Comparison of the substrate specificities of human liver cytochrome P450s 2C9 and 2C18: application to the design of a specific substrate of CYP 2C18

Biochemistry. 1999 Jun 15;38(24):7828-36. doi: 10.1021/bi9903289.

Abstract

A series of 2-aroylthiophenes derived from tienilic acid by replacement of its OCH2COOH substituent with groups bearing various functions have been synthesized and studied as possible substrates of recombinant human liver cytochrome P450s 2C9 and 2C18 expressed in yeast. Whereas only compounds bearing a negative charge acted as substrates of CYP 2C9 and were hydroxylated at position 5 of their thiophene ring at a significant rate, many neutral 2-aroylthiophenes were 5-hydroxylated by CYP 2C18 with kcat values of >2 min-1. Among the various compounds that were studied, those bearing an alcohol function were the best CYP 2C18 substrates. One of them, compound 3, which bears a terminal O(CH2)3OH function, appeared to be a particularly good substrate of CYP 2C18. It was regioselectively hydroxylated by CYP 2C18 at position 5 of its thiophene ring with a KM value of 9 +/- 1 microM and a kcat value of 125 +/- 25 min-1, which are the highest described so far for a CYP 2C. A comparison of the oxidations of 3, by yeast-expressed CYP 1A1, 1A2, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, and 3A5, showed that only CYP 2C8, 2C18, and 2C19 were able to catalyze the 5-hydroxylation of 3. However, the catalytic efficiency of CYP 2C18 for that reaction was considerably higher (kcat/KM value being 3-4 orders of magnitude larger than those found for CYP 2C8 and 2C19). Several human P450s exhibited small activities for the oxidative O-dealkylation of 3. The four recombinant CYP 2Cs were the best catalysts for that reaction (kcat between 1 and 5 min-1) when compared to all the P450s that were tested, even though it is a minor reaction in the case of CYP 2C18. All these results show that compound 3 is a new, selective, and highly efficient substrate for CYP 2C18 that should be useful for the study of this P450 in various organs and tissues. They also suggest some key differences between the active sites of CYP 2C9 and CYP 2C18 for substrate recognition.

Publication types

  • Comparative Study

MeSH terms

  • Aryl Hydrocarbon Hydroxylases*
  • Cytochrome P-450 CYP1A1 / metabolism
  • Cytochrome P-450 CYP1A2 / metabolism
  • Cytochrome P-450 CYP2C19
  • Cytochrome P-450 CYP2D6 / metabolism
  • Cytochrome P-450 CYP2E1 / metabolism
  • Cytochrome P-450 CYP3A
  • Cytochrome P-450 Enzyme System / chemistry*
  • Cytochrome P-450 Enzyme System / genetics
  • Cytochrome P-450 Enzyme System / metabolism*
  • Humans
  • Hydroxylation
  • Liver / enzymology*
  • Liver / metabolism
  • Mixed Function Oxygenases / metabolism
  • Nuclear Magnetic Resonance, Biomolecular
  • Oxidation-Reduction
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Spectrophotometry, Ultraviolet
  • Steroid 16-alpha-Hydroxylase*
  • Steroid Hydroxylases / chemistry
  • Steroid Hydroxylases / genetics
  • Steroid Hydroxylases / metabolism*
  • Structure-Activity Relationship
  • Substrate Specificity
  • Thiophenes / chemistry
  • Thiophenes / metabolism
  • Ticrynafen / chemical synthesis*
  • Ticrynafen / metabolism*

Substances

  • Recombinant Proteins
  • Thiophenes
  • Cytochrome P-450 Enzyme System
  • Mixed Function Oxygenases
  • Steroid Hydroxylases
  • Cytochrome P-450 CYP2E1
  • Aryl Hydrocarbon Hydroxylases
  • CYP1A2 protein, human
  • CYP2C18 protein, human
  • CYP2C19 protein, human
  • CYP3A protein, human
  • Cytochrome P-450 CYP1A1
  • Cytochrome P-450 CYP1A2
  • Cytochrome P-450 CYP2C19
  • Cytochrome P-450 CYP2D6
  • Cytochrome P-450 CYP3A
  • Steroid 16-alpha-Hydroxylase
  • Ticrynafen