency as compared to cinnamate hydroxylation. 7-Methoxycoumarin is also an alternative substrate for some animal P450s [49]. Its dealkylation results from the hydroxylation of the a-carbon of the alkyl side-chain.…”

Section: Discussionmentioning

confidence: 99%

Catalytic Properties of the Plant Cytochrome P450 CYP73 Expressed in Yeast

Pierrel

Batard

Kazmaier³

et al. 1994

European Journal of Biochemistry

114

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have been investigated. Microsomes from transformed yeast catalysed trans-cinnamate hydroxylation with high efficiency. CYP73 was highly specific for its natural substrate, and did not catalyse oxygenation of p-coumarate, benzoate, ferulate, naringenin or furanocoumarins. No metabolism of terpenoids or fatty acids, known substrates of plant P450s, was observed. CYP73 however demethylated the natural coumarin herniarin into umbelliferone. In addition, it was shown to oxygenate five xenobiotics and mechanism-based inactivators, including the herbicide chlorotoluron. All substrates of CYP73 were small planar aromatic molecules. Comparison of the kinetic parameters of CYP73 for its various substrates showed that, as expected, cinnamate was by far the best substrate of this P450. The physiological and toxicological significance of these observations are discussed.Cytochromes P450 form a large superfamily of several hundreds to several thousands of hemoproteins, involved in oxygen activation and oxygen tranfer into lipophilic molecules. They all share some sequence identity related to their common catalytic properties, i.e. heme and oxygen binding, electron transfer and oxygen activation [l]. Differences in their primary sequences usually reflect variations in specificity for substrates oxygenated. These differences may concern more than 80% of the amino acid sequence. However, the modification of a single amino acid residue can be sufficient to completely alter the substrate specificity of the enzyme 121.Full-length amino acid sequences presently available for plant P450s share less than 30% identity. This almost certainly implies great differences in the structures of their substrate binding sites. However, no relation between the structure of the enzymes and their catalytic activities has yet been established. CYP73 is the first DNA sequence coding for a plant P450 with an identified physiological function to have been isolated 131. It catalyses the 4-hydroxylation of trunscinnamic acid into p-coumaric acid. This hydroxylation is ductase (4-hydroxylating) (EC 1.14.13.11).the second reaction in the phenylpropanoid pathway. Cinnamate 4-hydroxylase (CA4H) is thus an obligatory step for the biosynthesis of lignin, a major component of the earth's total biomass. It is involved in the formation of most of the phenylpropanoid derivatives, essential for plant development, pigmentation and defense against both ultraviolet light and pathogens. Recent data [4-61 suggest that CA4H plays a central role in the regulation of the phenylpropanoid pathway. We, therefore, decided to establish more precisely which molecules are transformed by the enzyme or interfere with its catalytic activity. The determination of a population of substrates was also intended to provide information for analysis of the structurelfunction relationship of this plant P450. We devised an optimized system for the expression of CYP73 in Saccharomyces cerevisae [7]. This system provided yeast microsomes for which the only detectable P450 was CYP73. It routine...

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“…These properties enable testing larger numbers of experimental conditions with cost effective, higher-throughput methodologies. Indeed, fluorometric P450 substrates have been used for many applications in toxicology and drug discovery and development (Ullrich and Weber, 1972;Burke et al, 1985;DeLuca et al, 1988;White, 1988;Mayer et al, 1990;Buters et al, 1993;Kennedy and Jones, 1994;Crespi et al, 1997;Onderwater et al, 1999;Price et al, 2000;Renwick et al, 2000). Some fluorometric probes have been reported to be selective for various P450 isoforms in liver microsomes, including 7-methoxyresorufin and 7-ethoxyresorufin for CYP1A2 (Burke et al, 1994), 3-(3,4,-difluorobenzyloxy)-5,5-dimethyl-4-(4-methyl-sulfonylphenyl)-5H-furan-2-one for CYP3A4/5 (Chauret et al, 1999), and 2,5-bis(trifluoromethyl)-7-benzyloxy-4-trifluoromethylcoumarin for CYP3A4 (Renwick et al, 2001).…”

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confidence: 99%

Cytochrome P450 Fluorometric Substrates: Identification of Isoform-Selective Probes for Rat CYP2D2 and Human CYP3A4.

Stresser¹,

Turner²,

Blanchard³

et al. 2002

Drug Metabolism and Disposition

155

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ABSTRACT:We have tested a panel of 29 cDNA-expressed rat and human enzymes with 9 fluorometric substrates to determine the P450 isoform selectivity in the catalysis of the substrates to fluorescent products. The substrates examined were dibenzyl fluorescein, 7-benzyloxyquinoline (BQ), 3-cyano-7-ethoxycoumarin, 3-cyano-7-methoxycoumarin, 7-methoxy-4-trifluoromethylcoumarin, 3- Cytochrome P450 (P450 1 ) enzymes are the principal enzymes that catalyze the metabolism of drugs and other xenobiotics. Analysis of drug metabolism by the cytochrome P450 system has become an important part of the drug discovery/development process, and numerous assay methodologies have been developed. Cytochrome P450 activity assays which have fluorometric endpoints are advantageous in that they offer high sensitivity and are often direct and homogeneous assays. These properties enable testing larger numbers of experimental conditions with cost effective, higher-throughput methodologies. Indeed, fluorometric P450 substrates have been used for many applications in toxicology and drug discovery and development (Ullrich and

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7-alkoxyquinolines: New fluorescent substrates for cytochrome p450 monooxygenases

Cited by 49 publications

References 20 publications

Enzyme-Selective Effects of Nitric Oxide on Affinity and Maximum Velocity of Various Rat Cytochromes P450

Enzyme-Selective Effects of Nitric Oxide on Affinity and Maximum Velocity of Various Rat Cytochromes P450

Catalytic Properties of the Plant Cytochrome P450 CYP73 Expressed in Yeast

Cytochrome P450 Fluorometric Substrates: Identification of Isoform-Selective Probes for Rat CYP2D2 and Human CYP3A4.

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