Structure-activity relationships (SAR) for cytochrome P4502C9
In this project, an SAR approach was used to assess the putative active site interactions, using analogues of phenytoin (a co-regulated substrate), sulfaphenazole (CYP2C9-specific inhibitor) and bis-triazole antifungals (thought to exhibit less specific inhibition). Ki values were determined for the inhibition of tolbutamide methylhydroxylation. N2 of phenytoin is a postulated H-bond donor. Substitution (CH3 or NH2), reduced inhibitory potency from 46 μM to 74 μM and 98 μM, respectively. Inhibition was competitive. Removal of a phenyl ring removed inhibitory potential, suggesting that an aromatic interaction (π-π stacking) is more influential than the H-bonding. Replacement with a fused-ring structure enhanced potency (Ki = 25 μM). Inhibition was non-competitive, which may be explained by the overall bulk of these analogues, together with less directional lipophilic/π-π stacking interactions. A putative active site model could feature a H-bond acceptor site, a lipophilic pocket, haem interaction with the site of oxidation, and π-π stacking with an appropriate phenyl ring. Sulfaphenazole and methysulfaphenazole (CH2 instead of NH2) were the most potent analogues (Ki values of 0.82 μM and 0.39 μM). Removal of the pyrazole group reduced potency (Ki = 91 μM), as a CYP2C9-haem interaction was prevented (type II difference spectra). Additionally, the N-phenyl function may undergo hydrophobic binding and/or π-π stacking. The bis-triazoles also produced type II spectra, which indicates a haem-ligand interaction (N4 triazole lone pair electrons). Inhibition was non-competitive. The triazole was the dominant determinant of potency, as seen by relatively small decreases in potency on substitution of a proposed H-bonding site. In conclusion, the above work indicates interactions consistent with previous CYP2C9 active site models. SAR data suggest a predominance of lipophilic and π-π stacking interactions, and CYP2C9-haem liganding (if appropriate). Hydrogen-bonding also has a significant role.