Effect of drug substrates on the reduction of hepatic microsomal cytochrome P-450 by NADPH

Gigon, Philippe L.; Gram, Theodore E.; Gillette, James R.

doi:10.1016/0006-291x(68)90514-7

Cited by 134 publications

(37 citation statements)

References 3 publications

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“…It was shown that the inhibition was, in the case of hydroxylation of ethyl morphine, correlated with an inhibition of the reduction of the cytochrome-P-450 substrate complex. This is in agreement with the previous finding that the rate-limiting step in hydroxylation of ethyl morphine is the rate of reduction of the cytochrome-P-450 complex [3]. However, other steps than reduction of the cytochrome P-450 -substrate complex might involvc participation of water or protons and it can not be concluded from the present work that the rate-limiting step in the 6P-hydroxylation of tcstosterone and 26-hydroxylation of 5,!…”

Section: Wisupporting

confidence: 90%

“…On the basis of the finding that some type I compounds accelerate the reduction of cytochrome P-450 by NADPH in a carbon-monoxide atmosphere [3,41] it has been suggested that the rate-limiting step in the hydroxylation of type I compounds is the rate of reduction of cytochrome P-450 substrate complex, whereas some other step might be rate-limiting in the hydroxylation of type11 compounds [42]. Of the different substrates listed in Table 3, only testosterone, taurochenodeoxycholic acid and taurodeoxycholic acid have been tested with respect to spectral changes when mixed with microsomes.…”

Section: Wimentioning

confidence: 99%

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On the Rate-Limiting Step in Microsomal Hydroxylation of Steroids

Björkhem

1972

Eur J Biochem

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The rate-limiting step in the microsomal, NADPH-dependent 24-and 26-hydroxylations of 5@-cholestane-3a,7aI,12a-triol, 60-hydroxylation of testosterone and 16n-hydroxylation of pregnenolone has been studied. Possible isotope effects were determined when the hydrogen in the substrate which is removed in the hydroxylation was substituted with deuterium; when the 4A-or 4B-hydrogen in NADPH was substituted with deuterium ; and when the incubation was carried out in deuterated water. 3. When the reactions were carried out in deuterated water, there was no significant inhibition of 24-hydroxylation of 5@-cholestane-3a,7a, 12a-tri01, whereas 16a-hydroxylation of pregnenolone was inhibited by 150/,, 6t3-hydroxylation of testosterone by 30°/, and 26-hydroxylation of 50-cholestane-3a,7a, 12a-trio1 by 60°/,.As splitting of a C-2H bond in the rate-limiting step is expected to decrease the rate of the overall reaction to less than one-half, it was concluded that the rate-limiting step in the 16a-hydroxylation of pregnenolone and 24-hydroxylation of 5/?-cholestane-3a,7a,l2a-triol is the splitting of the C-H bond in the substrate. Some other step is rate-limiting in the 6@-hydroxylation of testosterone and the 26-hydroxylation of 5,4cholestane-3a,7a, 12n-triol. I n the case of 26-hydroxylation of 5@-cholestane-3~,7a,12a-triol, water might participate in the rate-limiting step by hydration or protonolysis of the enzyme. It is suggested that a common feature of hydroxylation in which splitting of the C-H bond in the substrate is rate-limiting is a relatively low sensitivity towards carbon monoxide.The rate-limiting step in most microsomal hydroxylations has not been defined. Kinetic studies have shown that in microsomal NADPH-dependent hydroxylations of some drugs, the rate of reduction Enzymes. 3a-Hydroxysteroid dehydrogenase, fa-hydroxysteroid: NADP oxidoreductase (EC 1.1.1.50).of cytochrome P-450 -substrate complex [2-41 or the rate of oxygen activation [4] may be rate-limiting in the over-all hydroxylation reaction. I n recent work from this laboratory [5,6], it was suggested that breaking of the C-H bond in the substrate might be a rate-limiting step in the microsomal hydroxylation of some steroids and fatty acids. It was shown that 7a-hydroxylation of [7a-3H]-plus [24-14C]-labeled taurodeoxycholic acid and 9-hydroxylation of [9-2H]decanoic acid were accompanied by a kinetic isotope effect of such magnitude that it was likely that splitting of the C-3H and C 2 H bonds repectively was rate-limiting. On the other hand,

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Section: Wisupporting

confidence: 90%

Section: Wimentioning

confidence: 99%

On the Rate-Limiting Step in Microsomal Hydroxylation of Steroids

Björkhem

1972

Eur J Biochem

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“…We also excluded seven drugs that were cost prohibitive, and two compounds that absorb in the 370-to 470-nm region and would be difficult to evaluate in the spectral binding assay. We next excluded 21 compounds whose structures have hindered nitrogens, as a hindered nitrogen is unlikely to coordinate the heme iron and form the N-Fe bond that often inhibits P450s (Gigon et al, 1968). The remaining compounds were categorized into four groups, as follows: nucleotide derivatives; platinum-containing, antibreast cancer drugs; aromatase inhibitors; and others.…”

Section: Resultsmentioning

confidence: 99%

Marketed Drugs Can Inhibit Cytochrome P450 27A1, a Potential New Target for Breast Cancer Adjuvant Therapy

2015

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Cytochrome P450 CYP27A1 is the only enzyme in humans converting cholesterol to 27-hydroxycholesterol, an oxysterol of multiple functions, including tissue-specific modulation of estrogen and liver X receptors. Both receptors seem to mediate adverse effects of 27-hydroxycholesterol in breast cancer when the levels of this oxysterol are elevated. The present work assessed druggability of CYP27A1 as a potential antibreast cancer target. We selected 26 anticancer and noncancer medications, most approved by the Food and Drug Administration, and evaluated them first in vitro for inhibition of purified recombinant CYP27A1 and binding to the enzyme active site. Six strong CYP27A1 inhibitors/binders were identified. These were the two antibreast cancer pharmaceuticals anastrozole and fadrozole, antiprostate cancer drug bicalutamide, sedative dexmedetomidine, and two antifungals ravuconazole and posaconazole. Anastrozole was then tested in vivo on mice, which received subcutaneous drug injections for 1 week. Mouse plasma and hepatic 27-hydroxycholesterol levels were decreased 2.6-and 1.6-fold, respectively, whereas plasma and hepatic cholesterol content remained unchanged. Thus, pharmacologic CYP27A1 inhibition is possible in the whole body and individual organs, but does not negatively affect cholesterol elimination. Our results enhance the potential of CYP27A1 as an antibreast cancer target, could be of importance for the interpretation of Femara versus Anastrozole Clinical Evaluation Trial, and bring attention to posaconazole as a potential complementary anti-breast cancer medication. More medications on the US market may have unanticipated off-target inhibition of CYP27A1, and we propose strategies for their identification.

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“…[More specifically, P450 isoform differences arise because the rate of reduction is more dependent on reduction potential than spin state (Ost et al, 2003). ]Generally, nitrogen-containing heme ligands, which bind more avidly than oxygen-containing ligands, are more often inhibitors than substrates (Gigon et al, 1968). Hence, many substrates (i.e., type I ligands) actually displace the heme water ligand, shifting the iron spin equilibrium toward the high-spin form, whereas nitrogen heterocycles and anilines (i.e., type II ligands) can replace the water ligand to stabilize the low-spin form.…”

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confidence: 99%

Visible Spectra of Type II Cytochrome P450-Drug Complexes: Evidence that “Incomplete” Heme Coordination Is Common

Locuson

Hutzler²,

Tracy³

2007

Drug Metab Dispos

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ABSTRACT:The visible spectrum of a ligand-bound cytochrome P450 is often used to determine the nature of the interaction between the ligand and the P450. One particularly characteristic form of spectra arises from the coordination of nitrogen-containing ligands to the P450 heme iron. These type II ligands tend to be inhibitors because they stabilize the low reduction potential P450 and prevent oxygen binding to the heme. Yet, several type II ligands containing aniline, imidazole, and triazole moieties are also known to be substrates of P450, although P450 binding spectra are not often scrutinized to make this distinction. Therefore, the three nitrogenous ligands aniline, imidazole, and triazole were used as binding spectra standards with purified human CYP3A4 and CYP2C9, because their small size should not present any steric limitations in their accessing the heme prosthetic group. Next, the spectra of P450 with drugs containing the three nitrogenous groups were collected for comparison. The absolute spectra demonstrated that the red-shift of the low-spin Soret band is mostly dependent on the electronic properties of the nitrogen ligand since they tended to match their respective standards, aniline, imidazole, and triazole. On the other hand, difference spectra seemed to be more sensitive to the steric properties of the ligand because they facilitated comparison of the spectral amplitudes achieved with the drugs versus those with the standard nitrogen ligands. Therefore, difference spectra may help reveal "weak" coordination to the heme that results from suboptimal orientation or ligand binding to more remote locations within the P450 active sites.When studying substrate binding to the cytochromes P450 (P450s), the heme prosthetic group often serves as a very useful chromophore that can be exploited for purposes of characterization. With P450s, the heme is bound as in a b-type cytochrome except that the iron atom is liganded to a single Cys side chain as found in only a few other heme-containing proteins (e.g., chloroperoxidase, nitric oxide-synthase, and prostacyclin synthase). Because oxidation of P450-bound substrate occurs at the heme, certain aspects of substrate binding can be monitored readily with a spectrophotometer. Most commonly, ligand titration experiments are carried out to determine a spectral dissociation constant (K S ) for a P450 substrate or inhibitor, but little attention is paid to the signature manners in which the P450 heme spectra are altered (reviewed by Jefcoate, 1978). In particular, there seems to be an oversimplification regarding coordination of P450 substrates to the heme iron. This interaction stabilizes the low-spin iron configuration and thus is assumed to prevent catalysis since it is the high-spin iron that is more conducive to reduction by P450 reductase. [More specifically, P450 isoform differences arise because the rate of reduction is more dependent on reduction potential than spin state (Ost et al., 2003).]Generally, nitrogen-containing heme ligands, which bind more avidl...

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Effect of drug substrates on the reduction of hepatic microsomal cytochrome P-450 by NADPH

Cited by 134 publications

References 3 publications

On the Rate-Limiting Step in Microsomal Hydroxylation of Steroids

On the Rate-Limiting Step in Microsomal Hydroxylation of Steroids

Marketed Drugs Can Inhibit Cytochrome P450 27A1, a Potential New Target for Breast Cancer Adjuvant Therapy

Visible Spectra of Type II Cytochrome P450-Drug Complexes: Evidence that “Incomplete” Heme Coordination Is Common

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