Frederick Sweet

Hydrogen bonding in steroidogenesis: Studies on new heterocyclic analogs of estrone that inhibit human estradiol 17β-dehydrogenase

New heterocyclic analogs of estrone are reported that inhibit estradiol 17 beta-dehydrogenase (E2-17 beta DH) from human placenta. The inhibitors are efficiently synthesized in two steps from estrone (or its 3-O-methyl ether), giving fully characterized analogs with pyrazole or isoxazole fused to the 16,17-position on the D ring. Dixon plots of enzyme kinetic data show the heterocyclic steroids are competitive inhibitors of E2-17 beta DH. Correlating molecular structures of the inhibitors with their Ki-values yields a pattern suggesting intermolecular hydrogen bonding stabilizes the [(pyrazole)inhibitor-E2-17 beta DH] complexes. A free energy difference of 2.74 Kcal/mol calculated from Ki-value differences between hydrogen bonded (4.08 microM) and non-bonded (425 microM) [inhibitor-E2-17 beta DH] complexes is in the range for intermolecular hydrogen bonding. We conclude that specific intermolecular hydrogen bonds stabilize [hydroxysteroid-enzyme] complexes, thereby making important contributions to the affinity between hydroxysteroids and steroid-specific enzymes of steroidogenesis.

Purification of 20α-hydroxysteroid oxidoreductase from bovine fetal erythrocytes

Abstract NADPH-dependent 20α-hydroxysteroid oxidoreductase (20α-HSD; EC 1.1.1.149) from bovine fetal erythrocytes was obtained for the first time free of hemoglobin by a new 2,500-fold purification scheme. This was achieved by a sequence of calcium phosphate gel adsorption, ammonium sulfate fractionation, and affinity chromatography. The present results lead us to believe that the NADPH-dependent 3β-hydroxysteroid oxidoreductase activity, which was co-purified with 20α-activity, may originate at the active site of 20α-HSD (2).

Daunorubicin conjugated to a monoclonal anti-CA125 antibody selectively kills human ovarian cancer cells

Abstract The present study was designed to test the in vitro efficacy for human ovarian cancer cells of daunorubicin (DNR) conjugated to a monoclonal antibody (OC125). The OC125 antibody specifically binds to the antigenic protein CA125 from human ovarian carcinoma. New analogs of DNR containing various linker groups were conjugated to mouse monoclonal anti-CA125 antibody (DNR-OC125); nonspecific murine IgG 1 (DNR-IgG 1 ); or bovine serum albumin (DNR-BSA). The DNR-protein conjugates were all stable for several days in neutral solutions at room temperature. The DNR-OC125 conjugates selectively killed dividing cell populations but not nondividing cell populations of two human ovarian cancer cell lines (SK-OV-3 or OVCAR-3) that express the CA125 antigen. Equivalent concentrations of DNR-IgG 1 or DNR-BSA conjugates were neither toxic to the dividing nor the nondividing populations of SK-OV-3 or OVCAR-3 cells. Only those DNR-protein conjugates linked to OC125 were cytotoxic to dividing cell populations of both cell lines. This indicates that cytotoxicity is dependent on OC125 antibody-CA125 antigen binding which concentrates DNR on the ovarian cancer cells. We advance the hypothesis that following antibody-antigen binding, DNR is released from the conjugate and it intercalates in DNA by a mechanism similar to that of the unmodified DNR. The new DNR-OC125 conjugate may be useful for delivering DNR to ovarian tumors that express the CA125 antigen because the drugantibody conjugates (1) retain the cytotoxic characteristics of the unmodified drug: (2) specifically kill the human ovarian cancer cells that express the CA125 antigen; and (3) are completely stable for days in neutral solutions at room temperature.

Synthesis of an affinity chromatography column designed for recovery of labile proteins.

Summary An affinity chromatography column has been synthesized in which llα-hydroxyprogesterone hemisuccinate is covalently bound to the stationary matrix through a disulfide linkage. This column was designed to explore the feasibility of recovering proteins which are not stable under conventional recovery conditions and which are stabilized by β-mercaptoethanol. 20β-Hydroxysteroid dehydrogenase (E.C.1.1.1.53) was adsorbed on a column of llα-hydroxyprogesterone ll-hemisuccinyl-bis-β-aminoethyl disulfideagarose in the presence of NAD+. After washing the column free of non-specific protein, the enzyme was recovered simply by inclusion of β-mercaptoethanol in the buffer.

Complete 1H and 13C NMR spectra of pregnenolone

Assignments for signals from 1H and 13C in the NMR spectra of pregnenolone (1), 16-dehydropregnenolone (2), and the 3-acetate of 1 (3) were validated by two-dimensional correlated spectroscopy (2D COSY) and heteronuclear single quantum coherence (HSQC). The narrow band of overlapping signals from H-7, H-2, and H-4 was resolved by exploiting three-bond coupling in the 2D COSY spectra and heteronuclear correlation. Assignments were based on high intensity cross peaks from long-range coupling by H-18 with H-17 and H-12 (axial). Similar cross peaks were observed for H-17 with H-21. Low intensity cross peaks were seen for H-4 coupled with H-6 and H-7, and also H-16 (quasi-axial) with H-14 of 1 and 3. Assignments based on 2D COSY spectra were confirmed by correlation peaks from HSQC. This now corrects the earlier conflicts among assignments reported for 13C signals of 1 and 3. Accurate assignments were similarly derived for signals from C-2, C-7, C-8, and C-21 of 1 and 3, and C-15 and C-16 of 1, 2, and 3. The complete sets of 1H and 13C NMR data for pregnenolone, pregnenolone 3-acetate, and 16-dehydropregnenolone serve as reference standards.

[33] Synthesis and use of affinity labeling steroids for analysis of macromolecular steroid-binding sites

Publisher Summary Affinity labeling (site-directed irreversible binding) of a macromolecular steroid binding site requires synthesis of appropriate steroid derivatives bearing reagent groups capable of reacting with amino acid residues present at that site. Concentration of the reagent group at the binding site by the reversible binding step of the steroid moiety favors covalent bond formation at the binding site as compared to the protein molecule in general. The basic scheme is indicated in this chapter. Reversible binding of steroid and protein is contrasted with the situation where the steroid bears a reagent group (X) capable of reacting with an amino acid residue (Y) at the binding site. A typical sequence of events is to select an inacromolecule for study, pick a steroid that has high affinity for the steroid binding site, and modify the steroid by adding a reagent group. The reagent groups are essentially small molecules capable of alkylating various amino acid residues in the protein. Many of these are delineated in an earlier volume in this series dealing with protein modification. If the derivative forms a covalent bond with the macromolecule in question, one may ascertain the existence of the primary reversible binding step. Ultimately, with strategically selected derivatives, it should be possible to delineate the topography of the steroid-binding site under study, permanently occupy such sites on receptors (with exclusion of natural steroids), and determine whether cytoplasmic steroid receptors play an obligatory and final role in the mechanism of steroid action. This chapter summarizes the information that can be gained by this technique and delineates synthesis and application of several model compounds.

Synthesis of 21-acryloxyprogesterone, 21-bromoacetoxyprogesterone and 11α-bromoacetoxyprogesterone for affinity labeling

Abstract Esters of 21- and 11α-hydroxyprogesterone capable of alkylating amino acids are prepared by reaction of acrylyl chloride or bromoacetyl bromide with the appropriate hydroxysteroid in dimethylformamide. Alternatively bromoacetic acid can be condensed with a steroidal alcohol in the presence of dicyclohexylcarbodiimide. Although the steroidal acrylate and bromoacetates all alkylate certain amino acids only the bromoacetates inactivate 20β-hydroxysteroid dehydrogenase by affinity labeling.

Boron estrogens: Synthesis, biochemical and biological testing of estrone and estradiol-17β 3-carboranylmethyl ethers

Synthesis, biochemical and biological testing of the first carborane derivatives of estrogens are described. Estrone 3-carboranylmethyl ether was synthesized in two steps from estrone. Reduction of estrone 3-carboranylmethyl ether with sodium borohydride provided estradiol-17 beta 3-carboranylmethyl ether. Enzyme kinetic measurements showed that estrone 3-carboranylmethyl ether is a substrate for human placental 17 beta-hydroxysteroid dehydrogenase with Km = 5 x 10(-6)M, and Vmax = 0.016 mumol min-1 microgram -1. The relative affinity constant of estradiol-17 beta 3-carboranylmethyl ether for rat uterine estrogen receptor was 0.5 (compared with a value of 100 for estradiol-17 beta). Consistent with its low affinity for estrogen receptor, the dose-dependent uterotropic response to estradiol-17 beta 3-carboranylmethyl ether in castrated female rats was one sixtieth that of estradiol-17 beta. None of the tested rats had a toxic reaction to estradiol-17 beta 3-carboranylmethyl ether. These results demonstrate that exceptionally stable carborane derivatives of estrogens can be synthesized with preservation of their biochemical and biological properties. Boron-containing estrogens may be useful for thermal neutron capture therapy of cancers with estrogen receptors to concentrate boron in the cell nucleus.

Isolation of 3β,20α-hydroxysteroid oxidoreductase from sheep fetal blood

3 beta,20 alpha-Hydroxysteroid oxidoreductase has been isolated from ovine fetal blood by a 2,370-fold purification scheme of ammonium sulfate fractionation, calcium phosphate gel adsorption, affinity chromatography, and fast performance liquid chromatography. A new high performance liquid chromatography-based assay for measuring 20 alpha-reductase activity is described. The enzyme is a monomer with a molecular weight of 35,000 and uses NADPH as a cofactor for reductase activity. It reduces progesterone to 4-pregnen-20 alpha-ol-3-one or 5 alpha-dihydrotestosterone to 5 alpha-androstan-3 beta,17 beta-diol with kinetic characteristics of Km = 30.8 microM and Vmax = 0.7 nmol min-1 (nmol of enzyme)-1 or Km = 74 microM and Vmax = 1.3 nmol min-1 (nmol of enzyme)-1, respectively. 5 alpha-Dihydrotestosterone competitively inhibits 20 alpha-reductase activity with a Ki value of 102 microM.

Bifunctional enzyme activity at the same active site: Competitive inhibition kinetics with 3α/20β-hydroxysteroid dehydrogenase

20 beta-Hydroxy-5 alpha-pregnan-3-one (HPO) is a competitive inhibitor of reduction by 3 alpha/20 beta-hydroxysteroid dehydrogenase (3 alpha/20 beta-HSD; E.C.1.1.1.53) of 17 beta-hydroxy-5 alpha-androstan-3-one (DHT; 3 alpha-activity; Ki = 4.6x10(-5)M), and of 6 beta-acetoxyprogesterone (6 beta-AP; 20 beta-activity; Ki = 4.34x10(-5)M). HPO and DHT inhibit affinity alkylation of 3 alpha/20 beta-HSD by 6 beta-bromoacetoxyprogesterone (6 beta-BAP). The facts that 1) enzyme 3 alpha-activity and 20 beta-activity are both competitively inhibited by HPO with practically identical Ki-values, 2) 6 beta-BAP is solely a 20 beta-activity substrate for 3 alpha/20 beta-HSD, 3) one mole of 6 beta-BAP reacts with one mole of 3 alpha/20 beta-HSD to simultaneously inactivate 3 alpha- and 20 beta-activity, and 4) inactivation of 3 alpha/20 beta-HSD by 6 beta-BAP is inhibited by DHT (a C19-steroid) or HPO (a C21-steroid), support the view that the same active site of 3 alpha/20 beta-HSD possesses both 3 alpha- and 20 beta-activity. Bifunctional activity at the same active site is considered for other steroid-specific enzymes in female mammalian reproductive systems.