Human Placental Enzyme Research Articles

New tamoxifen analogs for breast cancer therapy: synthesis, aromatase inhibition and in silico studies

A new class of tamoxifen analogues, using McMurry reaction conditions, is described. The scheme involved the conversion of ketoprofen (6) into amide derivatives 7 and 8, by coupling with N1,N1 -substituted propan-1,3-diamine derivatives in the presence DIC and HOB. Treatment of 7 and 8 with various ketones under McMurry reaction conditions afforded the tamoxifen analogues 9–16. All the analogues were screened in vitro for their aromatase inhibitory and antiproliferative activity against MCF-7 breast cancer cells. Compounds 10, 11 and 12 showed a potent activity against MCF-7 cell lines breast cancer with IC50 values of 0.070, 0.042 and 0.077 µM of selectivity index (SI) 3.0, 2.5 and 2.6, respectively. Further, 12 exhibited potent activity against estrogen receptor (14.7 ± 2.4 nM), while compound 10 was the most active analogues against aromatase with IC50 of (0.070 nM). Furthermore, all new compounds were docked into human placental aromatase enzyme and estrogen receptor and showed very good correlations with experimental IC50. Therefore, we can consider these designed compounds as starting scaffold to design an efficient drug against estrogen receptor and aromatase. Communicated by Ramaswamy H. Sarma

Synthesis, anticancer evaluation, and docking studies of some novel azo chromene derivatives

AbstractWe have described a simple, convenient, and high‐yielding one‐pot synthesis of novel azo chromene derivatives via a three‐component reaction of various azo aldehydes with dimedone and malononitrile using 10 mol% of 1,8‐diazabicyclo[5.4.0]undec‐7‐ene (DBU) as catalyst and ethanol as solvent at reflux condition. All the synthesized compounds have been characterized using Fourier‐transform infrared spectroscopy (FT‐IR), 1H NMR, 13C NMR, and HR‐MS spectra and molecular docking was performed to explore new inhibitors of human placental aromatase cytochrome P450 and cyclooxygenase‐2 enzymes. Of all the compounds docked, compound (E)‐2‐amino‐4‐(4,4‐dimethyl‐2,6‐dioxocyclohexyl)‐6‐((3‐methoxyphenyl)diazenyl‐4H‐chromene‐3‐carbonitrile (4o) showed good binding affinity with the active site of human placental aromatase cytochrome P450 enzyme (PDB: 3EQM) with inhibition constant (Ki) 1.66 nM and compound 4o also showed good binding affinity with the active site of cyclooxygenase‐2 enzyme (PDB: 6COX) with inhibition constant (Ki) 367.17 pM. In vitro anti‐cancer activity studies against MCF‐7 cells were also performed for compounds 4o, anastrozole and celecoxib. Compound 4o showed an effective cytotoxicity at 19.8 μg/ml compared to anastrozole and celecoxib (24.7 and 26.2 μg/ml).

Synthesis, Docking Studies and Biological Activity of New Benzimidazole- Triazolothiadiazine Derivatives as Aromatase Inhibitor.

In the last step of estrogen biosynthesis, aromatase enzyme catalyzes the conversion of androgens to estrogens. Aromatase inhibition is an important way to control estrogen-related diseases and estrogen levels. In this study, sixteen of benzimidazole-triazolothiadiazine derivatives have been synthesized and studied as potent aromatase inhibitors. First, these compounds were tested for their anti-cancer properties against human breast cancer cell line (MCF-7). The most active compounds 5c, 5e, 5k, and 5m on MCF-7 cell line were subject to further in vitro aromatase enzyme inhibition assays to determine the possible mechanisms of action underlying their activity. Compound 5e showed slight less potent aromatase inhibitory activity than that of letrozole with IC50 = 0.032 ± 0.042 µM, compared to IC50 = 0.024 ± 0.001 µM for letrozole. Furthermore, compound 5e and reference drug letrozole were docked into human placental aromatase enzyme to predict their possible binding modes with the enzyme. Finally, ADME parameters (absorption, distribution, metabolism, and excretion) of synthesized compounds (5a–5p) were calculated by QikProp 4.8 software.

Zearalenone metabolism in human placental subcellular organelles, JEG-3 cells, and recombinant CYP19A1.

Zearalenone (ZEN) and its derivative, zearalanone (ZAN), are endocrine disruptive mycotoxins produced by Fusarium species. We investigated the human placental metabolism of ZEN and ZAN invitro in JEG-3 cells, human term placental subcellular fractions and recombinant enzymes. Human placental enzymes were capable of metabolizing ZEN and ZAN to their primary OH-metabolites which have higher affinity for estrogen receptors than their parent compounds. These metabolites may interfere with physiological placental estrogen signaling and thus disrupt the progress of gestation.

ChemInform Abstract: Triazole Ring‐Opening Leads to the Discovery of Potent Nonsteroidal 17β‐Hydroxysteroid Dehydrogenase Type 2 Inhibitors.

AbstractA small library of novel non‐steroidal and non‐chiral 17β‐HSD2 inhibitors is designed, synthesized and evaluated in a cell‐free assay using human placental microsomal enzyme.

Extracellular purine and pyrimidine catabolism in cell culture

The presence of purines and pyrimidines bases, nucleosides, and nucleotides in the culture medium has shown to differently affect the growth of a Chinese hamster ovary (CHO) cell line producing the secreted form of the human placental alkaline phosphatase enzyme (SEAP; Carvalhal et al., Biotech Prog. 2003;19:69-83). CHO, BHK, as well as Sf9 cell growth was clearly reduced in the presence of purines but was not affected by pyrimidines at the concentrations tested. The knowledge about the mechanisms by which nucleotides exert their effect when present outside the cells remains very incomplete. The catabolism of both extracellular purines and pyrimidines was followed during the culture of CHO cells. Purines/pyrimidines nucleotides added at a concentration of 1 mM to the culture medium decreased to negligible concentrations in the first 2 days. Purine and pyrimidine catabolism originated only purinic and pyrimidic end-products, respectively. The comparison between AMP catabolism in serum-free cultures (CHO cells expressing Factor VII and Sf9 cells) and in cultures containing serum (CHO cells expressing SEAP and BHK cells expressing Factor VII) showed that AMP extracellular catabolism is mediated by both cells and enzymes present in the serum. This work shows that the quantification of purines and pyrimidines in the culture medium is essential in animal cell culture optimization. When using AMP addition as a chemical cell growth strategy for recombinant protein production improvement, AMP extracellular concentration monitoring allows the optimization of the multiple AMP addition strategy for a prolonged cell culture duration with high specific productivity.

523: Metabolism of 17-hydroxyprogesteronecaproate (17-P) by human placental mitochondrial and microsomal enzymes

523: Metabolism of 17-hydroxyprogesteronecaproate (17-P) by human placental mitochondrial and microsomal enzymes

Identification of the major human hepatic and placental enzymes responsible for the biotransformation of glyburide

One of the factors affecting the pharmacokinetics (PK) of a drug during pregnancy is the activity of hepatic and placental metabolizing enzymes. Recently, we reported on the biotransformation of glyburide by human hepatic and placental microsomes to six metabolites that are structurally identical between the two tissues. Two of the metabolites, 4- trans-(M1) and 3- cis-hydroxycyclohexyl glyburide (M2b), were previously identified in plasma and urine of patients treated with glyburide and are pharmacologically active. The aim of this investigation was to identify the major human hepatic and placental CYP450 isozymes responsible for the formation of each metabolite of glyburide. This was achieved by the use of chemical inhibitors selective for individual CYP isozymes and antibodies raised against them. The identification was confirmed by the kinetic constants for the biotransformation of glyburide by cDNA-expressed enzymes. The data revealed that the major hepatic isozymes responsible for the formation of each metabolite are as follows: CYP3A4 (ethylene-hydroxylated glyburide (M5), 3- trans-(M3) and 2- trans-(M4) cyclohexyl glyburide); CYP2C9 (M1, M2a (4- cis-) and M2b); CYP2C8 (M1 and M2b); and CYP2C19 (M2a). Human placental microsomal CYP19/aromatase was the major isozyme responsible for the biotransformation of glyburide to predominantly M5. The formation of significant amounts of M5 by CYP19 in the placenta could render this metabolite more accessible to the fetal circulation. The multiplicity of enzymes biotransforming glyburide and the metabolites formed underscores the potential for its drug interactions in vivo.

363: Identification of the major human hepatic and placental enzymes responsible for the metabolism of glyburide

A clinical trial to determine the pharmacokinetics of glyburide in treatment of gestational diabetes is underway. Previously, two metabolites of glyburide were identified namely, 4-trans-(M1) and 3-cis-(M2b) hydroxycyclohexyl glyburide. Recently, we reported on the identification and kinetics for formation of five additional and identical metabolites by human hepatic and placental microsomes. However, the major product/metabolite formed was different: M1 by hepatic and M5 (ethyl-hydroxy glyburide) by placental microsomes.

Placental Expression and Molecular Characterization of Aromatase Cytochrome P450 in the Spotted Hyena (Crocuta crocuta)

At birth, the external genitalia of female spotted hyenas (Crocuta crocuta) are the most masculinized of any known mammal, but are still sexually differentiated. Placental aromatase cytochrome P450 (P450arom) is an important route of androgen metabolism protecting human female fetuses from virilization in utero. Therefore, placental P450arom expression was examined in spotted hyenas to determine levels during genital differentiation, and to compare molecular characteristics between the hyena and human placental enzymes. Hyena placental P450arom activity was determined at gestational days (GD) 31, 35, 45, 65 and 95 (term, 110), and the relative sensitivity of hyena and human placental enzyme to inhibition by the specific inhibitor, Letrozole, was also examined. Expression of hyena P450arom in placenta was localized by immuno-histochemistry, and a full-length cDNA was cloned for phylogenetic analysis. Aromatase activity increased from GD31 to a peak at 45 and 65, apparently decreasing later in gestation. This activity was more sensitive to inhibition by Letrozole than was human placental aromatase activity. Expression of P450arom was localized to syncytiotrophoblast and giant cells of mid-gestation placentas. The coding sequence of hyena P450arom was 94% and 86% identical to the canine and human enzymes respectively, as reflected by phylogenetic analyses. These data demonstrate for the first time that hyena placental aromatase activity is comparable to that of human placentas when genital differentiation is in progress. This suggests that even in female spotted hyenas clitoral differentiation is likely protected from virilization by placental androgen metabolism. Decreased placental aromatase activity in late gestation may be equally important in allowing androgen to program behaviors at birth. Although hyena P450arom is closely related to the canine enzyme, both placental anatomy and P450arom expression differ. Other hyaenids and carnivores must be investigated to determine the morphological and functional ancestral state of their placentas, as it relates to evolutionary relationships among species in this important taxonomic group.

Designing Protein Therapies: Lessons Learned from Cerezyme® for Gaucher Disease

The development of carbohydrate-modified human glucocerebrosidase (Ceredase® and Cerezyme® [Genzyme Corporation, Cambridge, Massachusetts]) for the treatment of Gaucher disease has transformed the lives of patients and led to the development of enzyme replacement therapies (ERTs) for many other lysosomal storage disorders (LSDs). Gaucher disease and the use of Cerezyme as an ERT serve as a useful model for the design of protein and other therapies for the treatment of LSDs. Like other LSDs, Gaucher disease is a progressive and lethal metabolic disorder that results in irreversible endorgan damage when untreated. Early intervention is key to preventing irreversible complications. Several hundred different mutations have been identified that give rise to Gaucher disease,1 but there is still no reliable correlation between genotype and phenotype. Response to therapy is organ specific; that is, liver and spleen demonstrate a rapid response at a relatively low dose2 but a higher dose, earlier intervention, and long-term therapy are necessary to demonstrate benefit for the skeletal disease. Gaucher disease differs from other LSDs in the low percentage of patients that develop antibodies3; this reflects the fact that many patients produce residual, though inactive or partially active, protein. Cerezyme has an excellent safety and efficacy record, demonstrated over 15 years of clinical use.4 Nevertheless, we have evaluated alternative production methods in an attempt to improve mannose receptor targeting and enzyme delivery to target tissues. To evaluate the effect of increased amounts of terminal mannose residues on the uptake and biodistribution of human glucocerebrosidase, we produced human glucocerebrosidase with mannose chain lengths of 3 to 9 residues.5 Because Cerezyme differs from the human placental enzyme used in the production of Ceredase by 1 amino acid, we also evaluated the effect of that amino acid difference on uptake and biodistribution (even though no clinical difference has been observed between Cerezyme and Ceredase).6 Although terminal mannose is essential for efficient uptake into target cells, more mannose does not alter the uptake or biodistribution of human glucocerebrosidase. More mannose did, however, increase binding to the mannose-binding lectin in vitro, which could have negative consequences on clearance and uptake in vivo. The single amino acid difference between Cerezyme and placental glucocerebrosidase had no effect on half-life, uptake, or tissue targeting.5 In addition to engineering the carbohydrate residues on glucocerebrosidase, it is also possible to engineer the protein backbone to improve biological properties. Although the crystal structure of glucocerebrosidase has been determined,7 it is of little use for predicting how to improve the protein. It does not explain why most mutations cause disease or how the protein is activated by saposin C or phospholipids and is inconsistent with biochemical data. This most likely reflects the fact that the crystal structure is just a static image of a dynamic molecule. The use of competitive inhibitors as chemical chaperones has recently been proposed as a therapeutic strategy.8 This is based on the hypothesis that mutations which cause LSDs result in misfolded proteins, which are degraded by the proteasome, an effect that is likely to be mutation specific. We have studied the effect of chemical chaperones on the uptake and stabilization of Cerezyme as well as recombinantly produced N370S mutant glucocerebrosidase.9 These studies demonstrated that the increased enzyme levels observed are due to lysosomal stabilization. Chaperones, which are competitive inhibitors, remain bound in the lysosome and inhibit enzyme activity, as also demonstrated by Steet et al.10 Because these competitive inhibitors tend to have the same pH optima as the natural substrate—that is, they bind better at low pH—there is likely to be a narrow (if any) therapeutic window. As our results demonstrate, the effects of chaperones on protein levels reported in the literature can be accounted for by reduced lysosomal degradation. Gaucher disease is the first and most effectively treated of the LSDs, and Cerezyme is the gold standard against which to measure future LSD therapies. Alternative manufacturing platforms are unlikely to offer any improvement over Cerezyme. Small molecule chaperones are at best likely to be limited to a few of the several hundred disease-causing mutations. More promising approaches for the future include the development of a secondgeneration substrate inhibition therapy with improved specificity and reduced adverse effects, as well as gene therapy for LSDs with central nervous system involvement. Clinical Therapeutics/Volume 29, Supplement C, 2007

Peroxisome Proliferator-Activated Receptor δ Suppresses 11β-Hydroxysteroid Dehydrogenase Type 2 Gene Expression in Human Placental Trophoblast Cells

Accumulating evidence suggests that the human placental enzyme 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) plays a key role in fetal development by controlling fetal exposure to maternal glucocorticoids. Recently, the nuclear peroxisome proliferator-activated receptor delta (PPAR delta) has been found to be the most abundantly expressed PPAR subtype in the human placenta, but its function in this organ is unknown. Given that PPAR delta-null mice exhibited placental defects and consequent intrauterine growth restriction, the present study was undertaken to examine the hypothesis that PPAR delta regulates human placental function in part by targeting 11beta-HSD2. Using cultured human trophoblast cells as a model system, we demonstrated that 1) the putative PPAR delta agonist carbaprostacyclin (cPGI2) reduced 11beta-HSD2 activity as well as 11beta-HSD2 expression at both protein and mRNA levels; 2) GW610742 (a selective PPAR delta agonist) mimicked the effect of cPGI2, whereas indomethacin (a known ligand for PPARalpha and PPAR gamma) had no effect; 3) the cPGI2-induced down-regulation of 11beta-HSD2 mRNA did not require de novo protein synthesis; 4) cPGI2 suppressed HSD11B2 promoter activity, but did not alter the half-life of 11beta-HSD2 mRNA; and 5) the inhibitory effect of cPGI2 on HSD11B2 promoter activity was abrogated in trophoblast cells cotransfected with a dominant negative PPAR delta mutant. Taken together, these findings suggest that activation of PPAR delta down-regulates HSD11B2 gene expression in human trophoblast cells, and that this effect is mediated primarily at the transcriptional level. Thus, the present study reveals 11beta-HSD2 as an additional target for PPAR delta and identifies a molecular mechanism by which this nuclear receptor may regulate human placental function.

Native human nitric oxide sensitive guanylyl cyclase: purification and characterization

The only published report of the purification of native human soluble guanylyl cyclase (sGC) used placenta as starting material. This enzyme preparation showed low fold-activation by NO and a maximal absorption of the prosthetic heme-group at 417 nm indicative of a prosthetic heme-group in a hexa-coordinate state. These data are in contrast to what has subsequently been found for the recombinant human enzymes. Apart from this placental enzyme preparation, a native functional human NO-sensitive sGC has not been successfully purified. The aim of the current study was to purify and characterize native human sGC from another source, to see whether the discrepancies between native and recombinant sGC seen for placenta are a general phenomenon. We chose human platelets as starting material since the properties of this enzyme are directly relevant for the development of innovative antiplatelet and antianginal drugs. Our results indicate that the native platelet enzyme exists as a highly NO-sensitive, heterodimeric enzyme with an α 1 and β 1 subunit. In contrast to the native human placental enzyme and in accordance with the human recombinant enzymes, the native human platelet enzyme contains a ferrous, penta-coordinate heme group. To our knowledge this is the first report of the successful purification and characterization of the native human nitric oxide sensitive α 1/β 1 isoform of sGC which is widely expressed in the cardiovascular system and is an important target of innovative drugs.

Final report on the safety assessment of human placental protein, hydrolyzed human placental protein, human placental enzymes, human placental lipids, human umbilical extract, placental protein, hydrolyzed placental protein, placental enzymes, placental lipids, and umbilical extract.

Various proteins, lipids, or other extracts from human or other animal placentas are described as cosmetic ingredients. Human Placental Protein comprises protein derived from human placentas. Placental Protein is derived from animal placentas. Similarly, Human Placental Lipids and Placental Lipids are the lipid fractions from the same source materials. Hydrolyzed Human Placental Protein and Hydrolyzed Placental Protein are produced from the respective protein extracts by acid, enzyme, or other hydrolysis methods. Human Placental Enzymes and Placental Enzymes are enzymes obtained by aqueous extraction of human or other animal placental material. Human Umbilical Extract and Umbilical Extract are unspecified extracts of material from human or other animal umbilical cords. Different materials called Human Placental Extracts and Placental Extracts, assumed to contain estrogenic hormones or other biologically active substances, are not recognized as cosmetic ingredients, even though the use of these ingredients in cosmetics have been reported to the Food and Drug Administration (FDA). Human-derived ingredients are prohibited from use under the provisions of the European Union cosmetics directive based on concerns about transmission of human spongiform encephalopathies and viral diseases, for example, human immunodeficiency virus (HIV). Umbilical Extract has precedent for unrestricted use in Japan, except for certain products. Most of these ingredients are described as hair-conditioning agents and miscellaneous skin-conditioning agents, although the umbilical extracts function as biological additives in cosmetics. Of the human-derived ingredients, only Human Placental Protein is currently reported to be used. Animal-derived placental proteins, hydrolyzed proteins, lipids, and enzymes were all currently reported to be used. No current uses of the umbilical extracts were reported. Most of the available data relates to placental derivatives that appear to have estrogenic or other biological activity. The one clinical study that appears to utilize proteinaceous material only reported no irritant reaction. Clearly, the available data are insufficient to support safety of these ingredients in cosmetics. The additional data needed include (1) skin sensitization at concentration of use; (2) gross pathology and histopathology in skin and other major organ systems associated with repeated exposures, and dermal reproductive and developmental toxicity data; (3) photosensitization; (4) one genotoxicity assay in a mammalian system; if positive, then a 2-year dermal carcinogenicity study using National Toxicology Program (NTP) methods may be needed; (5) ocular toxicity, if available. Any studies should be done on all ingredients unless chemical analysis data show similarity among ingredients. Because there is confusion and concern about the use of substances with estrogenic or other biological activity in cosmetic formulations, it was concluded that none of these ingredients used in cosmetics should deliver any metabolic/endocrine activity. In addition, any current use of these ingredients should be free of detectable pathogenic viruses or infectious agents.

Biography of Tor Norseth

Various proteins, lipids, or other extracts from human or other animal placentas are described as cosmetic ingredients. Human Placental Protein comprises protein derived from human placentas. Placental Protein is derived from animal placentas. Similarly, Human Placental Lipids and Placental Lipids are the lipid fractions from the same source materials. Hydrolyzed Human Placental Protein and Hydrolyzed Placental Protein are produced from the respective protein extracts by acid, enzyme, or other hydrolysis methods. Human Placental Enzymes and Placental Enzymes are enzymes obtained by aqueous extraction of human or other animal placental material. Human Umbilical Extract and Umbilical Extract are unspecified extracts of material from human or other animal umbilical cords. Different materials called Human Placental Extracts and Placental Extracts, assumed to contain estrogenic hormones or other biologically active substances, are not recognized as cosmetic ingredients, even though the use of these ingredients in cosmetics have been reported to the Food and Drug Administration (FDA). Human-derived ingredients are prohibited from use under the provisions of the European Union cosmetics directive based on concerns about transmission of human spongiform encephalopathies and viral diseases, for example, human immunodeficiency virus (HIV). Umbilical Extract has precedent for unrestricted use in Japan, except for certain products. Most of these ingredients are described as hair-conditioning agents and miscellaneous skin-conditioning agents, although the umbilical extracts function as biological additives in cosmetics. Of the human-derived ingredients, only Human Placental Protein is currently reported to be used. Animal-derived placental proteins, hydrolyzed proteins, lipids, and enzymes were all currently reported to be used. No current uses of the umbilical extracts were reported. Most of the available data relates to placental derivatives that appear to have estrogenic or other biological activity. The one clinical study that appears to utilize proteinaceous material only reported no irritant reaction. Clearly, the available data are insufficient to support safety of these ingredients in cosmetics. The additional data needed include (1) skin sensitization at concentration of use; (2) gross pathology and histopathology in skin and other major organ systems associated with repeated exposures, and dermal reproductive and developmental toxicity data; (3) photosensitization; (4) one genotoxicity assay in a mammalian system; if positive, then a 2-year dermal carcinogenicity study using National Toxicology Program (NTP) methods may be needed; (5) ocular toxicity, if available. Any studies should be done on all ingredients unless chemical analysis data show similarity among ingredients. Because there is confusion and concern about the use of substances with estrogenic or other biological activity in cosmetic formulations, it was concluded that none of these ingredients used in cosmetics should deliver any metabolic/endocrine activity. In addition, any current use of these ingredients should be free of detectable pathogenic viruses or infectious agents.

Some 3-(4-Aminophenyl)pyrrolidine-2,5-diones as All- trans -retinoic Acid Metabolising Enzyme Inhibitors (RAMBAs)

A series of (±) -3-(4-aminophenyl) pyrrolidin-2,5-diones substituted in the 1-, 3- or 1,3- position with an aryl or long chain alkyl function are weak inhibitors of the metabolism of all-trans retinoic acid (RA) by rat liver microsomes (68-75% inhibition) compared with ketoconazole (85%). Further studies with the 1-cyclohexyl analogue (1) (IC 50 = 98.8 μM, ketoconazole, 22.15 μM) showed that it was not stereoselective in its inhibition. (±) - (1) was not an inhibitor of pig brain microsomal enzyme (ketoconazole, IC 50 = 20.9 μM), had little effect on human liver microsomal enzyme (19.3%, ketoconazole, 81.6%) or human placental microsomal enzyme (9.8%, ketoconazole 73.9%) but was a weak inhibitor of human and rat skin homogenates (52.6% and IC 50 = 211.6 μM respectively; ketoconazole, 38.8% and 85.95 μM). In RA-induced cell cultures of human male genital fibroblasts and HaCat cells, (±) - (1) was a weak inhibitor (c. 53% at 200 μM) whereas ketoconazole showed high potency (c. 65% at 0.625 μM and 0.25 μM respectively). The nature of the induced target enzyme is discussed.

Overexpression of wild type and SeCys/Cys mutant of human thioredoxin reductase in E. coli: the role of selenocysteine in the catalytic activity

In contrast to Escherichia coli and yeast thioredoxin reductases, the human placental enzyme contains an additional redox center consisting of a cysteine-selenocysteine pair that precedes the C-terminal glycine residue. This reactive selenocysteine-containing center imbues the enzyme with its unusually wide substrate specificity. For expression of the human gene in E. coli, the sequence corresponding to the SECIS element required for selenocysteine insertion in E. coli formate dehydrogenase H was inserted downstream of the TGA codon in the human thioredoxin reductase gene. Omission of this SECIS element from another construct resulted in termination at UGA. Change of the TGA codon to TGT gave a mutant enzyme form in which selenocysteine was replaced with cysteine. The three gene products were purified using a standard isolation protocol. Binding properties of the three proteins to the affinity resins used for purification and to NADPH were similar. The three proteins occurred as dimers in the native state and exhibited characteristic thiolate-flavin charge transfer spectra upon reduction. With DTNB as substrate, compared to native rat liver thioredoxin reductase, catalytic activities were 16% for the recombinant wild type enzyme, about 5% for the cysteine mutant enzyme, and negligible for the truncated enzyme form.

Identification of a meander region proline residue critical for heme binding to cytochrome P450: implications for the catalytic function of human CYP4B1.

Alignment of xenobiotic-metabolizing P450 protein sequences highlights an invariant proline residue in the meander region two amino acids N-terminal to the distal arginine of the putative ERR triad thought to be important for heme binding. This occurs as a serine in the sequences derived from human CYP4B1 gDNA and both human lung and placental CYP4B1 cDNAs. Reversion of this serine to the conserved proline residue (Ser427 --> Pro) by site-directed mutagenesis conferred the ability to incorporate heme on the human placental enzyme. Mutation of the corresponding proline in rabbit CYP4B1 (Pro422 --> Ser) abolished heme incorporation. Membrane preparations of human CYP4B1(Pro) and rabbit CYP4B1(Pro), but not the corresponding CYP4B1(Ser) variants, supported lauric acid hydroxylation preferentially at the omega-position. Purified, reconstituted human CYP4B1(Pro) and rabbit CYP4B1(Pro) formed 12-hydroxylauric acid at rates of 17-21 min-1, and both enzymes were also C-8 to C-10 fatty acid omega-hydroxylases preferentially, with total rates of hydroxylation decreasing in the order C-12 > C-10 > C-9 > C-8. Finally, neither human nor rabbit CYP4B1(Pro) formed detectable levels of any hydroxylated testosterone metabolites. Therefore, the presence of a consensus Pro-X-Arg motif is critical for incorporation of the heme prosthetic group in human and rabbit CYP4B1 proteins expressed in insect cells. Native human CYP4B1, expressed in vivo, is likely to be functionally impaired if Pro427 is required for holoenzyme expression in mammalian cells.

Molecular Cloning and Expression of Rat Liver Aminopeptidase B

We isolated, by immunological screening of a Uni-ZAP XR cDNA library constructed from rat liver mRNAs, a cDNA clone with 2212 base pairs encoding aminopeptidase B (EC 3.4.11.6). The open reading frame encodes a 649-amino acid protein with a theoretical molecular mass of 72,545 Da and bears the consensus sequence of the zinc metalloexopeptidases, indicating that the enzyme belongs to this family, which includes aminopeptidase A, aminopeptidase N, and leukotriene-A4 hydrolase. Escherichia coli SOLR cells infected with the pBluescript phagemid excised from the Uni-ZAP XR vector containing the aminopeptidase B cDNA had a high L-arginyl-beta-naphthylamidase activity. The recombinant protein was purified to homogeneity from the recombinant E. coli extracts. The enzyme had Cl--dependent aminopeptidase activity specifically restricted to the Arg and Lys derivatives and contained 1 mol of zinc per mol of the enzyme.

Synthesis and biological evaluation of 3-(prop-2-enyl)- and 3-(prop-2-ynyl)pyrrolidine-2,5-dione derivatives as potential aromatase inhibitors.

3-(4'-Aminophenyl)pyrrolidine-2,5-dione (WSP3), a known reversible inhibitor of P450 aromatase, was modified using molecular graphics and our model of reversible inhibitor and substrate binding to resemble 10 beta-prop-2-ynylestr-4-ene-3,17-dione (PED), a mechanism-based inactivator of the enzyme. The analogues prepared were 3-substituted 3-(prop-2-enyl) or 3-(prop-2-ynyl) pyrrolidine-2,5-diones and their N-alkyl derivatives. The reported compounds demonstrated no irreversible (time-dependent) inhibition of the human placental P450 aromatase enzyme. However, some reversible activity was seen in several of the 3-(prop-2-ynyl) compounds.