Analysis of catecholamines and related substances using porous graphitic carbon as separation media in liquid chromatography-tandem mass spectrometry

Capillary porous graphitic carbon (PGC) columns have been utilized for separation of several catecholamines and related compounds (i.e. L-tyrosine, L-DOPA, 3-O-methyl-DOPA, dopamine, 3,4-dihydroxy-phenyl-acetic acid (DOPAC), homovanillic acid, noradrenaline, vanillomandelic acid and adrenaline) on-line with electrospray ionization tandem mass spectrometry (ESI-MS/MS). The use of a mobile phase without ion-pairing agents and with high content of organic modifier facilitated the coupling to the selective and sensitive mass spectrometric detection. Minimum detectable sample concentration (MDC sample) for noradrenaline, dopamine and L-tyrosine in a standard solution was estimated to 3, 10 and 30 nM, respectively (3 S/N corresponds to MDQ for L-tyrosine of approximately 8 x 10(-14)mol). The developed strategy was applied for analysis of brain tissue, i.e. a substantia nigra (ns) sample.     

A novel method for analysis of nuclear receptor function at natural promoters: peroxisome proliferator-activated receptor gamma agonist actions on aP2 gene expression detected using branched DNA messenger RNA quantitation

Peroxisome proliferator-activated receptor-gamma (PPARgamma), a member of the nuclear hormone receptor superfamily, plays an essential role in the mediation of the actions of antidiabetic drugs known as thiazolidinediones (TZDs). PPARgamma activates many target genes involved in lipid anabolism including the adipocyte fatty acid binding protein (aP2). In this study, induction of aP2 gene expression by PPARgamma agonists was examined in both cultured cells and diabetic mice using branched DNA (bDNA)-mediated mRNA quantitation. bDNA technology allows for the direct measurement of a particular mRNA directly within cellular lysate using a 96-well plate format in a time frame comparable to a reporter gene assay. In cultured human subcutaneous preadipocytes, the TZDs, troglitazone and BRL-49653, both rapidly induced aP2 mRNA as detected with the bDNA method. In these cells, the effect of BRL-49653 on aP2 mRNA levels was detectable as early as 30 min after treatment (47% increase) and was maximal after 24 h of treatment (12-fold increase). The effects of troglitazone on aP2 mRNA induction were similar to those of BRL-49653 except that the maximal level of induction was consistently lower (e.g. 24 h treatment = 4-fold increase). Dose-response relationships for both of the TZDs were also determined using the 24-h treatment time point. EC50s for both BRL-49653 and troglitazone were estimated to be 80 nM and 690 nM, respectively. A natural PPARgamma ligand, 15-deoxy-delta12,14-PGJ2, was also active in this assay with a maximal induction of aP2 mRNA of approximately 5-fold when tested at 1 microM. Since the PPARgamma:retinoid X receptor (RXR) heterodimer has been characterized as a permissive heterodimer with respect to RXR ligands, the ability of 9-cis-retinoic acid (9-cis-RA) to induce aP2 mRNA was examined. Although 9-cis-RA had very low efficacy (2-fold induction), the maximal effect was reached at 100 nM. No synergism or additivity in aP2 mRNA induction was detected when 9-cis-RA was included with either of the TZDs used in this study. Significant induction of aP2 mRNA in bone marrow of db/db mice treated with either troglitazone or BRL-49653 was also detected, indicating that the bDNA assay may be a simple method to monitor nuclear receptor target gene induction in vivo.     

The importance of L-phenylalanine transport and its autocrine turnover to L-tyrosine for melanogenesis in human epidermal melanocytes.

A comparative study of (14)C-labeled l-phenylalanine and (3)H-labeled L-tyrosine uptake in cultures of human melanocytes (n = 8) and keratinocytes (n = 2) identified a significantly more rapid active transport for L-phenylalanine in melanocytes, whereas the slower uptake of L-tyrosine followed a concentration-dependent gradient, confirming facilitated diffusion rather than active transport. In addition, a significantly more efficient autocrine turnover of L-phenylalanine to L-tyrosine via intracellular phenylalanine hydroxylase was demonstrated in melanocytes. The incorporation of the newly synthesized radiolabeled L-tyrosine was directly followed into the end product melanin. The presence of L-phenylalanine in the culture medium produced 40% more melanin compared to an equivalent concentration of L-tyrosine alone. The transport of extracellular L-phenylalanine and its intracellular metabolism to L-tyrosine via intracellular phenylalanine hydroxylase are coupled to calcium uptake/efflux, whereas L-tyrosine uptake is calcium independent. Taken together, our results identified for the first time the importance of autocrine calcium-dependent active l-phenylalanine uptake/turnover in melanocytes as a major pathway for melanogenesis.     

Strategies for the labeling of halogen-substituted peroxisome proliferator-activated receptor gamma ligands: potential positron emission tomography and single photon emission computed tomography imaging agents

Well-known as an important regulator of lipid metabolism and adipocyte differentiation, the peroxisome proliferator-activated receptor gamma (PPARgamma) also has potential use as a target for antitumor therapy in certain cancers. To develop agents for radionuclide imaging PPARgamma in vivo, we synthesized fluorine, bromine, and iodine-substituted analogs (1-3) of a high-affinity benzophenone-tyrosine PPARgamma ligand; all three analogs retain very high affinity for the PPARgamma receptor. In preparation for the synthesis of these PPARgamma ligands in radiolabeled form, we have synthesized two types of precursors: (a) an aryltributylstannane (9), from which the bromine and iodine-substituted analogs (2 and 3) can readily be prepared by electrophilic destannylation, and (b) three diaryliodonium tosylate derivatives (12a-c), precursors for nucleophilic aromatic fluorination using fluoride ion. Conditions were developed whereby the thiophenyliodonium tosylate (12c) underwent nucleophilic aromatic substitution with fluoride ion, efficiently and in short reaction times, to produce the desired fluorine-substituted target compound 1. These reactions laid the groundwork for producing these three PPARgamma ligands in radiolabeled form; in addition, our use of diaryliodonium ion precursors for aromatic fluorination in this series provides an example that should encourage application of this approach for radiofluorination of more complicated radiopharmaceuticals.     

Modular engineering of L-tyrosine production in Escherichia coli

Efficient biosynthesis of L-tyrosine from glucose is necessary to make biological production economically viable. To this end, we designed and constructed a modular biosynthetic pathway for L-tyrosine production in E. coli MG1655 by encoding the enzymes for converting erythrose-4-phosphate (E4P) and phosphoenolpyruvate (PEP) to L-tyrosine on two plasmids. Rational engineering to improve L-tyrosine production and to identify pathway bottlenecks was directed by targeted proteomics and metabolite profiling. The bottlenecks in the pathway were relieved by modifications in plasmid copy numbers, promoter strength, gene codon usage, and the placement of genes in operons. One major bottleneck was due to the bifunctional activities of quinate/shikimate dehydrogenase (YdiB), which caused accumulation of the intermediates dehydroquinate (DHQ) and dehydroshikimate (DHS) and the side product quinate; this bottleneck was relieved by replacing YdiB with its paralog AroE, resulting in the production of over 700 mg/liter of shikimate. Another bottleneck in shikimate production, due to low expression of the dehydroquinate synthase (AroB), was alleviated by optimizing the first 15 codons of the gene. Shikimate conversion to L-tyrosine was improved by replacing the shikimate kinase AroK with its isozyme, AroL, which effectively consumed all intermediates formed in the first half of the pathway. Guided by the protein and metabolite measurements, the best producer, consisting of two medium-copy-number, dual-operon plasmids, was optimized to produce >2 g/liter L-tyrosine at 80% of the theoretical yield. This work demonstrates the utility of targeted proteomics and metabolite profiling in pathway construction and optimization, which should be applicable to other metabolic pathways.     

Synthesis and Biological Activity of Some Bromophenols and Their Derivatives Including Natural Products

In addition to the first synthesis of the natural bromophenol butyl 2-(3,5-dibromo-4-hydroxyphenyl)acetate ( 1 ), indene derivatives 34 and 35 were synthesized from 3-phenylpropenal derivatives in BBr₃ medium. Five known natural bromophenols and some derivatives were synthesized by known methods. Cholinesterase (ChEs) inhibitors reduce the breakdown of acetylcholine and are used in the treatment of Alzheimer's disease (AD) and dementia symptoms. The inhibition effects of all obtained compounds were examined towards acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and α-glycosidase enzymes. All synthesized compounds demonstrated the strong inhibition effects against both cholinergic enzymes. For determination of Ki values of novel bromophenols Lineweaver-Burk graphs were obtained. Ki values were found in the ranging of 0.13–14.74 nM for AChE, 5.11–23.95 nM for BChE, and 63.96–206.78 nM for α-glycosidase, respectively. All bromophenols and their derivatives exhibit effective inhibition profile when compared to positive controls.     

Biosynthesis of catecholamines in organotypic cultures of peripheral autonomic nervous system: modifications by biopterin and other agents.

Organotypic cultures of chick-embryo sympathetic ganglion chains maintained in vitro for 3-4 weeks rapidly synthesized catecholamines, as demonstrated by the conversion of L-[U-14C]tyrosine to catechol derivatives and by histofluorescence assay. The biosynthesis of catechols from radioactive L-tyrosine leveled off at 6 hr of incubation and dropped slightly at 10 hr. The addition of DL-alpha-methyl-p-tyrosine to the culture medium did not affect protein synthesis, but produced a complete block in the synthesis of catecholamines from L-tyrosine, with consequent loss of fluorescence in the bodies and proximal processes of adrenergic neurons in 2 hr, and essentially complete loss in 6 hr. Our observations suggest that a major portion of the catecholamines were synthesized in the perikarya and transported via neuronal processes to their terminals. The addition of monoamine oxidase inhibitors to the incubation medium produced a moderate to pronounced increase in fluorescence; reserpine caused a rapid and profound loss of catecholamines. When added to the culture medium, crude biopterin produced an increase in the synthesis of catechol derivatives from radioactive L-tyrosine and a marked increase in fluorescence, beginning in the neuronal perikarya. This effect was completely blocked by DL-alpha-methyl-p-tyrosine. The mechanism of biopterin's action in the synthesis of catecholamines in cultures of sympathetic ganglia is not completely elucidated from these studies, but may be related to the role it plays as cofactor for tyrosine hydrocylase.     

Identification of putative metabolites of docosahexaenoic acid as potent PPARgamma agonists and antidiabetic agents

We found that putative metabolites of docosahexaenoic acid (DHA) are strong PPARgamma activators and potential antidiabetic agents. We designed DHA derivatives based on the crystal structure of PPARgamma, synthesized them and evaluated their activities in vitro and in vivo. The efficacy of 5E-4-hydroxy-DHA 2a as a PPARgamma activator was about fourfold stronger than that of pioglitazone. Furthermore, the 4-keto derivative (10b) showed antidiabetic activity in animal models without producing undesirable effects such as obesity and hepatotoxicity.     

C17,20-lyase inhibitors I. Structure-based de novo design and SAR study of C17,20-lyase inhibitors

Novel nonsteroidal C(17,20)-lyase inhibitors were synthesized using de novo design based on its substrate, 17 alpha-hydroxypregnenolone, and several compounds exhibited potent C(17,20)-lyase inhibition. However, in vivo activities were found to be short-lasting, and in order to improve the duration of action, a series of benzothiophene derivatives were evaluated. As a result, compounds 9h, (S)-9i, and 9k with nanomolar enzyme inhibition (IC(50)=4-9 nM) and 9e (IC(50)=27 nM) were identified to have powerful in vivo efficacy with extended duration of action. The key structural determinants for the in vivo efficacy were demonstrated to be the 5-fluoro group on the benzothiophene ring and the 4-imidazolyl moiety. Superimposition of 9k and 17 alpha-hydroxypregnenolone demonstrated their structural similarity and enabled rationalization of the pharmacological results. In addition, selected compounds were also identified to be potent inhibitors of human enzyme with IC(50) values of 20-30 nM.     

Functionalized congeners of tyrosine-based P2X(7) receptor antagonists: probing multiple sites for linking and dimerization

Chemically funtionalized analogues of antagonists of the P2X(7) receptor, an ATP-gated cation channel, were synthesized as tools for biophysical studies of the receptor. These functionalized congeners were intended for use in chemical conjugation with retention of biological potency. The antagonists were L-tyrosine derivatives, related to [N-benzyloxycarbonyl-O-(4-arylsulfonyl)-L-tyrosyl]benzoylpiperazine (such as MRS2409, 2). The analogues were demonstrated to be antagonists in an assay of human P2X(7) receptor function, consisting of inhibition of ATP-induced K(+) efflux in HEK293 cells expressing the recombinant receptor. The analogues were of the general structure R(1)-Tyr(OR(2))-piperazinyl-R(3), in which three positions (R(1)-R(3)) were systematically varied in structure through introduction of chemically reactive groups. Each of the three positions was designed to incorporate a 3- or 4-nitrophenyl group. The nitro groups were reduced using NaBH(4)-copper(II) acetylacetonate to amines, which were either converted to the isothiocyanate groups, as potential affinity labels for the receptor, or acylated, as models for conjugation. An alternate route to N(alpha)-3-aminobenzyloxycarbonyl functionalization was devised. The various positions of functionalization were compared for effects on biological potency, and the R(2) and R(3) positions were found to be most amenable to derivatization with retention of high potency. Four dimeric permutations of the antagonists were synthesized by coupling each of the isothiocyanate derivatives to either the precursor amine or to other amine congeners. Only dimers linked at the R(2)-position were potent antagonists. In concentration-response studies, two derivatives, a 3-nitrobenzyloxycarbonyl derivative 18 and a 4-nitrotoluenesulfonate 26b, displayed IC(50) values of roughly 100 nM as antagonists of P2X(7) receptor-mediated K(+) flux.     

Pyrrolidinones as potent functional antagonists of the human melanocortin-4 receptor

A series of pyrrolidinones derived from phenylalaninepiperazines were synthesized and characterized as potent and selective antagonists of the melanocortin-4 receptor. In addition to their high binding affinities, these compounds displayed high functional potencies. 12a had a K(i) of 0.94 nM in binding and IC(50) of 21 nM in functional activity. 12a also demonstrated efficacy in a mouse cachexia model.     

Target genes of peroxisome proliferator-activated receptor gamma in colorectal cancer cells

Activation of the nuclear hormone peroxisome proliferator-activated receptor gamma (PPARgamma) inhibits cell growth and promotes differentiation in a broad spectrum of epithelial derived tumor cell lines. Here we utilized microarray technology to identify PPARgamma gene targets in intestinal epithelial cells. For each gene, the induction or repression was seen with two structurally distinct PPARgamma agonists, and the change in expression could be blocked by co-treatment with a specific PPARgamma antagonist. A majority of the genes could be regulated independently by a retinoid X receptor specific agonist. Genes implicated in lipid transport or storage (adipophilin and liver fatty acid-binding protein) were also activated by agonists of PPAR subtypes alpha and/or delta. In contrast, PPARgamma-selective targets included genes linked to growth regulatory pathways (regenerating gene IA), colon epithelial cell maturation (GOB-4 and keratin 20), and immune modulation (neutrophil-gelatinase-associated lipocalin). Additionally, three different genes of the carcinoembryonic antigen family were induced by PPARgamma. Cultured cells treated with PPARgamma ligands demonstrated an increase in Ca(2+)-independent, carcinoembryonic antigen-dependent homotypic aggregation, suggesting a potential role for PPARgamma in regulating intercellular adhesion. Collectively, these results will help define the mechanisms by which PPARgamma regulates intestinal epithelial cell biology.     

Effect of peroxisome proliferator activated receptor (PPAR)gamma agonists on prostaglandins cascade in joint cells

In response to inflammatory cytokines, chondrocytes and synovial fibroblasts produce high amounts of prostaglandins (PG) which self-perpetuate locally the inflammatory reaction. Prostaglandins act primarily through membrane receptors coupled to G proteins but also bind to nuclear Peroxisome Proliferator-Activated Receptors (PPARs). Amongst fatty acids, the cyclopentenone metabolite of PGD2, 15-deoxy-Delta12,14PGJ2 (15d-PGJ2), was shown to be a potent ligand of the PPARgamma isotype prone to inhibit the production of inflammatory mediators. As the stimulated synthesis of PGE2 originates from the preferential coupling of inducible enzymes, cyclooxygenase-2 (COX-2) and membrane PGE synthase-1 (mPGES-1), we investigated the potency of 15d-PGJ2 to regulate prostaglandins synthesis in rat chondrocytes stimulated with interleukin-1beta (IL-1beta). We demonstrated that 15d-PGJ2, but not the high-affinity PPARgamma ligand rosiglitazone, decreased almost completely PGE2 synthesis and mPGES-1 expression. The inhibitory potency of 15d-PGJ2 was unaffected by changes in PPARgamma expression and resulted from inhibition of NF-kappaB nuclear binding and IkappaBalpha sparing, secondary to reduced phosphorylation of IKKbeta. Consistently with 15d-PGJ2 being a putative endogenous regulator of the inflammatory reaction if synthesized in sufficient amounts, the present data confirm the variable PPARgamma-dependency of its effects in joint cells while underlining possible species and cell types specificities.     

Synthesis and biological activity of -tyrosine-based PPARγ agonists with reduced molecular weight

A series of PPARγ agonists were synthesized from -tyrosine that incorporated low molecular weight N-substituents. The most potent analogue, pyrrole (Scheme 1 and Scheme 2), demonstrated a K_i of 6.9 nM and an EC₅₀ of 4.7 nM in PPARγ binding and functional assays, respectively. Pyrrole (Scheme 1 and Scheme 2), which is readily synthesized from -tyrosine methyl ester in four steps, also demonstrated in vivo activity in a rodent model of Type 2 diabetes.     

Synthesis and structure-activity relationship of novel indene N-oxide derivatives as potent peroxisome proliferator activated receptor gamma (PPARgamma) agonists

A series of novel indene N-oxide derivatives were prepared by various synthetic methods and evaluated for their ability to activate PPARgamma. The best PPARgamma agonist in this series was 9h, which showed an EC(50) value of 15 nM.     

Synthesis and pharmacological evaluation of second-generation phosphatidic acid derivatives as lysophosphatidic acid receptor ligands

Short-chain phosphatidic acid derivatives, dioctanoyl glycerol pyrophosphate (DGPP 8:0, 1) and phosphatidic acid 8:0 (PA 8:0, 2), were previously identified as subtype-selective LPA(1) and LPA(3) receptor antagonists. Recently, we reported that the replacement of the phosphate headgroup by thiophosphate in a series of fatty alcohol phosphates (FAP) improves agonist as well as antagonist activities at LPA GPCR. Here, we report the synthesis of stereoisomers of PA 8:0 analogs and their biological evaluation at LPA GPCR, PPARgamma, and ATX. The results indicate that LPA receptors stereoselectively interact with glycerol backbone modified ligands. We observed entirely stereospecific responses by dioctyl PA 8:0 compounds, in which (R)-isomers were found to be agonists and (S)-isomers were antagonists of LPA GPCR. From this series, we identified compound 13b as the most potent LPA(3) receptor subtype-selective agonist (EC(50)=3 nM), and 8b as a potent and selective LPA(3) receptor antagonist (K(i)=5 nM) and inhibitor of ATX (IC(50)=600 nM). Serinediamide phosphate 19b was identified as an LPA(3) receptor specific antagonist with no effect on LPA(1), LPA(2), and PPARgamma.