Differential time-dependent inactivation of P450 3A4 and P450 3A5 by raloxifene

A key role for C239 in quenching reactive intermediates

Josh T. Pearson, Jan L. Wahlstrom, Leslie J. Dickmann, Santosh Kumar, James R. Halpert, Larry C. Wienkers, Robert S. Foti, Dan A. Rock

Research output: Contribution to journalArticle

35 Citations (Scopus)

Abstract

The role of C239 as the active-site residue responsible for forming the covalent linkage with raloxifene during P450 3A4 time-dependent inactivation (TDI) was recently identified. The corresponding residue in CYP3A5 is S239, and when the potential for TDI in P450 3A5 was investigated, only reversible inhibition was observed against midazolam and testosterone, with median inhibitory concentration (IC50) values of 2.4 and 2.9 μM, respectively. In a similar fashion, when C239 was replaced with alanine in P450 3A4, TDI was successfully engineered out, and the reversible inhibition was characterized by IC50 values of 3.7 and 3.5 μM against midazolam and testosterone, respectively. Metabolism studies confirmed that the reactive diquinone methide intermediate required for P450 3A4 inactivation formed in all of the P450 3A enzymes investigated. Furthermore, the absence of TDI in P450 3A5 led to an increase in the formation of GSH-related adducts of raloxifene compared with that for P450 3A4. Consequently, the absence of the nucleophilic cysteine leads to differential TDI and generation of reactive metabolites in the P450 3A enzyme, providing the foundation for pharmacogenetics that contributes to individual differences in susceptibility to adverse drug reactions.

Original languageEnglish (US)
Pages (from-to)1778-1786
Number of pages9
JournalChemical Research in Toxicology
Volume20
Issue number12
DOIs
StatePublished - Dec 1 2007
Externally publishedYes

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Midazolam
Testosterone
Quenching
Cytochrome P-450 CYP3A
Enzymes
Metabolites
Metabolism
Alanine
Cysteine
Cytochrome P-450 Enzyme System
Inhibitory Concentration 50
Pharmaceutical Preparations
Pharmacogenetics
Drug-Related Side Effects and Adverse Reactions
Individuality
Catalytic Domain
Raloxifene Hydrochloride
Inhibition (Psychology)

All Science Journal Classification (ASJC) codes

  • Toxicology

Cite this

Differential time-dependent inactivation of P450 3A4 and P450 3A5 by raloxifene : A key role for C239 in quenching reactive intermediates. / Pearson, Josh T.; Wahlstrom, Jan L.; Dickmann, Leslie J.; Kumar, Santosh; Halpert, James R.; Wienkers, Larry C.; Foti, Robert S.; Rock, Dan A.

In: Chemical Research in Toxicology, Vol. 20, No. 12, 01.12.2007, p. 1778-1786.

Research output: Contribution to journalArticle

Pearson, Josh T. ; Wahlstrom, Jan L. ; Dickmann, Leslie J. ; Kumar, Santosh ; Halpert, James R. ; Wienkers, Larry C. ; Foti, Robert S. ; Rock, Dan A. / Differential time-dependent inactivation of P450 3A4 and P450 3A5 by raloxifene : A key role for C239 in quenching reactive intermediates. In: Chemical Research in Toxicology. 2007 ; Vol. 20, No. 12. pp. 1778-1786.
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abstract = "The role of C239 as the active-site residue responsible for forming the covalent linkage with raloxifene during P450 3A4 time-dependent inactivation (TDI) was recently identified. The corresponding residue in CYP3A5 is S239, and when the potential for TDI in P450 3A5 was investigated, only reversible inhibition was observed against midazolam and testosterone, with median inhibitory concentration (IC50) values of 2.4 and 2.9 μM, respectively. In a similar fashion, when C239 was replaced with alanine in P450 3A4, TDI was successfully engineered out, and the reversible inhibition was characterized by IC50 values of 3.7 and 3.5 μM against midazolam and testosterone, respectively. Metabolism studies confirmed that the reactive diquinone methide intermediate required for P450 3A4 inactivation formed in all of the P450 3A enzymes investigated. Furthermore, the absence of TDI in P450 3A5 led to an increase in the formation of GSH-related adducts of raloxifene compared with that for P450 3A4. Consequently, the absence of the nucleophilic cysteine leads to differential TDI and generation of reactive metabolites in the P450 3A enzyme, providing the foundation for pharmacogenetics that contributes to individual differences in susceptibility to adverse drug reactions.",
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