Synthesis and cytogenetic studies of structure--biological activity relationship of esters of Hecogenin and aza-homo-Hecogenin with N,N-bis(2-chloroethyl)aminocinnamic acid isomers

The esters of Hecogenin and aza-homo-Hecogenin with N,N-bis(2-chloroethyl)aminocinnamic acid isomers have been prepared and their cytogenetic studies of structure-biological activity relationship were evaluated. The cytogenetic effects (sister chromatid exchanges (SCEs) induction and proliferation rate indices (PRIs) depression) by o-, m- and p-[N,N-bis(2-chloroethyl)amino] cinnamic acid were also investigated. Among the above compounds tested, those of the m-[N,N-bis(2-chloroethyl)amino] cinnamic acid and of the o-[N,N-bis(2-chloroethyl)amino] cinnamic acid ester of aza-homo-Hecogenin were more active in comparison to the others.     

Characterization and detection of lysine-arginine cross-links derived from dehydroascorbic acid

Covalently cross-linked proteins are among the major modifications caused by the advanced Maillard reaction. So far, the chemical nature of these aggregates is largely unknown. L-dehydroascorbic acid (DHA, 5), the oxidation product of L-ascorbic acid (vitamin C), is known as a potent glycation agent. Identification is reported for the lysine-arginine cross-links N6-[2-[(4-amino-4-carboxybutyl)amino]-5-(2-hydroxyethyl)-3,5-dihydro-4H-imidazol-4-ylidene]-L-lysine (9), N6-[2-[(4-amino-4-carboxybutyl)amino]-5-(1,2-dihydroxyethyl)-3,5-dihydro-4H-imidazol-4-ylidene]-L-lysine (11), and N6-[2-[(4-amino-4-carboxybutyl)amino]-5-[(1S,2S)-1,2,3-trihydroxypropyl]-3,5-dihydro-4H-imidazol-4-ylidene]-L-lysine (13). The formation pathways could be established starting from dehydroascorbic acid (5), the degradation products 1,3,4-trihydroxybutan-2-one (7, L-erythrulose), 3,4-dihydroxy-2-oxobutanal (10, L-threosone), and L-threo-pentos-2-ulose (12, L-xylosone) were proven as precursors of the lysine-arginine cross-links 9, 11, and 13. Products 9 and 11 were synthesized starting from DHA 5, compound N6-[2-[(4-amino-4-carboxybutyl)amino]-5-[(1S,2R)-1,2,3-trihydroxypropyl]-3,5-dihydro-4H-imidazol-4-ylidene]-L-lysine (16) via the precursor D-erythro-pentos-2-ulose (15). The present study revealed that the modification of lysine and arginine side chains by DHA 5 is a complex process and could involve a number of reactive carbonyl species.     

Chronic nicotine exposure selectively activates a carrier-mediated release of endogenous glutamate and aspartate from rat hippocampal synaptosomes

The effect of chronic nicotine treatment on the release of endogenous glutamate (GLU), aspartate (ASP) and GABA evoked in vitro by KCl, 4-aminopyridine (4AP) and nicotinic agonists in synaptosomes of rat hippocampus was investigated. Rats were chronically administered with nicotine bitartrate or saline vehicle each for 14 days using osmotic mini-pumps. Hippocampal synaptosomes were stimulated with KCl, 4AP, nicotine or with choline (Ch) and 5-iodo-A-85380 dihydrochloride (5IA85380). The GLU and ASP overflow evoked by Ch, nicotine, KCl and 4AP were increased in treated animals while the nicotine-evoked GABA overflow was reduced and that evoked by Ch, KCl and 4AP was unaffected. The 5IA85380-evoked overflow of the three aminoacids (AAs) was always reduced. The increase of ASP and GLU overflow evoked by KCl, 4AP or Ch was blocked by dl-threo-β-benzyloxyaspartic acid (dl-TBOA), a carrier transporter inhibitor, and by inhibitors of the Na(+)/Ca(2+) exchangers 2-[[4-[(4-nitrophenyl)methoxy]phenyl]methyl]-4-thiazolidinecarboxylic acid ethyl ester (SN-6) and 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea mesylate (KB-R7943). In conclusion long-term nicotine treatment may selectively increase GLU and ASP overflow elicited by KCl, 4AP and Ch through the activation of a carrier-mediated release mechanism and completely abolished the stimulatory effects of α4β2 nAChRs which modulate the release of all the three AA.     

Entamoeba histolytica: an ecto-phosphatase activity regulated by oxidation-reduction reactions

In this work, an ecto-phosphatase activity of Entamoeba histolytica was characterized using intact cells. This activity presented the following biochemical characteristics: (i) it hydrolyzes p-NPP with V(max) of 8.00+/-0.22 nmol p-NP x h(-1) x 10(-5) cells and K(m) of 2.68+/-0.25 mM; (ii) it is inhibited by acid phosphatase inhibitors, such as sodium molybdate (K(i)=1.70+/-0.24 microM) and sodium fluoride (K(i)=0.25+/-0.02 mM); (iii) it also showed high sensitivity to phosphotyrosine phosphatase inhibitors, such as sodium orthovanadate (K(i)=1.07+/-0.14 microM), bpV-PHEN (K(i)=0.38+/-0.02 microM) and mpV-PIC (K(i)=0.39+/-0.04 microM). Zn(2+), an oxidizing agent, decreased the enzymatic activity in 50%. DTT and GSH, two reducing agents, enhanced the activity twofold. The non-invasive E. histolytica and free-living E. moshkovskii were less efficient in hydrolyzing p-NPP than the pathogenic E. histolytica suggesting that this enzyme could represent a virulence marker for this cell.     

Participation of peroxisomal beta-oxidation system in the chain-shortening of a xenobiotic acyl compound

A drug, (E)-3-[4-(1-imidazolylmethyl)phenyl]-2-propenoic acid, was metabolized to 4-(1-imidazolylmethyl)benzoic acid in isolated hepatocytes of rats, which was enhanced markedly by the pretreatment of rats with clofibrate. With liver homogenates, the formation of the CoA-ester of this drug and its subsequent chain-shortening were demonstrated. In the series of these reactions, acyl-CoA synthetase, CoA, ATP and NAD were required, whereas cyanide did not inhibit the reaction. These results indicate that peroxisomes are capable of shortening the acyl side-chains of drugs by the beta-oxidation, giving an additional suggestion on the functions of peroxisomes.     

Reconstitution into liposomes of the B°-like glutamine-neutral amino acid transporter from renal cell plasma membrane

Na⁺ dependent [³H]glutamine uptake was found in liposomes reconstituted with solubilized rat kidney brush border in the presence of intraliposomal K⁺. The reconstituted system was optimised with respect to the critical parameters of the cyclic detergent removal procedure, i.e., the detergent used for the solubilization, the protein concentration, the detergent/phospholipid ratio and the number of passages through a single Amberlite column. Time dependent [³H]glutamine accumulation in proteoliposomes occurred only in the presence of external Na⁺and internal K⁺. The transporter showed low if there is any tolerance towards the substitution of Na⁺ or K⁺ for other cations. Valinomycin strongly stimulated the transport indicating that it is electrogenic. Intraliposomal glutamine had no effect. From the dependence of the transport rate on the Na⁺ concentration cooperativity index close to 1 was derived, indicating that 1 Na⁺ should be involved in the cotransport with glutamine. The electrogenicity of the transport originated from the Na⁺ transport. Optimal rate of 0.1 mM [³H]glutamine uptake was found in the presence of 50 mM intraliposomal K-gluconate. At higher K-gluconate concentrations the transport rate decreased. The activity of the reconstituted transporter was pH dependent with optimal function in the range pH 6.5-7.0. [³H]glutamine (and [³H]leucine) uptake was inhibited by all the neutral but not by the positively or negatively charged amino acids. The sulfhydryl reagents HgCl₂, mersalyl, p-hydroxymercuribenzoate and the substrate analogue 2-aminobicyclo[2,2,1]heptane-2-carboxylate strongly inhibited the transporter, whereas the amino acid analogue α-(methylamino)isobutyrate had no effect. The inhibition by mersalyl was protected by the presence of the substrate. On the basis of the Na⁺ dependence, the electrogenic transport mode and the specificity towards the amino acids, the reconstituted transporter was classified as B°-like.     

Absolute stereochemistry of methanopterin and 5,6,7,8-tetrahydromethanopterin

The configuration at the C-9 of methanopterin (MPT) has been determined by comparing the circular dichroism (CD) spectra of MPT and its hydrolytic fragment, 1-[4-[[1-(2-amino-7-methyl-4-hydroxy-6-pteridinyl)-ethyl]amino]phenyl]-1-deoxy-D -ribitol (HP-1), with the CD spectra of a series of model compounds of known stereochemistry. These compounds included (S)-6-[1-(4-carboxymethylanilino)ethyl]pterin, (S-6(1-hydroxyethyl)-7-methylpterin, (S-6-(1-hydroxyethyl)pterin, (R)-6-(1-phenoxyethyl)pterin, D (+)-neopterin, and L -biopterin. From this comparison it was concluded that MPT has the R configuration at C-9 and is thus configurationally related to D (+)-neopterin, which has the S configuration at C-1. From previous work establishing the relative stereochemistry at C-6, C-7, and C-9 of N⁵-N¹⁰-methenyl-5,6,7,8-tetrahydromethanopterin (N⁵-N¹⁰-methenyl-H₄MPT) as R, S, and R, respectively, it is clear that the remaining asymmetric carbons at C-6 and C-7 of H₄MPT have the S and S configuration, respectively. Comparison of these latter two positions to the equivalent carbons in 5,6,7,8-tetrahydrofolate (H₄folate) show that the steps involved in the biological reduction of MPT to H₄MPT occur with the same stereochemical outcome as those involved in the biological reduction of folate to H₄folate. © 1996 Wiley-Liss, Inc.     

Co-refolding of two peptide fragments derived from Agrobacterium tumefaciens beta-glucosidase with catalytic activity

Four sites of the non-homologous region (coding amino acid residues of 347, 421, 466 and 533) of a gene were randomly selected for splitting to investigate the function of β-glucosidase from Agrobacterium tumefaciens in the co-refolding of peptides into the catalytically active enzyme. As a result of gene splitting, four N- and C-terminal domain peptides were obtained as insoluble inclusion bodies. No catalytic activity was observed when these fragments refolded individually. However, a considerable amount of activity was restored when the two fragments derived from N- and C- terminal peptides were co-refolded together. The deletion of amino acid residues in the non-homologous region resulted in a complete loss of enzyme activity, which suggests that truncation of amino acids in this region strongly affects the co-refolding ability of the enzyme to maintain activity.     

Chemopreventive potential of 3-[2,6-bis(4-fluorophenyl)-3-methylpiperidin-4-ylideneamino]-2-thioxoimidazolidin-4-one on 7,12-dimethylbenz[a]anthracene (DMBA) induced hamster buccal pouch carcinogenesis

In the present work, a new bis heterocyclic compound comprising both the piperidone and thiohydantoin nuclei namely 3-[2,6-bis(4-fluorophenyl)-3-methylpiperidin-4-ylideneamino]-2-thioxoimidazolidin-4-one was synthesised and characterised with the help of mp, elemental analysis, FT-IR, MS and one-dimensional NMR (¹H and ¹³C) spectra. The inhibitory effect of 3-[2,6-bis(4-fluorophenyl)-3-methylpiperidin-4-ylideneamino]-2-thioxoimidazolidin-4-one on 7,12-dimethylbenz[a]anthracene (DMBA) induced buccal pouch carcinogenesis was investigated in Syrian male hamsters. All the hamsters that were painted with DMBA on their buccal pouches for 14 weeks developed squamous cell carcinoma. Administration of 3-[2,6-bis(4-fluorophenyl)-3-methylpiperidin-4-ylideneamino]-2-thioxoimidazolidin-4-one effectively suppressed the oral carcinogenesis initiated with the DMBA as revealed by a reduced incidence of neoplasms. Lipid peroxidation, glutathione (GSH) content and the activities of glutathione peroxidase (GPx), glutathione S-transferase (GST) were used to biomonitor the chemopreventive potential of 3-[2,6-bis(4-fluorophenyl)-3-methylpiperidin-4-ylideneamino]-2-thioxoimidazolidin-4-one. Lipid peroxidation was found to be significantly decreased, whereas GSH, GPx, GST and GGT were elevated in the oral mucosa of tumour bearing animals. Our data suggest that 3-[2,6-bis(4-fluorophenyl)-3-methylpiperidin-4-ylideneamino]-2-thioxoimidazolidin-4-one may exert its chemopreventive effects in the oral mucosa by modulation of lipid peroxidation, antioxidants and detoxification systems.     

Synthesis of anellated carbasugars from (--)-quinic acid

(3R,4R,5R)-3-[(tert-Butyl-dimethylsilyl)oxy]-4,5-(isopropylidenedioxy)-1-cyclohexanone (2) reacted with carbon disulfide and methyl iodide in the presence of sodium hydride to furnish (3R,4R,5R)-5-[(tert-butyl-dimethylsilyl)oxy]-3,4-(isopropylidenedioxy)-2-[bis(methylthio)methylene]-1-cyclohexanone (3). 2 and N,N-dimethylformamide dimethyl acetal afforded (2E,3R,4R,5R)-5-[(tert-butyl-dimethylsilyl)oxy]-2-(dimethylaminomethylene)-3,4-(isopropylidenedioxy)-1-cyclohexanone (4). These push-pull activated methylenecyclohexanones 3 and 4 underwent a ring closure reaction with hydrazine hydrate and methylhydrazine, respectively, to give pyrazoloanellated carbasugars. Treatment of 3 with formamidinium, acetamidinium and benzamidinium salts, respectively, in the presence of sodium methanolate yielded three (5R,6R,7R)-7-[(tert-butyl-dimethylsilyl)oxy]-5,6,7,8-tetrahydro-5,6-(isopropylidenedioxy)benzo[d]pyrimidines.     

Pyridinium-carbaldehyde: active Maillard reaction product from the reaction of hexoses with lysine residues

Besides the formation of the aminotriazine N6-[4-(3-amino-1,2,4-triazin-5-yl)-2,3-dihydroxybutyl]-L-lysine, the reaction of [1-13C]D-glucose with lysine and aminoguanidine leads to the generation of 6-[2-([[amino(imino)methyl]hydrazono]methyl)pyridinium-1-yl]-L-norleucine (14-13C1). The dideoxyosone N6-(2,3-dihydroxy-5,6-dioxohexyl)-L-lysine was shown to be a precursor in the formation of 14-13C1, which proceeds via the reactive carbonyl intermediate 6-(2-formylpyridinium-1-yl)-L-norleucine (13-13C1). In order to study the reactivity of 13-13C1, the model compound 1-butyl-2-formylpyridinium (18) was prepared in a two-step procedure starting from 2-pyridinemethanol. The reaction of the pyridinium-carbaldehyde 18 with L-lysine yielded the Strecker analogous degradation product 2-(aminomethyl)-1-butylpyridinium and another compound, which was shown to be as 1-butyl-2-[(2-oxopiperidin-3-ylidene)methyl]pyridinium. Reaction of 18 with the C-H acidic 4-hydroxy-5-methylfuran-3(2H)-one leads to the formation of the condensation product 1-butyl-2-[hydroxy-(4-hydroxy-5-methyl-3-oxofuran-2(3H)-ylidene)methyl]-pyridinium.     

R-state hemoglobin bound to heterotropic effectors: models of the DPG, IHP and RSR13 binding sites

We performed a docking study followed by a 500-ps molecular dynamics simulation of R-state human adult hemoglobin (HbA) complexed to different heterotropic effectors [2,3-diphosphoglycerate (DPG), inositol hexaphosphate (IHP), and 2-[4-[(3,5-dichlorophenylcarbamoyl)-]methyl]-phenoxy]-2-methylpropionic acid (RSR13)) to propose a molecular basis for recently reported interactions of effectors with oxygenated hemoglobin. The simulations were carried out with counterions and explicit solvation. As reported for T-state HbA, the effector binding sites are also located in the central cavity of the R-state and differ depending on effector anionic character. DPG and IHP bind between the alpha-subunits and the RSR13 site spans the alpha1-, alpha2- and beta2-subunits. The generated models provide the first report of the molecular details of R-state HbA bound to heterotropic effectors.     

Differential Vertical Movement of Nonvolatile Nematicides in Soil

When incorporated in the top 5-cm of Tifton sandy loam at 11.2 kg/ha in the field, B-68138 [ethyl 4-(methylthio)-m-tolyl isopropyl phosphoramidate] prevented galling of tomato roots by Meloidogyne sp. down to 20 cm. A similar application of 16.8 kg/ha of V-C 9-104 [0-ethyl S,S-dipropyl phosphorodithioate] was 99% effective down to 20 cm. Aldicarb [2-methyl-2-(methylthio) propionaldehyde 0-(methylcarbamoyl) oxime], B-25141 [0,0-diethyl 0-{p-(methylsulfinyl) phenyl}phosphorothioate], and carbofuran [2,3-dihydro-2,2-dimethyl-7-benzofuranyl methylcarbamate] prevented galling only in the zone of incorporation (top 5-cm of soil). When aldicarb (11.2 kg/ha) was applied to the surface of Ontario fine sandy loam contained in clay pots in the greenhouse, it prevented galling of tomato roots down to 20 cm deep. V-C 9-104 and B-68138, applied similarly, were 100% effective to a depth of 5 cm. B-25141 was 100% effective in the zone of incorporation only. D-1410 [S-methyl-l-(dimethylcarbamoyl)-N-l(methylcarbamoyl)oxy] thioformimidatel did not control 100% of the root-knot nematodes at any depth.     

Reactive nitrogen species derived activation of rat liver microsomal glutathione S-transferase

The effect of reactive nitrogen species on rat liver microsomal glutathione S-transferase (MGST1) was investigated using microsomes and purified MGST1. When microsomes or the purified enzyme were incubated with peroxynitrite (ONOO(-)), the GST activity was increased to 2.5-6.5 fold in concentration-dependent manner and a small amount of the MGST1 dimer was detected. MGST1 activity was increased by ONOO(-) in the presence of high amounts of reducing agents including glutathione (GSH) and the activities increased by ONOO(-) or ONOO(-) plus GSH treatment were decreased by 30-40% by further incubation with dithiothreitol (DTT, reducing disulfide) or by sodium arsenite (reducing sulfenic acid). Furthermore, GSH was detected by HPLC from the MGST1 which was incubated with ONOO(-) plus GSH or S-nitrosoglutathione followed by DTT treatment. In addition, the MGST1 activity increased by nitric oxide (NO) donors such as S-nitrosoglutathione, S-nitrosocysteine or the non-thiol NO donor 1-hydroxy-2-oxo-3 (3-aminopropyl)-3-isopropyl was restored by the DTT treatment. Since DTT can reduce S-nitrosothiol and disulfide bond to thiol, S-nitrosylation and a mixed disulfide bond formation of MGST1 were suggested. Thus, it was demonstrated that MGST1 is activated by reactive nitrogen species through a forming dimeric protein, mixed disulfide bond, nitrosylation and sulfenic acid.     

Structural Basis for Featuring of Steroid Isomerase Activity in Alpha Class Glutathione Transferases

Glutathione transferases (GSTs) are abundant enzymes catalyzing the conjugation of hydrophobic toxic substrates with glutathione. In addition to detoxication, human GST A3-3 displays prominent steroid double-bond isomerase activity; e.g. transforming Δ⁵-androstene-3-17-dione into Δ⁴-androstene-3-17-dione (AD). This chemical transformation is a crucial step in the biosynthesis of steroids, such as testosterone and progesterone. In contrast to GST A3-3, the homologous GST A2-2 does not show significant steroid isomerase activity. We have solved the 3D structures of human GSTs A2-2 and A3-3 in complex with AD. In the GST A3-3 crystal structure, AD was bound in an orientation suitable for the glutathione (GSH)-mediated catalysis to occur. In GST A2-2, however, AD was bound in a completely different orientation with its reactive double bond distant from the GSH-binding site. The structures illustrate how a few amino acid substitutions in the active site spectacularly alter the binding mode of the steroid substrate in relation to the conserved catalytic groups and an essentially fixed polypeptide chain conformation. Furthermore, AD did not bind to the GST A2-2-GSH complex. Altogether, these results provide a first-time structural insight into the steroid isomerase activity of any GST and explain the 5000-fold difference in catalytic efficiency between GSTs A2-2 and A3-3. More generally, the structures illustrate how dramatic diversification of functional properties can arise via minimal structural alterations. We suggest a novel structure-based mechanism of the steroid isomerization reaction.     

Identification of galloylated propelargonidins and procyanidins in buckwheat grain and quantification of rutin and flavanols from homostylous hybrids originating from F. esculentumxF. homotropicum

The flavonoid rich grain of buckwheat (Fagopyrum esculentum Moench, Fam. Polygonaceae) is of high nutritional value. With the aim to improve its agronomic productivity, cultivars were crossed with the wild species F. homotropicum which, however, differs in its flavonoid content. The intention of this work was to determine the flavonoid composition in developed interspecific hybrids and to elucidate the proanthocyanidin structures. Seven compounds were purified from methanol extracts of buckwheat (Fagopyrum esculentum Moench) grains by Sephadex LH-20 column chromatography. Beside the procyanidin epicatechin-[4-8]-epicatechin-3-O-(3,4)-dimethylgallate the following propelargonidins were identified: epiafzelechin-[4-6]-epicatechin, epiafzelechin-[4-8]-epiafzelechin-[4-8]-epicatechin, epiafzelechin-[4-8]-epicatechin-3-O-(3,4-dimethyl)-gallate, epiafzelechin-[4-8]-epiafzelechin-[4-8]-epicatechin-3-O-(3,4-dimethyl)-gallate, epiafzelechin-[4-8]-epicatechin-3-O-4-methyl-gallate and epiafzelechin-[4-8]-epicatechin-p-OH-benzoate on the basis of HPLC and LC-MS/MS.     

Crystallographic and Functional Characterization of the Fluorodifen-inducible Glutathione Transferase from Glycine max Reveals an Active Site Topography Suited for Diphenylether Herbicides and a Novel L-site

Glutathione transferases (GSTs) from the tau class (GSTU) are unique to plants and have important roles in stress tolerance and the detoxification of herbicides in crops and weeds. A fluorodifen-induced GST isoezyme (GmGSTU4-4) belonging to the tau class was purified from Glycine max by affinity chromatography. This isoenzyme was cloned and expressed in Escherichia coli, and its structural and catalytic properties were investigated. The structure of GmGSTU4-4 was determined at 1.75 Å resolution in complex with S-(p-nitrobenzyl)-glutathione (Nb-GSH). The enzyme adopts the canonical GST fold but with a number of functionally important differences. Compared with other plant GSTs, the three-dimensional structure of GmGSTU4-4 primarily shows structural differences in the hydrphobic substrate binding site, the linker segment and the C-terminal region. The X-ray structure identifies key amino acid residues in the hydrophobic binding site (H-site) and provides insights into the substrate specificity and catalytic mechanism of the enzyme. The isoenzyme was highly active in conjugating the diphenylether herbicide fluorodifen. A possible reaction pathway involving the conjugation of glutathione with fluorodifen is described based on site-directed mutagenesis and molecular modeling studies. A serine residue (Ser13) is present in the active site, at a position that would allow it to stabilise the thiolate anion of glutathione and enhance its nucleophilicity. Tyr107 and Arg111 present in the active site are important structural moieties that modulate the catalytic efficiency and specificity of the enzyme, and participate in k_cat regulation by affecting the rate-limiting step of the catalytic reaction. A hitherto undescribed ligand-binding site (L-site) located in a surface pocket of the enzyme was also found. This site is formed by conserved residues, suggesting it may have an important functional role in the transfer and delivery of bound ligands, presumably to specific protein receptors.     

Molecular probes for the A2A adenosine receptor based on a pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine scaffold

Pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine derivatives such as SCH 442416 display high affinity and selectivity as antagonists for the human A_2A adenosine receptor (AR). We extended ether-linked chain substituents at the p-position of the phenyl group using optimized O-alkylation. The conjugates included an ester, carboxylic acid and amines (for amide condensation), an alkyne (for click chemistry), a fluoropropyl group (for ¹⁸F incorporation), and fluorophore reporter groups (e.g., BODIPY conjugate 14, K_i 15 nM). The potent and A_2AAR-selective N-aminoethylacetamide 7 and N-[2-(2-aminoethyl)-aminoethyl]acetamide 8 congeners were coupled to polyamidoamine (PAMAM) G3.5 dendrimers, and the multivalent conjugates displayed high A_2AAR affinity. Theoretical docking of an AlexaFluor conjugate to the receptor X-ray structure highlighted the key interactions between the heterocyclic core and the binding pocket of the A_2AAR as well as the distal anchoring of the fluorophore. In conclusion, we have synthesized a family of high affinity functionalized congeners as pharmacological probes for studying the A_2AAR.     

Differential nucleotide excision repair susceptibility of bulky DNA adducts in different sequence contexts: hierarchies of recognition signals

The structural origin underlying differential nucleotide excision repair (NER) susceptibilities of bulky DNA lesions remains a challenging problem. We investigated the 10S (+)-trans-anti-[BP]-N(2)-2'-deoxyguanosine (G*) adduct in double-stranded DNA. This adduct arises from the reaction, in vitro and in vivo, of a major genotoxic metabolite of benzo[a]pyrene (BP), (+)-(7R,8S,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, with the exocyclic amino group of guanine. Removal of this lesion by the NER apparatus in cell-free extracts has been found to depend on the base sequence context in which the lesion is embedded, providing an excellent opportunity for elucidating the properties of the damaged DNA duplexes that favor NER. While the BP ring system is in the B-DNA minor groove, 5' directed along the modified strand, there are orientational distinctions that are sequence dependent and are governed by flanking amino groups [Nucleic Acids Res.35 (2007), 1555-1568]. To elucidate sequence-governed NER susceptibility, we conducted molecular dynamics simulations for the 5'-...CG*GC..., 5'-...CGG*C..., and 5'-...TCG*CT... adduct-containing duplexes. We also investigated the 5'-...CG*IC... and 5'-...CIG*C... sequences, which contain "I" (2'-deoxyinosine), with hydrogen replacing the amino group in 2'-deoxyguanosine, to further characterize the structural and dynamic roles of the flanking amino groups in the damaged duplexes. Our results pinpoint explicit roles for the amino groups in tandem GG sequences on the efficiency of NER and suggest a hierarchy of destabilizing structural features that differentially facilitate NER of the BP lesion in the sequence contexts investigated. Furthermore, combinations of several locally destabilizing features in the hierarchy, consistent with a multipartite model, may provide a relatively strong recognition signal.