Pharmacology of novel steroidal inhibitors of cytochrome P45017α (17α-hydroxylase/C17–20 lyase)

Medical or surgical castration for the treatment of prostatic cancers prevents androgen production by the testes, but not by the adrenals. Inhibition of the key enzyme for androgen biosynthesis, cytochrome P450_17α, could prevent androgen production from both sources. The in vivo effects of 17-(3-pyridyl)androsta-5,16-dien-3β-ol (CB7598) and 17-(3-pyridyl)androsta-5,16-dien-3-one (CB7627), novel potent steroidal inhibitors of this enzyme, on WHT mice were compared with those of castration and two clinically active compounds, ketoconazole and flutamide. Flutamide and surgical castration caused significant reductions in the weights of the ventral prostate and seminal vesicles. CB7598, in its 3β-O-acetate form (CB7630), and CB7627 caused significant reductions in the weights of the ventral prostate, seminal vesicles, kidneys and testes when administered once daily for 2 weeks. Ketoconazole, given on the same schedule, caused no reductions. Plasma testosterone was reduced to 0.1 nM by CB7630, despite a 3- to 4-fold increase in the plasma level of luteinizing hormone. Adrenal weights were unchanged following treatment with CB7630 or CB7627 but were markedly increased following ketoconazole, indicating no inhibition of corticosterone production by these steroidal compounds. These results indicate that CB7598, CB7630 or CB7627 may be useful in the treatment of hormone-dependent prostatic cancers.     

The differential regulation of aldosterone output in hamster adrenal by angiotensinII and adrenocorticotropin

Aldosterone was isolated from hamster adrenal cells and was identified by high performance liquid chromatography and thermospray mass spectroscopy analysis. Basal outputs from adrenal cell suspensions were of the same order of magnitude, 8.4 ± 1.9 ng and 8.0 ± 0.7 ng/2 h/50,000 cells, for aldosterone and corticosteroid, respectively. The outputs of aldosterone and corticosteroid increased with K⁺ concentrations to reach maxima of 3.3- and 1.6-fold at 10 meq/l of K⁺. Angiotensin_II (A_II) produced dose-dependent increases in aldosterone and corticosteroid outputs with maxima of 3- and 4-fold, respectively. In contrast, ACTH induced relatively no changes in aldosterone output, whereas dose-dependent increases in corticosteroid output were found. In time study experiments, with 10⁻⁸ M A_II, aldosterone and corticosteroid outputs were maximally increased after 1 h (6-fold) and 3 h (1.8-fold), respectively. At 10⁻⁸ M, ACTH had a small stimulatory effect on aldosterone output after 6 h, whereas it provoked a gradual increase in corticosteroid output (up to 7-fold after 8 h of incubation). The effects of A_II and ACTH on adrenal cytochrome P-450_11β involved in the last steps of aldosterone formation were evaluated by c combined in vivo andin vitro experiments. The P-450_11β mRNA level was increased by a low sodium intake but not by a 24 h ACTH stimulus. These results taken together indicate that ACTH and A_II differentially regulate P-450_11β. It is postulated that these two regulatory peptides regulate the hamster adrenal steroidogenesis by different P-450 genes.     

Bacterial Expression and Purification of the Arabidopsis NADPH–Cytochrome P450 Reductase ATR2

An N-terminally modified form of the Arabidopsis NADPH–cytochrome P450 ATR2 (ATR2mod) was expressed from the tactac promoter in Escherichia coli to obtain high yields of the enzyme. The N-terminal modification eliminates the predicted chloroplast transit peptide of ATR2 allowing for more efficient expression. ATR2mod was purified from membrane extracts using a 2′,5′-ADP–agarose affinity column. The specific activity of the purified ATR2mod for cytochrome c reduction was 9.4 μmol min⁻¹ mg⁻¹ and the K_m for cytochrome c reduction was 15 ± 2 μM. The purified NADPH–cytochrome P450 reductase was able to support function of CYP79B2.     

Human prostatic steroid 5α-reductase isoforms—A comparative study of selective inhibitors

The present study describes the independent expression of the type 1 and 2 isoforms of human 5α-reductase in the baculovirus-directed insect cell expression system and the selectivity of their inhibition. The catalytic properties and kinetic parameters of the recombinant isozymes were consistent with published data. The type 1 isoform displayed a neutral (range 6–8) pH optimum and the type 2 isoform an acidic (5–6) pH optimum. The type 2 isoform had higher affinity for testosterone than did the type 1 isoform (K_m = 0.5 and 2.9 μM, respectively). Finasteride and turosteride were selective inhibitors of the type 2 isoform (K_i (type 2) = 7.3 and 21.7 nM compared to K_i (type 1) = 108 and 330 nM, respectively). 4-MA and the lipido-sterol extract of Serenoa repens (LSESr) markedly inhibited both isozymes (K_i (type 1) = 8.4 nM and 7.2 μg/ml, respectively; K_i (type 2) = 7.4 nM and 4.9 μg/ml, respectively). The three azasteroids were competitive inhibitors vs substrate, whereas LSESr displayed non-competitive inhibition of the type 1 isozyme and uncompetitive inhibition of the type 2 isozyme. These observations suggest that the lipid component of LSESr might be responsible for its inhibitory effect by modulating the membrane environment of 5α-reductase. Partially purified recombinant 5α-reductase type 1 activity was preserved by the presence of lipids indicating that lipids can exert either stimulatory or inhibitory effects on human 5α-reductase.     

Resistance to Imidazoles and Triazoles in Saccharomyces cerevisiae as a New Dominant Marker

The imidazole and triazole fungicides inhibit cytochrome P450 14α-lanosterol demethylase (P450_14DM) implicated in the ergosterol biosynthesis pathway, which is specific to fungi and yeasts. Two plasmids were obtained which allow triazole and imidazole resistance in Saccharomyces cerevisiae . The low copy number plasmid (pFD 383) encodes cytochrome P450 C14α lanosterol demethylase under the control of phospho-glycerate-kinase promoter. S. cerevisiae transformed by the pFD 383 plasmid are resistant to imidazoles and triazoles. Moreover, this transformed strain shows increased levels of P450_14DM mRNA and of cytochrome P450. A second low copy number plasmid (pFD 384) carries a mutant cytochrome P450 14α lanosterol demethylase gene, which increases imidazole and triazole resistance. These constructions can be used on a dominant selection marker to transform wild-type yeasts and to confer imidazole and triazole resistance in industrial fermentation.     

Novel aromatase and 5α-reductase inhibitors

Inhibitors of aromatase and 5α-reductase may be of use for the therapy of postmenopausal breast cancer and benign prostatic hyperplasia, respectively. FCE 27993 is a novel steroidal irreversible aromatase inhibitor structurally related to exemestane (FCE 24304). The compound was found to be a very potent competitive inhibitor of human placental aromatase, with a K_i of 7.2 nM (4.3 nM for exemestane). In preincubation studies with placental aromatase FCE 27993, like exemestane, was found to cause time-dependent inhibition with a higher rate of inactivation ( ) and a similar K_i(inact) (56 vs 66 nM). The compound was found to have a very low binding affinity to the androgen receptor (RBA 0.09% of dihydrotestosterone) and, in contrast to exemestane, no androgenic activity up to 100 mg/kg/day s.c. in immature castrated rats. Among a series of novel 4-azasteroids with fluoro-substituted-17β-amidic side chains, three compounds, namely FCE 28260, FCE 28175 and FCE 27837, were identified as potent in vitro and in vivo inhibitors of prostatic 5α-reductase. Their IC₅₀ values were found to be 16, 38 and 51 nM for the inhibition of the human enzyme, and 15, 20 and 60 nM for the inhibition of the rat enzyme, respectively. When given orally for 7 days in castrated and testosterone (Silastic implants) supplemented rats, the new compounds were very effective in reducing prostate growth. At a dose of 0.3 mg/kg/day inhibitions of 42, 36 and 41% were caused by FCE 28260, FCE 28175 and FCE 27837, respectively.     

Purification and characterization of liver microsomal cytochrome P-450 from untreated male rats

Different forms of cytochrome P-450 from untreated male rats were simultaneously purified to homogeneity using the HPLC technique. The absorption maximum, molecular weight, NH₂-terminal sequence and catalytic activity of them were determined. The NH₂-terminal sequences of six forms of cytochrome P-450 (designated P450 UT-1, UT-2, UT-4, UT-5, UT-7 and UT-8) indicate that these cytochrome P-450 isozymes are of different molecular species. The hydrophobicity values of the NH₂-terminal sequences of P450 UT-1 and P450 UT-8 were lower than that of other forms. P450 UT-8 has the highest molecular weight, 54 000, of the six forms of P-450. P450 UT-2 was active in demethylation of benzphetmaine, 450 UT-4 was active in the metabolism of 7-ethoxycoumarin and p-nitroanisole. P450 UT-1 ad P450 UT-2 were active in the 2α- and 16α-hydroxylation of testosterone, whereas P450 UT-4 was active in the 6β-, 7α- and 15α-hydroxylation of the same steroid. We believe that P450 UT-1, P450 UT-7 and P450 UT-8 are as yet unrecognized forms of cytochrome P-450.     

Novel in vitro and in vivo inhibitors of prolyl endopeptidase

Inhibition of prolyl endopeptidase by Z-cyclohexyl prolinal and Z-indolinyl prolinal occurs with slow, tight binding inhibition and K_i values of 2 – 3 nM. In vivo enzyme inhibition is also observed with a half time for recovery of enzyme activity of 3 – 4 h.Inhibition of prolyl endopeptidase by Z-cyclohexyl prolinal and Z-indolinyl prolinal occurs with slow, tight binding inhibition and K_i values of 2 – 3 nM. In vivo enzyme inhibition is also observed with a half time for recovery of enzyme activity of 3 – 4 h.     

The Interaction of Synthetic Decapeptide SLTCLVKGFY with Human T Lymphocytes

The synthetic peptide SLTCLVKGFY, corresponding to the 364–373 amino acid sequence of the human IgG heavy chain (Immunorphin), was found to compete with [¹²⁵I] -endorphin for binding by high-affinity receptors on T lymphocytes isolated from the blood of healthy donors (K _i0.6 nM). The fragments 3–10, 4–10, 5–10, and 6–10 of Immunorphin also inhibited the binding (K _i2.2, 3.4, 8.0, and 15 nM, respectively). Specificity of these receptors was studied: they turned out to be insensitive to naloxone and [Met]enkephaline and, therefore, are not opioid. The K _dvalues of the specific binding of ¹²⁵I-labeled Immunorphin and its 6–10 fragment to the receptor were found to be 7.4 and 36.3 nM, respectively.     

Palmitic Acid Analogs Exhibit Nanomolar Binding Affinity for the HIV-1 CD4 Receptor and Nanomolar Inhibition of gp120-to-CD4 Fusion

Background

We recently reported that palmitic acid (PA) is a novel and efficient CD4 fusion inhibitor to HIV-1 entry and infection. In the present report, based on in silico modeling of the novel CD4 pocket that binds PA, we describe discovery of highly potent PA analogs with increased CD4 receptor binding affinities (K_d) and gp120-to-CD4 inhibition constants (K_i). The PA analogs were selected to satisfy Lipinski''s rule of drug-likeness, increased solubility, and to avoid potential cytotoxicity.

Principal Findings

PA analog 2-bromopalmitate (2-BP) was most efficacious with K_d ∼74 nM and K_i ∼122 nM, ascorbyl palmitate (6-AP) exhibited slightly higher K_d ∼140 nM and K_i ∼354 nM, and sucrose palmitate (SP) was least efficacious binding to CD4 with K_d ∼364 nM and inhibiting gp120-to-CD4 binding with K_i ∼1486 nM. Importantly, PA and its analogs specifically bound to the CD4 receptor with the one to one stoichiometry.

Significance

Considering observed differences between K_i and K_d values indicates clear and rational direction for improving inhibition efficacy to HIV-1 entry and infection. Taken together this report introduces a novel class of natural small molecules fusion inhibitors with nanomolar efficacy of CD4 receptor binding and inhibition of HIV-1 entry.     

1-Methoxy-, 1-deoxy-11-hydroxy- and 11-Hydroxy-1-methoxy-Δ-tetrahydrocannabinols: new selective ligands for the CB2 receptor

Three series of new cannabinoids were prepared and their affinities for the CB₁ and CB₂ cannabinoid recptors were determined. These are the 1-methoxy-3-(1′,1′-dimethylalkyl)-, 1-deoxy-11-hydroxy-3-(1′,1′-dimethylalkyl)- and 11-hydroxy-1-methoxy-3-(1′,1′-dimethylalkyl)-Δ⁸-tetrahydrocannabinols, which contain alkyl chains from dimethylethyl to dimethylheptyl appended to C-3 of the cannabinoid. All of these compounds have greater affinity for the CB₂ receptor than for the CB₁ receptor, however only 1-methoxy-3-(1′,1′-dimethylhexyl)-Δ⁸-THC (JWH-229, 6e) has effectively no affinity for the CB₁ receptor (K_i=3134±110 nM) and high affinity for CB₂ (K_i=18±2 nM).     

Selective κ Opioid Antagonists nor-BNI,GNTI and JDTic Have Low Affinities for Non-Opioid Receptors and Transporters

Background

Nor-BNI, GNTI and JDTic induce selective κ opioid antagonism that is delayed and extremely prolonged, but some other effects are of rapid onset and brief duration. The transient effects of these compounds differ, suggesting that some of them may be mediated by other targets.

Results

In binding assays, the three antagonists showed no detectable affinity (K _i≥10 µM) for most non-opioid receptors and transporters (26 of 43 tested). There was no non-opioid target for which all three compounds shared detectable affinity, or for which any two shared sub-micromolar affinity. All three compounds showed low nanomolar affinity for κ opioid receptors, with moderate selectivity over μ and δ (3 to 44-fold). Nor-BNI bound weakly to the α_2C-adrenoceptor (K _i = 630 nM). GNTI enhanced calcium mobilization by noradrenaline at the α_1A-adrenoceptor (EC₅₀ = 41 nM), but did not activate the receptor, displace radioligands, or enhance PI hydrolysis. This suggests that it is a functionally-selective allosteric enhancer. GNTI was also a weak M₁ receptor antagonist (K _B = 3.7 µM). JDTic bound to the noradrenaline transporter (K _i = 54 nM), but only weakly inhibited transport (IC₅₀ = 1.1 µM). JDTic also bound to the opioid-like receptor NOP (K _i = 12 nM), but gave little antagonism even at 30 µM. All three compounds exhibited rapid permeation and active efflux across Caco-2 cell monolayers.

Conclusions

Across 43 non-opioid CNS targets, only GNTI exhibited a potent functional effect (allosteric enhancement of α_1A-adrenoceptors). This may contribute to GNTI''s severe transient effects. Plasma concentrations of nor-BNI and GNTI may be high enough to affect some peripheral non-opioid targets. Nonetheless, κ opioid antagonism persists for weeks or months after these transient effects dissipate. With an adequate pre-administration interval, our results therefore strengthen the evidence that nor-BNI, GNTI and JDTic are highly selective κ opioid antagonists.     

Purification and characterization of a low molecular weight cysteine proteinase inhibitor from bovine muscle

A low-M_r tight binding proteinase inhibitor was purified from bovine muscle by alkaline denaturation of cysteine proteinases, gel filtration on Sexphadex G-75 and affinity chromatography on carboxymethyl-papain-Sepharose. Chromatofocusing separated three isoforms which are similar in their M_r of about 14 000, their stability with heating at 80°C and their inhibitory activity towards cathepsin H, cathepsin B and papain. The equilibrium constants (K_i) were determined for these three cysteine proteinases but for cathepsin H, association (k_ass) and dissociation (k_diss) rate constants were also evaluated. K_i values of 56 nM and 8.4 nM were found for cathepsin B and cathepsin H, respectively. For papain, K_i was in the range of 0.1–1 nM. The kinetic features of enzyme-inhibitor binding suggest a possible role for this low-M_r protein inhibitor in controlling ‘in vivo’ cathepsin H proteolytic activity. With regard to cathepsin B, such a physiological role was less evident.     

Purification of Cytochrome P450 and Ferredoxin, Involved in Bisphenol A Degradation, from Sphingomonas sp. Strain AO1

In a previous study (M. Sasaki, J. Maki, K. Oshiman, Y. Matsumura, and T. Tsuchido, Biodegradation 16:449-459, 2005), the cytochrome P450 monooxygenase system was shown to be involved in bisphenol A (BPA) degradation by Sphingomonas sp. strain AO1. In the present investigation, we purified the components of this monooxygenase, cytochrome P450 (P450_bisd), ferredoxin (Fd_bisd), and ferredoxin reductase (Red_bisd). We demonstrated that P450_bisd and Fd_bisd are homodimeric proteins with molecular masses of 102.3 and 19.1 kDa, respectively, by gel filtration chromatography analysis. Spectroscopic analysis of Fd_bisd revealed the presence of a putidaredoxin-type [2Fe-2S] cluster. P450_bisd, in the presence of Fd_bisd, Red_bisd, and NADH, was able to convert BPA. The K_m and k_cat values for BPA degradation were 85 ± 4.7 μM and 3.9 ± 0.04 min⁻¹, respectively. NADPH, spinach ferredoxin, and spinach ferredoxin reductase resulted in weak monooxygenase activity. These results indicated that the electron transport system of P450_bisd might exhibit strict specificity. Two BPA degradation products of the P450_bisd system were detected by high-performance liquid chromatography analysis and were thought to be 1,2-bis(4-hydroxyphenyl)-2-propanol and 2,2-bis(4-hydroxyphenyl)-1-propanol based on mass spectrometry-mass spectrometry analysis. This is the first report demonstrating that the cytochrome P450 monooxygenase system in bacteria is involved in BPA degradation.     

Isolation and biosynthesis of 18-hydroxyprostaglandins E1 and E2 in human seminal fluid

18-Hydroxy-PGE₁ and 18-hydroxy-PGE₂ were identified in human seminal fluid by capillary gas-liquid chromatography-mass spectrometry. The levels of these prostaglandins was 1–2% of the corresponding 19-hydroxy-PGE compounds in human semen. 18Hydroxy-PGE₁ and 18-hydroxy-PGE₂ are likely formed by cytochrome P-450 in seminal vesicles in analogy with the 19-hydroxy-PGE compounds. This was supported by the finding that microsome of seminal vesicles of the cynomolgus monkey, Macaca fascicularis, supplemented with 1 nM NADPH, metabolized PGE₁ to both 19-hydroxy-PGE₁ (92%) and 18-hydroxy-PGE₁ (8%). The hydroxylation of prostaglandins in seminal vesicles of primates may thus show a high but not absolute specificity for the penultimate carbon of prostaglandins.     

Estrogen interaction with the anterior pituitary of female rats differential cytosol binding, nuclear translocation and stimulation of RNA synthesis by 17β-estradiol and tamoxifen

The interaction of tamoxifen (trans-1-(p-β-dimethylaminoethoxyphenyl)-1,2-diphenylbut-1-ene) with the cytosol estrogen receptor of the anterior pituitary of female rats was studied. No differences were recorded between incubations of cytosol samples with 17β-[³H]estradiol performed in the presence or absence of unlabeled 17β-estradiol and tamoxifen, respectively, thus suggesting that these interactions were at common receptor sites and excluding possible cooperative interactions. Competition experiments and Scatchard plot analysis of saturation experiments add further evidence for common receptor sites. A dissociation constant for tamoxifen of K_d = 2 nM was recorded. Tamoxifen was found to be bound to a moiety sedimenting in the 4–5 S region, on a 6–24% linear sucrose density gradient at low salt concentrations, whereas 17β-estradiol sedimented in the 8–9 S area. These data suggest possible conformational changes of the receptor in the presence of tamoxifen. Furthermore, nuclear estrogen receptor levels remained elevated for at least 80 h after the application of tamoxifen alone or in a combination with 17β-estradiol, and a concomitant inhibition of cytosol receptor replenishment was noted. Tamoxifen and 17β-estradiol, respectively, were found to stimulate progesterone receptor levels when applied through 5 days. Tamoxifen plus 17β-estradiol administration elevated progesterone receptor contents above those found for each of the two compounds alone. On the other hand, tamoxifen enhanced the 17β-estradiol-induced prolactin serum levels, but did not stimulate prolactin serum levels by itself. These data combine to suggest that tamoxifen interacts with common estrogen receptor sites at the rat anterior pituitary.     

Interaction of purified microsomal cytochrome P-450 with cytochrome b5

The interactions between purified microsomal cytochrome P-450 and cytochrome b₅ has been demonstrated by aqueous two-phase partition technique. Major forms of cytochrome P-450 induced by phenobarbital (P-450_LM2) and β-naphthoflavone (P-450_LM4) are almost exclusively distributed in the dextran-rich bottom phase (partition coefficient, K = 0.06), whereas NADPH-cytochrome P-450 reductase and cytochrome b₅ are mainly distributed in the polyethylene glycol-rich top phase (K = 3.5 and 2.5, respectively), when these enzymes were partitioned separately in the dextran-polyethylene glycol two-phase system. The mixing of P-450_LM with cytochrome b₅ changes the partition coefficients of both P-450_LM and cytochrome b₅ indicating that molecular interaction between P-450_LM and cytochrome b₅ occurred. Complex formation was also confirmed by optical absorbance difference spectral titration, and the stimulation of the P-450_LM-dependent 7-ethoxycoumarin and p-nitrophenetole O-deethylase activities by equal molar quantity of detergent-solubilized cytochrome b₅, but not trypsin-solubilized enzyme, in the reconstituted system. Cytochrome b₅ decreases the K_m's of both substrates for P-450_LM2-dependent O-deethylations and increases the V's of both reactions by two- to three-fold. This stimulatory effect requires the presence of phospholipid in the reconstituted enzyme system. These results suggest that cytochrome b₅ plays a role in some reconstituted drug oxidation enzyme systems and that molecular interactions among cytochrome P-450, reductase, and cytochrome b₅ are catalytically competent in the electron transport reactions.     

Discovery of potent carbonic anhydrase,acetylcholinesterase, and butyrylcholinesterase enzymes inhibitors: The new amides and thiazolidine‐4‐ones synthesized on an acetophenone base

Compounds containing nitrogen and sulfur atoms can be widely used in various fields, including industry, medicine, biotechnology, and chemical technology. Among them, amides of acids and heterocyclic compounds have an important place. These amides and thiazolidine‐4‐ones showed good inhibitory action against butyrylcholinesterase (BChE), acetylcholinesterase (AChE), and human carbonic anhydrase isoforms. AChE exists at high concentrations in the brain and red blood cells. BChE is an important enzyme that is plentiful in the liver, and it is released into the blood in a soluble form. They were demonstrated to have effective inhibition profiles with K_i values of 23.76–102.75 nM against hCA I, 58.92–136.64 nM against hCA II, 1.40–12.86 nM against AChE, and 9.82–52.77 nM against BChE. On the other hand, acetazolamide showed K_i value of 482.63 ± 56.20 nM against hCA I, and 1019.60 ± 163.70 nM against hCA II. Additionally, Tacrine inhibited AChE and BChE, showing K_i values of 397.03 ± 31.66 and 210.21 ± 15.98 nM, respectively.     

Purification of hepatic microsomal cytochromes P-450 from β-naphthoflavone-treated Atlantic cod (Gadus morhua), a marine teleost fish

Four isozymes of cytochrome P-450 were purified to varying degrees of homogeneity from liver microsomes of cod, a marine teleost fish. The cod were treated with β-naphthoflavone by intraperitoneal injection, and liver microsomes were prepared by calcium aggregation. After solubilization of cytochromes P-450 with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propansulfonate, chromatography on Phenyl-Sepharose CL-4B, and subsequently on DEAE-Sepharose, resulted in two cytochrome P-450 fractions. These were further resolved on hydroxyapatite into a total of four fractions containing different isozymes of cytochromes P-450. One fraction, designated cod cytochrome P-450c, was electrophoretically homogeneous, was recovered in the highest yield and constituted the major form of the isozymes. The relative molecular mass of this form (58 000) corresponds well with a protein band appearing in cod liver microsomes after treatment with β-naphthoflavone. Both cytochrome P-450c and a minor form called cytochrome P-450d (56 000) showed activity towards 7-ethoxyresorufin in a reconstituted system containing rat liver NADPH-cytochrome P-450 reductase and phospholipid. Differences between these two forms were observed in the rate and optimal pH for conversion of this substrate, and in optical properties. Rabbit antiserum to cod cytochrome P-450c did not show any cross-reactions with cod cytochrome P-450a (M_r 55 000) or cytochrome P-450d in Ouchterlony immunodiffusion, but gave a precipitin line of partial identify with cod cytochrome P-450b (M_r 54 000), possibly as a result of contaminating cytochrome P-450c in this fraction.