Verras Andreas, Kuntz Irwin D, Ortiz de Montellano Paul R
Department of Pharmaceutical Chemistry, University of California, 600 16th Street, San Francisco, CA 94143-2280, USA.
J Med Chem. 2004 Jul 1;47(14):3572-9. doi: 10.1021/jm030608t.
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.
DOCK程序的一个修改版本已被用于预测细胞色素P450cam及其L244A突变体的抑制剂。在计算机上设计了一个唑类化合物库，并筛选其与野生型P450cam的结合情况。合成了先导化合物，发现其能抑制野生型P450cam。为了测试我们设计能区分密切相关位点的配体的方法，将唑类化合物库对接至野生型P450cam及其L244A突变体的活性位点。预计L244A活性位点比野生型P450cam的活性位点略大。合成了预计仅对突变体具有高亲和力的配体，并用重组酶进行了测定。所有化合物均显示出对L244A酶的抑制作用（IC(50)=6 - 40 microM），而预计太大而无法与野生型结合的化合物显示出较差的抑制作用（IC(50)≥1 mM）。通过光谱分析表明结合模式与我们修改后的DOCK版本所预测的相似。IC(50)值与光谱K(s)值之间的差异表明光谱结合常数不能准确估计抑制活性。这项研究是关于P450酶的计算机辅助配体（药物）设计的首次报道，其中在结构建模中明确考虑了咪唑与血红素之间的配位键，由于唑类化合物被广泛用作P450酶抑制剂和药物，因此开辟了一条有前景的设计途径。
