Green tea polyphenols: novel and potent inhibitors of squalene epoxidase

The green tea gallocatechins, (-)-epigallocatechin-3-O-gallate (EGCG) (IC(50) = 0.69 microM), (-)-gallocatechin-3-O-gallate (GCG) (IC(50) = 0.67 microM), (-)-epicatechin-3-O-gallate (ECG) (IC(50) = 1.3 microM), and theasinensin A (IC(50) = 0.13 microM), were found to be potent and selective inhibitors of rat squalene epoxidase (SE), a rate-limiting enzyme of cholesterol biogenesis. On the other hand, flavan-3-ols without galloyl group at C-3 did not show significant enzyme inhibition. It was demonstrated for the first time that the cholesterol lowering effect of green tea may be attributed to their potent SE inhibition activities. Inhibition kinetics revealed that EGCG inhibited SE in noncompetitive (K(I) = 0.74 microM), and non-time-dependent manner. The potent enzyme inhibition would be caused by specific binding to the enzyme, and by scavenging reactive oxygen species required for the monooxygenase reaction.     

Green tea polyphenols as inhibitors of ribonuclease A

Ribonucleases (RNases), which are essential for cleavage of RNA, may be cytotoxic due to undesired cleavage of RNA in the cell. The quest for small molecule inhibitors of members of the ribonuclease superfamily has become indispensable with a growing number exhibiting unusual biological properties. Thus, inhibitors of RNases may serve as potential drug candidates. Green tea catechins (GTC), particularly its major constituent (-)-epigallocatechin-3-gallate (EGCG), have reported potential against cell proliferation and angiogenesis induced by several growth factors including angiogenin, a member of the RNase superfamily. This study reports the inhibition of bovine pancreatic ribonuclease A (RNase A) by EGCG and GTC. This has been checked qualitatively by an agarose gel based assay. Enzyme kinetic studies with cytidine 2',3' cyclic monophosphate as the substrate have also been conducted. Results indicate substantial inhibitory activity of a noncompetitive nature with an inhibition constant of approximately 80 microM for EGCG and approximately 100 microM for GTC measured in gallic acid equivalents.     

Green tea polyphenols for prostate cancer chemoprevention: A translational perspective

Every year nearly 200,000 men in the United States are diagnosed with prostate cancer (PCa), and another 29,000 men succumb to the disease. Within certain regions of the world population based studies have identified a possible role for green tea in the prevention of certain cancers, especially PCa. One constituent in particular, epigallocatechin-3-gallate also known as EGCG has been shown in cell culture models to decrease cell viability and promote apoptosis in multiple cancer cell lines including PCa with no effect on non-cancerous cell lines. In addition, animal models have consistently shown that standardized green tea polyphenols when administered in drinking water delay the development and progression of PCa. Altogether, three clinical trials have been performed in PCa patients and suggest that green tea may have a distinct role as a chemopreventive agent. This review will present the available data for standardized green tea polyphenols in regard to PCa chemoprevention that will include epidemiological, mechanism based studies, safety, pharmacokinetics, and applicable clinical trials. The data that has been collected so far suggests that green tea may be a promising agent for PCa chemoprevention and further clinical trials of participants at risk of PCa or early stage PCa are warranted.     

Synthetic analogs of green tea polyphenols as proteasome inhibitors

BACKGROUND: Animal, epidemiological and clinical studies have demonstrated the anti-tumor activity of pharmacological proteasome inhibitors and the cancer-preventive effects of green tea consumption. Previously, one of our laboratories reported that natural ester bond-containing green tea polyphenols (GTPs), such as (-)-epigallocatechin-3-gallate [(-)-EGCG] and (-)-gallocatechin-3-gallate [(-)-GCG], are potent and specific proteasome inhibitors. Another of our groups, for the first time, was able to enantioselectively synthesize (-)-EGCG as well as other analogs of this natural GTP. Our interest in designing and developing novel synthetic GTPs as proteasome inhibitors and potential cancer-preventive agents prompted our current study. MATERIALS AND METHODS: GTP analogs, (+)-EGCG, (+)-GCG, and a fully benzyl-protected (+)-EGCG [Bn-(+)-EGCG], were prepared by enantioselective synthesis. Inhibition of the proteasome or calpain (as a control) activities under cell-free conditions were measured by fluorogenic substrate assay. Inhibition of intact tumor cell proteasome activity was measured by accumulation of some proteasome target proteins (p27, I kappa B-alpha and Bax) using Western blot analysis. Inhibition of tumor cell proliferation and induction of apoptosis by synthetic GTPs were determined by G(1) arrest and caspase activation, respectively. Finally, inhibition of the transforming activity of human prostate cancer cells by synthetic GTPs was measured by a colony formation assay. RESULTS: (+)-EGCG and (+)-GCG potently and specifically inhibit the chymotrypsin-like activity of purified 20S proteasome and the 26S proteasome in tumor cell lysates, while Bn-(+)-EGCG does not. Treatment of leukemic Jurkat T or prostate cancer LNCaP cells with either (+)-EGCG or (+)-GCG accumulated p27 and IkappaB-alpha proteins, associated with an increased G(1) population. (+)-EGCG treatment also accumulated the pro-apoptotic Bax protein and induced apoptosis in LNCaP cells expressing high basal levels of Bax, but not prostate cancer DU-145 cells with low Bax expression. Finally, synthetic GTPs significantly inhibited colony formation by LNCaP cancer cells. CONCLUSIONS: Enantiomeric analogs of natural GTPs, (+)-EGCG and (+)-GCG, are able to potently and specifically inhibit the proteasome both, in vitro and in vivo, while protection of the hydroxyl groups on (+)-EGCG renders the compound completely inactive.     

Accurate assay of dopa decarboxylase by preventing nonenzymatic decarboxylation of dopa

The nonenzymatic decarboxylation of dopa was completely blocked by both 2-mercaptoethanol and EDTA together over the wide range of pH. This finding made it possible to measure the activity of dopa decarboxylase precisely even at an alkaline pH value. The pH optimum of dopa decarboxylase was found to be pH 7.0 and the Km value for dopa was determined to be 4 X 10(-5) M.     

Green tea polyphenols modulate insulin secretion by inhibiting glutamate dehydrogenase

Insulin secretion by pancreatic beta-cells is stimulated by glucose, amino acids, and other metabolic fuels. Glutamate dehydrogenase (GDH) has been shown to play a regulatory role in this process. The importance of GDH was underscored by features of hyperinsulinemia/hyperammonemia syndrome, where a dominant mutation causes the loss of inhibition by GTP and ATP. Here we report the effects of green tea polyphenols on GDH and insulin secretion. Of the four compounds tested, epigallocatechin gallate (EGCG) and epicatechin gallate were found to inhibit GDH with nanomolar ED(50) values and were therefore found to be as potent as the physiologically important inhibitor GTP. Furthermore, we have demonstrated that EGCG inhibits BCH-stimulated insulin secretion, a process that is mediated by GDH, under conditions where GDH is no longer inhibited by high energy metabolites. EGCG does not affect glucose-stimulated insulin secretion under high energy conditions where GDH is probably fully inhibited. We have further shown that these compounds act in an allosteric manner independent of their antioxidant activity and that the beta-cell stimulatory effects are directly correlated with glutamine oxidation. These results demonstrate that EGCG, much like the activator of GDH (BCH), can facilitate dissecting the complex regulation of insulin secretion by pharmacologically modulating the effects of GDH.     

Structure based design of novel irreversible FAAH inhibitors

Fatty acid amide hydrolase (FAAH) has attracted significant attention due to its promise as an analgesic target. This has resulted in the discovery of numerous chemical classes as inhibitors of this potential therapeutic target. In this paper we disclose a new series of novel FAAH irreversible azetidine urea inhibitors. In general these compounds illustrate potent activity against the rat FAAH enzyme. Our SAR studies allowed us to optimize this series resulting in the identification of compounds 13 which were potent inhibitors of both human and rat enzyme. This series of compounds illustrated good hydrolase selectivity along with good PK properties.     

Rational design of novel irreversible inhibitors for human arginase

Parasites have developed a variety of strategies for invading hosts and escaping their immune response. A common mechanism by which parasites escape nitric oxide (NO) toxicity is the activation of host arginase. This activation leads to a depletion of l-arginine, which is the substrate for NO synthase, resulting in lower levels of NO and increased production of polyamines that are necessary for parasite growth and differentiation. For this reason, small molecule inhibitors for arginase show promise as new anti-parasitic chemotherapeutics. However, few arginase inhibitors have been reported. Here, we describe the discovery of novel irreversible arginase inhibitors, and their characterization using biochemical, kinetic, and structural studies. Importantly, we determined the site on human arginase that is labeled by one of the small molecule inhibitors. The tandem mass spectra data show that the inhibitor occupies the enzyme active site and forms a covalent bond with Thr135 of arginase. These findings pave the way for the development of more potent and selective irreversible arginase inhibitors.     

The genetics of dopa decarboxylase in Drosophila melanogaster

Of 204 mutations located in the 8–12 band Df(2L)130 region, 37B9-C1,2;37D1-2, 199 have been assigned to twelve lethal genes and one visible gene (hook). The 13 genes are not evenly distributed. Twelve, (possibly all thirteen) are in the seven band region 37B10-C4 giving a gene-to-band ratio of almost two. Only one gene, 1(2)37Cf, may be in the four band region 37C5-7, and none are localized in band 37D1. In situ hybridization places the dopa decarboxylase structural gene, Ddc, in or very close to band 37C1,2 (Hirsh and Davidson, 1981). The methyl dopa hypersensitive gene, 1(2) amd, is 0.002 map units distal to Ddc. Df(2L)VA17, 37C1,2; 37F5-38A1 may actually break in the 37C1,2 singlet. It places six genes, hook, 1(2)amd, and four lethal genes, in a maximum of five bands, 37B10, 11, 12, 13 and perhaps part of the 37C1,2 singlet and localizes six genes, Ddc plus five lethal genes, in a maximum of three bands; probably part of the 37C1,2 singlet plus bands, C3, and C4. Wild type activity of five of twelve lethal genes is necessary for female fertility. — Band 37C5 puffs at the time of pupariation; Puff Stages 8–10. Twelve of eighteen alleles of 1(2)37Cf havs been examined as heterozygotes over CyO and none affect the appearance of a homozygous 37C5 puff. — Of the 204 mutations considered here only one Ddc ^p1, affects the function of more than one gene. It eliminates Ddc ⁺ and l(2) 37Ca ⁺ function and at 30 ° C reduces l(2)37Ce ⁺ function. It is not a deficiency but could be a polar mutant.Prof. Beermann's co-authors are very pleased to dedicate this paper to him in honor of his sixtieth birthday and in recognition of his seminal, most significant, extensive, and authoritive contributions on the functional organization of chromosomes     

A kinetic analysis of Drosophila melanogaster dopa decarboxylase

The kinetic mechanism of dopa decarboxylase (3,4-dihydroxy-L-phenylalanine carboxy-lyase, EC 4.1.1.28) was investigated in Drosophila melanogaster. Based on initial velocity and product inhibition studies, an ordered reaction is proposed for dopa decarboxylase. This kinetic mechanism is interpreted in the context of measured enzyme activities and the catecholamine pools in Drosophila. The 1(2)amd gene is immediately adjacent to the gene coding for dopa decarboxylase (Ddc) and determines hypersensitivity to alpha-methyldopa in Drosophila. Dopa decarboxylase does not decarboxylate alpha-methyldopa and hence does not generate a toxic product capable of inhibiting 1(2)amd gene function. We propose that the 1(2)amd gene is involved with an unknown catecholamine pathway involving dopa but not dopamine.     

Analysis of tea polyphenols

Tea is the most highly consumed beverage in the world, other than water. However, unlike water, tea contains substantial amounts of polyphenols that have unique biological activities and may be responsible for many of the health benefits of tea. As a result, it is essential to be able to measure the various tea-associated polyphenols. Total polyphenol content is currently measured by using methodology based on reducing activity. Several HPLC systems with detectors that, collectively, have wide ranges in sensitivity have been developed for analysis of individual flavonoids in tea and biological samples, and for theaflavins in tea. Catechins also have been measured in plasma by solid phase extraction, addition of a chromophore, and colorimetric quantification. Except for theaflavins in tea, routine and robust methods for the measurement of polyphenol condensation products (dimers and thearubigens) in tea and biological samples have not been developed. Although in vitro and animal studies suggest substantial metabolism of flavonoids in the gastrointestinal tract, only a single HPLC procedure has been assembled for monitoring the metabolic products of quercetin in urine of human subjects.     

Green tea polyphenols reduce body weight in rats by modulating obesity-related genes

Beneficial effects of green tea polyphenols (GTP) against obesity have been reported, however, the mechanism of this protection is not clear. Therefore, the objective of this study was to identify GTP-targeted genes in obesity using the high-fat-diet-induced obese rat model. A total of three groups (n?=?12/group) of Sprague Dawley (SD) female rats were tested, including the control group (rats fed with low-fat diet), the HF group (rats fed with high-fat diet), and the HF+GTP group (rats fed with high-fat diet and GTP in drinking water). The HF group increased body weight as compared to the control group. Supplementation of GTP in the drinking water in the HF+GTP group reduced body weight as compared to the HF group. RNA from liver samples was extracted for gene expression analysis. A total of eighty-four genes related to obesity were analyzed using PCR array. Compared to the rats in the control group, the rats in the HF group had the expression levels of 12 genes with significant changes, including 3 orexigenic genes (Agrp, Ghrl, and Nr3c1); 7 anorectic genes (Apoa4, Cntf, Ghr, IL-1β, Ins1, Lepr, and Sort); and 2 genes that relate to energy expenditure (Adcyap1r1 and Adrb1). Intriguingly, the HF+GTP group restored the expression levels of these genes in the high-fat-induced obese rats. The protein expression levels of IL-1β and IL-6 in the serum samples from the control, HF, and HF+GTP groups confirmed the results of gene expression. Furthermore, the protein expression levels of superoxide dismutase-1 (SOD1) and catechol-O-methyltransferase (COMT) also showed GTP-regulated protective changes in this obese rat model. Collectively, this study revealed the beneficial effects of GTP on body weight via regulating obesity-related genes, anti-inflammation, anti-oxidant capacity, and estrogen-related actions in high-fat-induced obese rats.     

Cloning and expression analysis of a novel tissue-specific dopa decarboxylase mRNA splicing variant in Bombyx mori

Dopa decarboxylase (DDC) protein is involved in the synthesis of dopamine and serotonin. Here, we show that in the silkworm Bombyx mori, a novel DDC splicing variant is selectively expressed in the brain and subesophageal ganglia. In Drosophila melanogaster, a neuron-specific isoform of DDC is known to be alternatively spliced in a similar manner.     

Hormonal regulation of dopa decarboxylase during a larval molt

Cuticular sclerotization in insects requires dopamine derivatives and thus the presence of dopa decarboxylase (DDC), the enzyme which converts dopa to dopamine. During the last half of the larval molt of the tobacco hornworm, Manduca sexta, beginning at 16 hr after head capsule slippage, the epidermal DDC activity increased fourfold. By contrast, allatectomized larvae which were destined to produce a melanized cuticle showed a sevenfold increase. This increase in DDC activity was prevented by infusion of 20-hydroxyecdysone (20HE) into the larva, indicating that the fall of the ecdysteroid titer is necessary for the increase. In vitro 20HE also prevented the increase in a dose-dependent manner when the epidermis was explanted at 16 hr after head capsule slippage but had less effect on epidermis explanted 3 hr later. Both 5 micrograms/ml alpha-amanitin and 100 micrograms/ml cycloheximide also prevented the increase. Application of juvenile hormone I showed that the critical period for determination of the level of the later increase in DDC activity was about 4 hr after head capsule slippage at the peak of the ecdysteroid titer. Apparently then the rise and fall of ecdysteroid regulate different aspects of DDC synthesis, the rise determining its later appearance and the fall timing this appearance.