Studies of the active site of cytochrome P-450scc with a high-affinity spin-labeled inhibitor.

The intramolecular site of P-450scc for conversion of cholesterol to pregnenolone involves a substrate site, an active site, and a site for transmission of electrons. The substrate site was studied with a high-affinity, high-potency nitroxide spin-labeled inhibitor of cholesterol side-chain cleavage. This substance, 17 alpha-hydroxy-11-deoxycorticosterone nitroxide (SL-V), has an affinity comparable to that of the most active substrate inhibitors ever reported and 2-50 times greater than that of the natural substrate cholesterol. Competition experiments with cholesterol and its analogues confirmed that SL-V binds reversibly to the substrate site. Titration experiments showed a single binding site on the P-450 molecule. The substrate site is on the apoprotein and has little or no direct interaction with the heme. Spin-spin interactions between the Fe3+ and side-chain or A-ring spin-labeled groups could not be demonstrated, which is consistent with carbons 22 and 20 being closest to the heme iron. We postulate that substrate disrupts a histidine nitrogen coordination with the heme iron and induces conformational changes in the apoprotein. These changes lead to increased affinity for iron-sulfur protein.

Inhibition of iron-sulfur protein-mediated reduction of cytochrome P-450SCC by specific antibodies.

The mechanism of inhibition of cholesterol side-chain cleavage by specific antibodies was studied systematically. The antibodies had no effect on substrate binding as determined by optical spectroscopy or on the heme environment of the cytochrome P-450 insofar as was detectable by electron paramagnetic resonance spectroscopy. They did not bind to either iron-sulfur protein or its reductase. The antibodies had no effect on chemical reduction of the P-450 or on P-450-CO complex formation. They did inhibit the NADPH-dependent reduction of P-450 and subsequent formation of the P-450-CO complex. This inhibitory effect was concentration dependent and was correlated with the inhibitory effect of the antibodies on enzymatic cholesterol side-chain cleavage. Similar results were obtained using Fab fragments. These results indicate that the antibodies inhibit side-chain cleavage by binding to a region close to the iron-sulfur protein-binding site, thereby preventing transfer of reducing electrons to the cytochrome P-450.

Molecular interactions of progesterone analogues with rabbit uterine cytoplasmic receptor.

Binding energies of progesterone analogues with single modifications were calculated from their affinities for the cytosolic receptor of rabbit uteri. The effects of individual substituents were analyzed in terms of hydrogen bonds, van der Waals' forces, and hydrophobic interactions. Binding to the receptor is attributed to hydrogen bonds involving the ketones at carbons 3 and 20, and van der Waals' interactions at carbons 2, 4, 7, 9, 12, 18, and 19 at which positions the separation of the steroid from the receptor appears to be about 0.1 nm or less. Greater separation occurs at carbons 6, 11, 14, 15, 16, and 21. The receptor probably has a hydrogen acceptor approximating the 11 beta position of the bound steroid. The enthalpy of binding of the progesterone molecule is about 26 kcal/mol but on the basis that the two hydrogen bonds contribute about 6 kcal/mol and each of the van der Waals' attractions about 1 kcal/mol, the sum of the individual bonds totals only about 20 kcal/mol. The difference of 6 kcal/mol is attributed to intrareceptor bonds that are established after a change in receptor conformation is initiated by progesterone binding. This change in conformation fixes the steroid in its protein niche and retards dissociation. We speculate that this alteration in conformation is related to "activation" and possibly other functions of the complex.

Tamoxifen and aminoglutethimide in advanced breast cancer.

Tamoxifen (TAM), a standard endocrine treatment for advanced breast cancer, probably acts by competing for the estrogen receptor protein in the breast tumor cells. If so, resistance to TAM may be a function of the level of the available endogenous estrogen. Inhibition of estrogen synthesis by aminoglutethimide may therefore facilitate the action of the antiestrogen. To test this hypothesis, the two agents were given concurrently (a) to patients who had become resistant to TAM and (b) to patients who had never received TAM in a randomized cross-over study against TAM alone. Patients with estrogen receptor protein-negative disease were excluded. Estrogen and aminoglutethimide levels were measured serially. In the first study, four of 26 patients experienced partial responses, and four of 26, stabilization of their disease. In the randomized study, four of 11 patients on the combination and three of nine on TAM alone had responses. Two patients on the combination and three on TAM alone had stabilization of disease. In the first group, the low rate of response may be attributed to extensive prior treatment. In the randomized study, there is presently no clear advantage for one treatment, and overall, there was no statistically significant correlation between degree of estrogen suppression, aminoglutethimide level, and response. The findings do not exclude the possibility that these agents may act in breast cancer by mechanisms other than inhibition of estrogen receptor.

Basic studies on aminoglutethimide.

Aminoglutethimide (AG), a bicyclic substance with two optically active isomers, inhibits the cholesterol side-chain cleavage (SCC) and aromatase systems by blocking the terminal cytochrome P-450s. AG interacts with these cytochromes to induce typical type II, low-spin spectra; the amount of spectral change correlates directly with the amount of enzymatic inhibition. AG acts by preventing reduction of the cytochrome, an obligate step preceding oxygenation of substrate. The apparent Ki of D(+)-AG is about 2.5 times less than the Ki of L(-)-AG in inhibiting SCC and about 40 times less in inhibiting aromatase. The spectral Ks ratios of the D- and L-enantiomers for P-450scc and PU-450 aromatase are 2.5 and 5, respectively, in the presence of substrate. The slope of the substrate concentration versus inhibition curves is less than 0.1 and extends over 2 logarithmic units of substrate concentration. By in vivo rabbit and rat assays for SCC, D-AG is 5 and 25 times more potent than L-AG. In women, AG rapidly lowers estrogen and progesterone levels in the luteal phase of the menstrual cycle and in early pregnancy, eliciting a reflex release of luteinizing hormone in the luteal phase. In postmenopausal, adrenalectomized, ovariectomized women, AG prevents the aromatization of administered androstenedione. In inhibition studies in women, plasma steroid concentrations were inversely proportional to AG levels, which were about 20 to 30 microM. This level of AG, which approximates the apparent Kis of the SCC and aromatase systems, causes a decrease in hormone levels of about 50%.

Inhibition of cholesterol side chain cleavage by active site directed antibody to corpus luteum cytochrome P-450.

Goat antibody IgG produced against bovine corpus luteum mitochondrial cytochrome P-450 (P-450scc) associated with cholesterol side chain cleavage (CSCC) was used to compare immunological characteristics of mitochondrial cytochrome P-450s from the bovine adrenal cortex (BAM), bovine corpus luteum (BCLM), and human placenta (HPM). In Ouchterlony double diffusion, anti-P450scc produced a single band with BAM and BCLM P-450scc, but not with HPM P-450scc or BAM P-450 11 beta. Appropriate concentrations of this anti-P-450scc IgG inhibited the conversion of cholesterol to pregnenolone in BCLM and BAM preparations equivalently, but inhibition of placental P-450scc was considerably less. The addition of BCLM iron sulfur protein and iron sulfur protein reductase to HPM P-450scc increased CSCC approximately 5-fold. Under these conditions, anti-P-450scc inhibited CSCC in HPM. Solubilized and sonicated BCLM preparations were inhibited equivalently but more than whole mitochondria. Addition of anti-P-450scc IgG to BAM increased 11 beta-hydroxylation activity in concentration-dependent fashion. It appears that the cytochrome P-450sccs from BAM and BCLM are very similar if not identical, but immunologically different from HPM P-450scc. The BAM P-450 11 beta is immunochemically distinct from BCLM P-450scc. The CSCC and 11 beta-hydroxylation systems of the adrenal are intimately linked because inhibition of P-450scc markedly stimulated 11 beta-hydroxylation. Finally, the inhibition of CSCC activity of BAM, BCLM, and HPM P-450 indicates that the antigenic effect is directed toward the active site.

Effects of cholesterol side chain cleavage inhibitors on 11 beta-hydroxylation: linkage of mitochondrial oxygenase systems.

Potent inhibitors of cholesterol side chain cleavage were tested for inhibition of 11 beta-hydroxylation of 11-deoxycortisol by bovine adrenal cortex mitochondria. Compounds which inhibited 11 beta-hydroxylation were metyrapone, 4-phenylimidazole, 1-benzylimidazole, 17 beta-ureido - 1, 4- androstadien-3-one. SU-8000, 4-methylaminoglutethimide, and 20 alpha-hydroxycholestrol. Compounds which did not inhibit 11 beta-hydroxylation at concentrations of 0.5 mM were d-aminoglutethimide tartrate, 1-aminoglutethimide tartrate, N-methylaminoglutethimide, 16 alpha-methylpregnenolone, 16 beta-methylpregnenolone, 20-tolylpregnenediol, 16 alpha-chloropregnenolone-3-acetate, 16 alpha-benzyloxpregnenolone-3-acetate and cyanoketone. The results obtained indicate that aminoglutethimide and its congeners, the 16-halogenated and 16-benzoylated derivatives of pregnenolone and cyanoketone are specific inhibitors of cholesterol side chain cleavage enzyme. The two mitochondrial steroid oxyganase systems are linked through their competition for a single electron source.

Temperature dependence of kinetic interactions between progesterone and the uterine cytoplasmic receptor.

The temperature dependence of the rates of dissociation and association for progesterone-receptor interactions was measured over the temperature range of 0-20 degrees C. The dissociation process is biphasic indicating that either two forms of receptor are present or that the binding of progesterone to the receptor is a concatenated reaction. The enthalpy of activation for the dissociation of progesterone from the receptor is about 26-28 kcal/mol and the entropic energy of activation is about -5 kcal/mol. The enthalpy of activation for the association of these molecules is about 3 kcal/mol and the entropic energy of activation is about 6 kcal/mol. These data are consistent with a model of progesterone binding to the receptor that includes hydrogen bonds between each of the two ketone groups and hydrogen donors on the receptor protein and involves van der Waals' interactions, due to the close proximity of the receptor binding site to a large fraction of the progesterone surface.

Modification of enzymatic activity and difference spectra of cytochrome P-450 from various sources by cholesterol side chain cleavage inhibitors.

Several groups of compounds were tested for their ability to inhibit cholesterol side chain cleavage and induce spectral change in cytochrome P-450 from bovine corpus luteum, bovine adrenal cortex, and human placental mitochondria. The substances tested include: steroids, pyridines, glutarimides, anilines and imidazoles. Good correlation was found between spectral change and enzymatic inhibition, especially in the corpus luteum which has only a single P-450-linked steroid hydroxylase. The cholesterol side chain cleavage enzyme systems from each of the three sources appear to have similar affinities for the inhibitors, which adds further support to the concept that these cytochrome P-450s are functionally identical.

Purification and characterization of mitochondrial cytochrome P-450 associated with cholesterol side chain cleavage from bovine corpus luteum.

The cytochrome P-450scc associated with cholesterol side chain cleavage was purified to homogeneity from bovine corpora lutea mitochondria by fractional column chromatography on various ion exchange columns and on a heptyl-Sepharose hydrophobic column. Purification was 75-fold, recovery was 10%, the specific concentration of P-450 was about 15 nmol/mg of protein, and the specific content of heme was 16 nmol/mg of protein. The purified protein migrated as a single band on native polyacrylamide gel electrophoresis and as a single major band with an insignificant minor band (less than 1%) on SDS-gel electrophoresis. The molecular weight of the major band was 48,000. The optical absorption spectra of the preparation exhibited Soret maxima at 416, 416, and 450 nm for the oxidized form, reduced form, and CO-reduced complex, respectively. Apparently homogeneous enzymes were purified with a molecular weight 2, 4, 6, and 8 times the molecular weight of the single unit. The enzymatic activities of the reconstituted systems under optimal conditions were 16 nmol of cholesterol cleaved/mol of P-450. The 2-unit protein was about 5% as active either because it is an incomplete portion of the natural enzyme or because the molecular environmental conditions were not optimal. No differences in biophysical characteristics between cytochrome P-450sec from corpus luteum and adrenal sources could be identified.

Stereoselective inhibition of aromatase by enantiomers of aminoglutethimide.

The dextrorotatory enantiomer of aminoglutethimide is 38 times more potent than the levoenantiomer in inhibiting aromatization of testosterone by human placental microsomes. The spectral affinity constant for microsomal cytochrome P-450 is 36 times greater for the d-enantiomer. Enzymatic inhibition and affinity are highly correlated for each of the isomers as well as for the racemic mixture. Spectral analysis of the interactions of the inhibitors with the substrate supports the evidence for participation of cytochrome P-450 in aromatization.

Comparison of luteolytic potencies of aminoglutethimide enantiomers in the rabbit and rat.

The luteolytic potency of D- and L-aminoglutethimide (D- and L-AG) was compared in in vivo assays in the rat and rabbit. By assay of plasma progesterone depletion in the rabbit, the potency of L-AG relative to D-AG was 0.21. By the plasma procedure in the rat, the relative potencies of L-AG and of the racemic mixture to D-AG were 0.04 and 0.37, respectively. By the ovarian progesterone depletion method, the L-form had very little activity and the DL-mixture was half as active as the D-isomer. Thus, in both species, almost all of the activity of the racemic mixture results from the content of D-AG. Interpretation of paradoxical data implies that in the rat, L-AG may inhibit liver degradation of progesterone at levels which do not modify secretion from the corpus luteum.

Interaction of cyanoketone and other steroid nitriles with cytochrome oxidase, hemoglobin, and cytochrome P-450.

The inhibitory activity of cyanoketone (CNK; 2alpha-cyano-4,4,17alpha-trimethyl-17beta-hydroxy-5-androsten-3-one), was investigated for enzymes of the respiratory chain and cholesterol side chain cleavage (CSCC). In bovine corpus luteum mitochondria incubated with [26-14C]cholesterol, 500 micron CNK caused 90% inhibition of pregnenolone synthesis. Comparable results were obtained with adrenal and placental mitochondria. Addition of CNK to bovine corpus luteum mitochondria or to cytochrome P-450 purified from this source elicited a concentration-dependent, reverse type I difference spectrum with an absorption maximum at about 423 nm and a minimum at about 395 nm, confirming binding to oxidized cytochrome P-450. This spectral change resembles those of steroids which inhibit CSCC. In mitochondrial preparations, CNK induced a second peak at about 445 nm. This peak was similar to that elicited by the interaction of potassium cyanide with cytochrome a3 when the former is added to rabbit heart mitochondria which are devoid of P-450. Like cyanide, CNK block mitochondrial respiration at the cytochrome oxidase site, and induced spectral changes in human hemoglobin. Therefore, this peak at 445 nm probably represents the interaction of CNK with oxidized cytochrome a3. Several other steroid nitriles had little, if any, effect on CSCC activity, nor did they induce spectral changes with cytochrome oxidase or hemoglobin. It appears that the steroid configuration of CNK is responsible for the binding to P-450 and inhibition of CSCC, whereas the binding to cytochrome a3 and hemoglobin and the inhibitory effect on electron transfer are probably related to the cyano group of CNK.

Stereoselective inhibition of cholesterol side chain cleavage by enantiomers of aminoglutethimide.

Because aminoglutethimide is a potentially important drug in the treatment of certain maligancies as well as fertility control, its stereoisomers were studied for binding to corpus luteum mitochondrial cytochrome P-450 and inhibition of cholesterol side chain cleavage. The binding affinity, determined from induced spectral changes, is 2.6 times greater for the d- than for the l-isomer. In the enzyme assay, the d-isomer is 2.5 times more potent as an inhibitor of cholesterol side chain cleavage than is the l-isomer. The extent of inhibition and the change in the absorptivity of the P-450-inhibitor complex are linearly related for both chiral and racemic forms. Thus, the active center of the enzyme is stereoselective for the enantiomers of aminoglutethimide.

Ligand modification of corpus luteum mitochondrial cytochrome P-450 spectra and cholesterol monooxygenation: an assay of enzyme-specific inhibitors.

Absorbance changes in the spectrum of cytochrome P-450 were related to the inhibition of [26-14C]cholesterol oxidation to [14C]isocaproate and pregnenolone in mitochondria from bovine corpus luteum produced by two types of ligands. Nitrogenous inhibitors, such as aminoglutethimide, elicit an absorption maximum at about 427 nm and a minimum at about 393 nm (type II), while steroidal inhibitors, such as (20R)-20-(p-tolyl)-5-pregnene-3beta,20-diol (20-tolyl-pregnenediol), cause difference spectra with maximum at about 420 nm and minimum at about 390 nm (reverse type I). The magnitude of spectral change and the amount of inhibition of pregnenolone synthesis by aminoglutethimide are closely correlated at concentrations ranging from 5 to 750 muM and by the model steroid, 20-tolyl-pregnenediol, at concentrations from 0.5 to 25 muM. The responses are concentration dependent and linear over the range of effective concentrations. The concentrations of inhibitors for the half-maximal inhibition of pregnenolone biosynthesis are identical with the concentrations producing half-maximal spectral changes within experimental error. Displacement of substrate from cytochrome P-450 and/or stabilization of the redox potential subsequent to to the ligation of heme iron is proposed as the specific mechanism of cholesterol side chain cleavage inhibition. Finding, together, the two procedures offer a sensitive, specific, and accurate means of screening inhibitors of the cholesterol side chain cleavage system.

Implication of ligand modified spectra of cytochrome P-450 associated with pregnenolone synthesis in mitochondria from corpus luteum.

The implications of ligand modified spectra of cytochrome P-450 in mitochondria from corpus luteum are considered. Mitochondria from bovine corpus luteum contain a single cytochrome P-450 which oxidizes cholesterol to pregnenolone and isocaproaldehyde. These mitochondria and the cytochrome P-450 purified from these mitochondria yield Type I spectral changes with substrates, reverse Type I spectral changes with certain steroid substrates, steroid products and unrelated steroid ligands. Nitrogenous ligands yield Type II spectral changes. Mitochondrial and purified cytochrome P-450 preparations are equivalent in this study. The inhibitory effects on the cholesterol monooxygenase are directly related to the spectral changes induced by Type II ligands. Lastly, it is suggested that a similar relationship exists with reverse Type I ligands.