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

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 mu M); it is much less potent toward CYP 2C8 and 2C18 (K-i = 63 and 29 mu M, 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 (K-s = 0.4 +/- 0.1 mu M). 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.