Olefin cross-metathesis (CM) is potentially an attractive method for generating dynamic combinatorial libraries (DCLs). In order for the CM reaction to be useful for DCL production, the course of the reaction and product distribution must be relatively insensitive to functionality remote from the reacting centers. We report on the CM of a series of allyl- and homoallylamides that are strongly dependent on remote functionality. This includes an unusual example of a cis-selective CM. [Reaction: see text]

The human type 1 (placenta, breast tumors) and type 2 (gonads, adrenals) isoforms of 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD) are key enzymes in biosynthesis of all active steroid hormones. Human 3β-HSD1 is a critical enzyme in the conversion of DHEA to estradiol in breast tumors and may be a major target enzyme for the treatment of breast cancer. 3β-HSD2 participates in the production of cortisol and aldosterone in the human adrenal gland. The goals of this project are to evaluate the role of the 2α-cyano group on trilostane (2α-cyano-4α,5α-epoxy-17β-ol-androstane-3-one) and determine which amino acids may be critical for 3β-HSD1 specificity. Trilostane without the 2α-cyano group, 4α,5α-epoxy-testosterone, was synthesized. Using our structural model of 3β-HSD1, trilostane or 4α,5α-epoxy-testosterone was docked in the active site using Autodock 3.0, and the potentially critical residues (Met187 and Ser124) were identified. The M187T and S124T mutants of 3β-HSD1 were created, expressed and purified. Dixon analyses of the inhibition of wild-type 3β-HSD1, 3β-HSD2, M187T and S124T by trilostane and 4α,5α-epoxy-testosterone suggest that the 2α-cyano group of trilostane is anchored by Ser124 in both isoenzymes. Kinetic analyses of cofactor and substrate utilization as well as the inhibition kinetics of M187T and the wild-type enzymes suggest that the 16-fold higher-affinity inhibition of 3β-HSD1 by trilostane may be related to the presence of Met187 in 3β-HSD1 and Thr187 in 3β-HSD2. This structure/function information may lead to the production of more highly specific inhibitors of 3β-HSD1 to block the hormone-dependent growth of breast tumors.

Human 3β-hydroxysteroid dehydrogenase/isomerase type 1 (3β-HSD1) is a critical enzyme in the conversion of DHEA to estradiol in breast tumors and may be a target enzyme for inhibition in the treatment of breast cancer in postmenopausal women. Human 3β-HSD2 participates in the production of cortisol and aldosterone in the human adrenal gland in this population. In our recombinant human breast tumor MCF-7 Tet-off cells that express either 3β-HSD1 or 3β-HSD2, trilostane and epostane inhibit the DHEA-induced proliferation of MCF-7 3β-HSD1 cells with 12-to 16-fold lower IC 50 values compared to the MCF-7 3β-HSD2 cells. The compounds also competitively inhibit purified human 3β-HSD1 with 12-to 16-fold lower K i values compared to the noncompetitive Ki values measured for human 3β-HSD2. Using our structural model of 3β-HSD1, trilostane or 17β-acetoxytrilostane was docked in the active site of 3β-HSD1, and Arg195 in 3β-HSD1 or Pro195 in 3β-HSD2 was identified as a potentially critical residue (one of 23 nonidentical residues in the two isoenzymes). The P195R mutant of 3β-HSD2 were created, expressed and purified. Kinetic analyses of enzyme inhibition suggest that the high-affinity, competitive inhibition of 3β-HSD1 by trilostane and epostane may be related to the presence of Arg195 in 3β-HSD1 vs Pro195 in 3β-HSD2.

Human 3β-hydroxysteroid dehydrogenase/isomerase type 1 (3β-HSD1) is a critical enzyme in the conversion of DHEA to estradiol in breast tumors and may be a target enzyme for inhibition in the treatment of breast cancer in postmenopausal women. Human 3β-HSD2 participates in the production of cortisol and aldosterone in the human adrenal gland in this population. In our recombinant human breast tumor MCF-7 Tet-off cells that express either 3β-HSD1 or 3β-HSD2, trilostane and epostane inhibit the DHEA-induced proliferation of MCF-7 3β-HSD1 cells with 12-to 16-fold lower IC 50 values compared to the MCF-7 3β-HSD2 cells. Trilostane and epostane also competitively inhibit purified human 3β-HSD1 with 12-fold to 16-fold lower K i values compared to the noncompetitive Ki values measured for human 3β-HSD2. Using our structural model of 3β-HSD1, trilostane was docked in the active site of 3β-HSD1, and Arg195 in 3β-HSD1 or Pro195 in 3β-HSD2 was identified as a potentially critical residue. The R195P-1 mutant of 3β-HSD1 and the P195R-2 mutant of 3β-HSD2 were created, expressed and purified. Kinetic analyses of enzyme inhibition suggest that the high-affinity, competitive inhibition of 3β-HSD1 by trilostane may be related to the presence of Arg195 in 3β-HSD1 vs Pro195 in 3β-HSD2. In addition, His156 in 3β-HSD1 may play a role in the higher affinity of 3β-HSD1 for substrates and inhibitors compared to 3β-HSD2 containing Try156. Structural modeling of the 3β-HSD1 dimer identified a possible interaction between His156 on one subunit and Gln105 on the other. Kinetic analyses of the H156Y-1, Q105M-1 and Q105M-2 support subunit interactions that contribute to the higher affinity of 3β-HSD1 for the inhibitor, epostane, compared to 3β-HSD2.

In post-menopausal women, human 3β-hydroxysteroid dehydrogenase type 1 (3β-HSD1) is a critical enzyme in the conversion of DHEA to estradiol in breast tumors, while 3β-HSD2 participates in the production of cortisol and aldosterone in the human adrenal gland. The goals of this project are to determine if Arg195 in 3β-HSD1 vs Pro195 in 3β-HSD2 in the substrate/inhibitor binding site is a critical structural difference responsible for the higher affinity of 3β-HSD1 for inhibitor and substrate steroids compared to 3β-HSD2 and whether Asp61, Glu192 and Thr8 are fingerprint residues for cofactor and substrate binding using site-directed mutagenesis. The R195P-1 mutant of 3β-HSD1 and the P195R-2 mutant of 3β-HSD2 have been created, expressed, purified and characterized kinetically. Dixon analyses of the inhibition of the R195P-1 mutant, P195R-2 mutant, wild-type 3β-HSD1 and wild-type 3β-HSD2 by trilostane has produced kinetic profiles that show inhibition of 3β-HSD1 by trilostane (Ki= 0.10 μM, competitive) with a 16-fold lower Ki and different mode than measured for 3β-HSD2 (Ki= 1.60 μM, noncompetitive). The R195P-1 mutation shifts the high-affinity, competitive inhibition profile of 3β-HSD1 to a low-affinity (trilostane Ki= 2.56 μM), noncompetitive inhibition profile similar to that of 3β-HSD2 containing Pro195. The P195R-2 mutation shifts the low-affinity, noncompetitive inhibition profile of 3β-HSD2 to a high affinity (trilostane Ki= 0.19 μM), competitive inhibition profile similar to that of 3β-HSD1 containing Arg195. Michaelis-Menten kinetics for DHEA, 16β-hydroxy-DHEA and 16α-hydroxy-DHEA substrate utilization by the R195P-1 and P195R-2 enzymes provide further validation for higher affinity binding due to Arg195 in 3β-HSD1. Comparisons of the Michaelis-Menten values of cofactor and substrate for the targeted mutants of 3β-HSD1 (D61N, D61V, E192A, T8A) clarify the functions of these residues as well.