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The enantioselectivity ratio ((+)-:(-)-forms) of three substituted 1-[(benzofuran-2-yl) phenylmethyl] imidazoles as inhibitors of aromatase (P450AROM) was 2.16, 12.3 and 1.0 for the 4-methyl-, 4-fluoro- and 4-chloro-substituted compounds, respectively. The (+/-)-compounds were all >1000 times more potent than (+/-)-aminoglutethimide (IC50 = 12 x 10(3) nM). High potency (5.3-65.0 nM) for all the enantiomers studied is unusual since activity usually resides in one form for chiral inhibitors of P450AROM. The 4-methyl derivative was fitted into the model [Furet, P., Batzl, C., Bhatnager, A.S., Francotte, E., Rihs, G. and Lang, M. (1993) J. Med. Chem. 36, pp. 1393-1400] for binding of S-(-)-fadrazole to the active site and the (R)- and (S)- forms both gave a good fitting pattern with (S)-(-)-fadrazole so accounting for their close activity. Docking of both forms into the active site model for P450AROM [Laughton, C.A., Zvelebil, M.J.J.M. and Neidel, S. (1993) J. Steroid Biochem. Mol. Biol. 44, pp. 399-407], using the orientation of (S)-(-)-fadrazole, gave similar strong binding along the position of the C and D rings of the steroid substrate and in the hydrophobic cavity below the A/B rings. The site was probed for group size accommodation using the less potent 4-phenyl analogue (IC50(+/-) = 242 nM): the (S)-form showed restricted access to the region under the A ring due to the extended bulk of the biphenyl group.

Flavone and isoflavone phytoestrogens are plant chemicals and are known to be competitive inhibitors of cytochrome P450 aromatase with respect to the androgen substrate. Aromatase is the enzyme that converts androgen to estrogen; therefore, these plant chemicals are thought to be capable of modifying the estrogen level in women. In this study, the inhibition profiles of four flavones [chrysin (5, 7-dihydroxyflavone), 7,8-dihydroxyflavone, baicalein (5,6,7-trihydroxyflavone), and galangin (3,5,7-trihydroxyflavone)], two isoflavones [genistein (4,5,7-trihydroxyisoflavone) and biochanin A (5,7-dihydroxy-4-methoxyisoflavone)], one flavanone [naringenin (4, 5,7-trihydroxyflavanone)], and one naphthoflavone (alpha-naphthoflavone) on the wild-type and six human aromatase mutants (I133Y, P308F, D309A, T310S, I395F, and I474Y) were determined. In combination with computer modeling, the binding characteristics and the structure requirement for flavone and isoflavone phytoestrogens to inhibit human aromatase were obtained. These compounds were found to bind to the active site of aromatase in an orientation in which rings A and C mimic rings D and C of the androgen substrate, respectively. This study also provides a molecular basis as to why isoflavones are significantly poorer inhibitors of aromatase than flavones.

We have evaluated the binding characteristics of three steroidal inhibitors [4-hydroxyandrostenedione (4-OHA), 7alpha-(4'-amino)phenylthio-1,4-androstadiene-3,17-dione (7alpha-APTADD), and bridge (2,19-methyleneoxy) androstene-3,17-dione (MDL 101,003)], four nonsteroidal inhibitors [aminoglutethimide (AG), CGS 20267, ICI D1033, and vorozole (R83842)], and two flavone phytoestrogens (chrysin, and 7,8-dihydroxyflavone) to aromatase through a combination of computer modeling and inhibitory profile studies on the wild-type and six aromatase mutants (I133Y, P308F, D309A, T310S, I395F, and I474Y). We have generated two aromatase models based on the x-ray structures of cytochrome P450-cam and cytochrome P450bm3, respectively. A major difference between the cytochrome P450cam-based and cytochrome P450bm3-based models is in the predicted lengths of helices F and G. In the cytochrome P450cam-based model, helices F and G lie antiparallel and extend across the active-site face of the molecule from one edge to the center, so that the carboxyl-terminal residues of helix F and the N-terminal residues of helix G make a major contribution to the structure of the active site. In the cytochrome P450bm3-based model, both helices are longer and so extend almost all the way across the active-site face of the molecule. Considering the size of the androgen substrate, we evaluated our results mainly based on the cytochrome P450cam model. The mutations involved in this study are thought to be at or near the proposed active site pocket. The inhibitory profile analysis has produced very interesting results and provided a molecular basis as to how seven aromatase inhibitors with different structures bind to the active site of aromatase. Furthermore, the investigation reveals that phytoestrogens bind to the active site of aromatase in a different orientation from that in the estrogen receptor.

Aromatase, a cytochrome P450, catalyzes three consecutive hydroxylation reactions converting C19 androgens to aromatic C18 estrogenic steroids. In this study, eight human aromatase mutants (I133Y, I133W, F235L, I395F, I474Y, I474W, I474M, and I474N) were prepared to evaluate the active site and a proposed hydrophobic pocket of the enzyme that exists in an aromatase model based on the X-ray structure of cytochrome P450cam. In addition, the binding characteristics of three steroidal inhibitors [4-hydroxyandrostenedione, 7alpha-(4'-amino)phenylthio-1,4-androstandiene-3,17-dione, and bridge (2,19-methyleneoxy)androstene-3,17-dione (MDL 101,003)] and four nonsteroidal inhibitors [aminoglutethimide, CGS 20267, ICI D1033, and vorozole (R83842)] were investigated through inhibitory profile studies on the eight new and three previously generated mutants (P308F, D309A, and T310S). The latter analyses have provided a molecular basis regarding how seven aromatase inhibitors with different structures bind to the active site of aromatase.

Derivatives of 4-pyridylacetic acid are known to be inhibitors of the cytochrome P450 enzymes aromatase and lyase (17 alpha-hydroxylase/C17-20lyase), and are therefore of interest in the treatment of hormone dependent breast and prostate cancers. We report the determination of the crystal structure of one such derivative, the 4-tert-butyl cyclohexyl ester, and molecular modeling studies on two related inhibitors, the cyclohexyl ester and its alpha-methyl derivative. These latter two compounds show a marked difference in their relative activities against aromatase and lyase. Two models are proposed for the interaction of these molecules with the target enzymes on the basis of their ability to adopt conformations that partially mimic steroid substrates. From these models an explanation can be advanced for the fact that, compared with the unmethylated analogue, the (racemic) alpha-methylated compound is seven times poorer as an inhibitor of aromatase but seven times better as an inhibitor of lyase. The model proposed for binding to aromatase places the alpha-carbon of the ester group in the position occupied by C(2) of steroid substrates. In contrast, that proposed for binding to lyase places this atom in the position occupied by C(17) of steroid substrates. The introduction of steric bulk at C(2) is known to be unfavorable for aromatase inhibition, while its introduction at C(17) may lead to a better mimicry of the steroid D-ring and so improve lyase inhibition.

Inhibitors of the cytochrome P450 enzyme aromatase, which is involved in the biosynthesis of estrogens from androgens, are of proven utility in the treatment of hormone-dependent breast cancer. The determination of the crystal structure of one such inhibitor, 3-ethyl-3-(4-pyridyl)piperidine-2,6-dione (2) and its 3-butyl analogue (3) is described. In the absence of three-dimensional structural information for the enzyme, conformational analysis and comparison with natural substrates has been performed in order to define possible "active" conformations. The enhanced inhibitory activity of 3 may be linked to hydrophobic interactions between the side chain and that portion of the enzyme that normally interacts with the B and C rings of a steroid substrate. Information gained from this study and previous studies by other workers has been combined in order to produce a hypothesis to explain the pattern of activity of N(1)-alkyl derivatives of 2. The successful application of this hypothesis to the prediction of the relative aromatase inhibitory activities of the two enantiomers of the N-octyl derivative (4) is described.