A modified version of the DOCK program has been used to predict inhibitors for cytochrome P450cam and its L244A mutant. A library of azole compounds was designed in silico and screened for binding to wild-type P450cam. Lead compounds were synthesized and found to inhibit wild-type P450cam. To test our approach to designing ligands that discriminate between closely related sites, the azole library was DOCKed into both the active sites of wild-type P450cam and its L244A mutant. The L244A active site is predicted to be slightly larger than that of wild-type P450cam. Ligands predicted to have a high affinity for the mutant alone were synthesized and assayed with the recombinant enzymes. All of the compounds showed inhibition of the L244A enzyme (IC(50) = 6-40 microM), and the compounds that were predicted to be too large to bind to the wild-type showed poor inhibition (IC(50) > or = 1 mM). The binding mode was shown to be similar to that predicted by our modified version of DOCK by spectroscopic analysis. A discrepancy between the IC(50) values and spectroscopic K(s) values indicates that the spectroscopic binding constants do not accurately estimate inhibitory activity. This study, the first report of computer-assisted ligand (drug) design for P450 enzymes in which the coordination bond between imidazole and the heme is explicitly considered in structural modeling, opens a promising design avenue because azole compounds are widely used as P450 enzyme inhibitors and drugs.