Interaction of sulfaphenazole derivatives with human liver cytochromes P450 2C: molecular origin of the specific inhibitory effects of sulfaphenazole on CYP 2C9 and consequences for the substrate binding site topology of CYP 2C9

Biochemistry. 1996 Dec 17;35(50):16205-12. doi: 10.1021/bi961950t.


The effects of sulfaphenazole, 1, on typical activities catalyzed by human cytochromes P450 of the 1A, 3A, and 2C subfamilies expressed in yeast were studied. 1 acts as a strong, competitive inhibitor of CYP 2C9 (K(i) = 0.3 +/- 0.1 microM); it is much less potent toward CYP 2C8 and 2C18 (K(i) = 63 and 29 microM, respectively) and fails to inhibit CYP 1A1, 1A2, 3A4, and 2C19. From difference visible spectroscopy experiments using microsomes of yeast expressing various human P450s, 1 selectively interacts only with CYP 2C9 with the appearance of a peak at 429 nm as expected for the formation of a P450 Fe(III)-nitrogenous ligand complex (Ks = 0.4 +/- 0.1 microM). Comparative studies of the spectral interaction and inhibitory effects of twelve compounds related to 1 with CYP 2C9 showed that the aniline function of 1 is responsible for the formation of the iron-nitrogen bond of the 429 nm-absorbing complex and is necessary for the inhibitory effects of 1. The study of two new compounds synthesized during this work, in which the N-phenyl group of 1 was replaced with either an ethyl group or a 3,4-dichlorophenyl group, showed that the presence of an hydrophobic substituent at position 1 of the pyrazole function of 1 is required for a strong interaction with CYP 2C9. A model for the binding of 1 in the CYP 2C9 active site is proposed; that takes into account three major interactions that should be at the origin of the high-affinity and specific inhibitory effects of 1 toward CYP 2C9: (i) the binding of its nitrogen atom to CYP 2C9 iron, (ii) an ionic interaction of its SO2N- anionic site with a cationic residue of CYP 2C9, and (iii) an interaction of its N-phenyl group with an hydrophobic part of the protein active site.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aryl Hydrocarbon Hydroxylases*
  • Binding Sites
  • Cloning, Molecular
  • Cytochrome P-450 Enzyme Inhibitors
  • Cytochrome P-450 Enzyme System / chemistry*
  • Cytochrome P-450 Enzyme System / metabolism*
  • Humans
  • Kinetics
  • Liver / enzymology*
  • Microsomes / enzymology
  • Models, Molecular
  • Molecular Structure
  • Protein Conformation
  • Recombinant Proteins / antagonists & inhibitors
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Saccharomyces cerevisiae
  • Spectrophotometry
  • Steroid 16-alpha-Hydroxylase*
  • Steroid Hydroxylases / antagonists & inhibitors
  • Steroid Hydroxylases / chemistry*
  • Steroid Hydroxylases / metabolism*
  • Structure-Activity Relationship
  • Substrate Specificity
  • Sulfaphenazole / analogs & derivatives*
  • Sulfaphenazole / chemical synthesis
  • Sulfaphenazole / metabolism
  • Sulfaphenazole / pharmacology*


  • Cytochrome P-450 Enzyme Inhibitors
  • Recombinant Proteins
  • Sulfaphenazole
  • Cytochrome P-450 Enzyme System
  • Steroid Hydroxylases
  • Aryl Hydrocarbon Hydroxylases
  • Steroid 16-alpha-Hydroxylase