Abstract

Noncovalent forces, other than hydrophobic interactions, are important determinants of substrate bias exhibited by some cytochromes P450. The CYP2C9 pharmacophore is proposed to include either an anionic group or hydrogen bond donor in addition to its hydrophobic groups. By constructing analogs of benzbromarone, evidence supporting the existence of a 2C9 anion-binding site was revealed. A nonsubstituted phenol analog was determined to have a pK_a of 8.4 and a K_i of 414 nM whereas those with dihalogenated benzoyl phenols had pK_a values between 4.2 to 5.2 and K_i values as low as 1 nM. The nonhalogenated, nonionizable analog is the poorest binder at 796 nM. The K_i range covers around three orders of magnitude with even the weakest binder being a more potent inhibitor than 2C9 substrate phenytoin. Thus, benzbromarone derivatives represent a class of molecules with the potential to reveal more structural details of the 2C9 active site.