Structure-activity relationships of neurokinin A (4-10) at the human tachykinin NK(2) receptor: the role of natural residues and their chirality

A structure-activity study of neurokinin A (NKA) (4-10) was performed to investigate the importance of residue and chirality for affinity and efficacy at the NK(2) receptor in human colon circular muscle. Two series of NKA(4-10) analogues were produced with either L-alanine or the D-enantiomer substituted. Their activities were determined in vitro by means of radioligand binding and isolated smooth muscle pharmacology. NKA was more potent than NKA(4-10) at the human, unlike the rabbit, NK(2) receptor. The contractile response of NKA(4-10) was unaffected by N-terminal acetylation. L-Ala substitution of Asp(4), Val(7), Leu(9), and Met(10) caused an 8- to 80-fold decrease, and substitution of Phe(6) caused a 5000-fold decrease in binding affinity (P < 0.01). Positions Ser(5) and Gly(8) were not significantly affected. In functional studies, a similar pattern was observed. The replacement of residues with their respective D-enantiomer drastically reduced binding affinity and functional potency, particularly at positions 6 and 7 (P < 0.05). NKA(4-10) analogues L-Ala(6), L-Ala(8), D-Phe(6), D-Val(7), and D-Met(10) were partial agonists. An excellent correlation was observed between binding and functional data (r = 0.95). A retro-inverso analogue of NKA(4-10) was inactive. In conclusion, the side chains of Asp(4), Phe(6), Val(7), Leu(9), and Met(10) are structurally important features of NKA(4-10) for agonist activity, and changes in amino acid chirality are detrimental to binding affinity and functional activity. Overall, our data are broadly similar to those of previous studies in the rat. However, at the human NK(2) receptor, unlike the rat, [Ala(8)]NKA(4-10) was an antagonist.     

Synthesis and Antimicrobial Activity of Some New 2-(3-(4-Aryl)-1-phenyl-1H-pyrazol-4-yl) Chroman-4-ones

Seven new 2-(3-(4-aryl)-1-phenyl-1H-pyrazol-4-yl) chroman-4-ones (4a-4g) have been synthesized by cyclization of 2-hydroxychalcone analogues of pyrazole 3a-3g using conc. HCl in acetic acid. The structures of the compounds 4a-4g were established by the combined use of (1)HNMR, IR and mass spectra. All the seven compounds were tested in vitro for their antibacterial activity against two Gram positive bacteria namely Staphylococcus aureus and Bacillus subtilis and two Gram negative bacteria Escherichia coli and Pseudomonas aeruginosa. The compounds 4b, 4c, 4e, 4f, 4g have displayed good antibacterial activity when compared with commercially available antibiotic, ciprofloxacin. These compounds also were screened for their antifungal activity against two ear pathogenic fungi, namely Aspergillus Niger and A. flavus. The compounds 4a, 4c, 4d, 4g exhibited good antifungal activity when compared with commercially available antifungal, fluconazole.     

R-(+)-[2,3-Dihydro-5-methyl-3-(4-morpholinylmethyl)-pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphtalenylmethanone (WIN-2) ameliorates experimental autoimmune encephalomyelitis and induces encephalitogenic T cell apoptosis: partial involvement of the CB(2) receptor

Many reports have shown that cannabinoids might be beneficial in the symptomatic treatment of multiple sclerosis (MS). We have investigated the therapeutic properties of the non-selective cannabinoid receptor agonist WIN-2 as a suppressive drug in the experimental autoimmune encephalomyelitis (EAE) model of MS. In the passive variety of EAE, induced in Lewis rats by adoptive transfer of myelin-reactive T cells, WIN-2 ameliorates the clinical signs and diminishes the cell infiltration of the spinal cord. Due to the involvement of cannabinoids in the regulation of cell death and survival, we investigated the effects of WIN-2 on the encephalitogenic T cell population. WIN-2 induced a profound increase of apoptosis in a dose- and time-dependent manner. The potential involvement of cannabinoid receptors (CB) was investigated by encephalitogenic T cell stimulation in the presence of the CB(1) (SR141716A) and CB(2) (SR144528) antagonists, pertussis toxin (PTX) and the inactive enantiomer WIN-3. WIN-2-induced apoptosis was partially blocked by SR144528 and PTX, whereas, WIN-3 only exerted a mild effect on cell viability. These results point to the partial involvement of CB(2) receptor together with other receptor-independent mechanism or by yet unknown cannabinoid receptors. Moreover, WIN-2 induced the extrinsic pathway of apoptosis, as shown by caspase-10 and -3 activation. These results suggest that cannabinoid-induced apoptosis of encephalitogenic T cells may cooperate in their anti-inflammatory action in EAE models. The partial involvement of CB(2) receptors in WIN-2 action may open new therapeutic doors in the management of MS by non-psychoactive selective cannabinoid agonists.     

咪苯嗪酮对花生四烯酸环氧酶途径代谢产物的选择性影响

兔iv咪苯嗪酮(CI—914)20 mg/kg,用RIA法测定其血浆中TXB_2和6—kcto—PGF_(1α)含量,给药后30 min和60 min.6—keto—PGF_(1α)/TXB_2比值显著升高(p<0.05).在用放射性TLC法进行的洗涤大鼠血小板~(14)C—AA代谢实验中,CI—914在2～500 μmol/L范围内以剂量依赖方式抑制大鼠血小板TXB_2生成,IC_(50)为51.5μmol/L,对HHT生成的抑制明显弱于对TXB_2生成的抑制作用;在相同剂量范围内,CI—914同时以剂量依赖方式使PGE_2,PGF_(2α)和PGD_2生成增加.在相同实验条件下,50μmol/L的已知的TXA_2合成酶抑制剂DAZ,与大剂量CI—914的实验结果类似.上述结果提示,CI—914对血小板TXA_2合成酶可能具有选择性抑制作用.     

Comparative pharmacology of human dopamine D(2)-like receptor stable cell lines coupled to calcium flux through Galpha(qo5)

The goal of this study was to develop a new approach to study the pharmacology of the dopamine D(4) receptor that could be used in comparative studies with dopamine D(2) and D(3) receptors. Stable HEK-293 cell lines co-expressing recombinant human D(2L), D(3) or D(4) receptors along with Galpha(qo5) cDNA were prepared. Dopamine induced a robust, transient calcium signal in these cell lines with EC(50)s for D(2L), D(3) and D(4) of 18.0, 11.9 and 2.2 nM, respectively. Reported D(4)-selective agonists CP226269 and PD168077 were potent, partial D(4) agonists exhibiting 31-1700-fold selectivity for D(4) over D(3) or D(2). Non-selective D(2)-like agonists apomorphine and quinpirole showed full efficacy but did not discriminate across the three receptors. D(3)-selective agonists 7-hydroxy-DPAT and PD128907 were potent but non-selective D(2)-like agonists. The reported D(3) partial agonist BP-897 exhibited minimal agonist activity at D(3) but was a potent D(3) antagonist and a partial D(4) agonist. Other D(2)-like antagonists, haloperidol, clozapine, and domperidone showed concentration-dependent inhibition of dopamine responses at all three receptors with K(i) ranging from 0.05 to 48.3 nM. The D(3) selective antagonist S33084 and D(4)-selective antagonist L-745870 were highly selective for D(3) and D(4) receptors with K(b) of 0.7 and 0.1 nM, respectively. Stable co-expression of D(2)-like receptors with chimeric Galpha(qo5) proteins in HEK-293 cells is an efficient method to study receptor activation in a common cellular background and an efficient method for direct comparison of ligand affinity and efficacy across human D(2L), D(3) and D(4) receptors.     

Discovery of 3-(4-hydroxybenzyl)-1-(thiophen-2-yl)chromeno[2,3-c]pyrrol-9(2H)-one as a phosphodiesterase-5 inhibitor and its complex crystal structure

Phosphodiesterase-5 (PDE5) inhibitors have been approved for the treatment of erectile dysfunction and pulmonary hypertension, but enthusiasm on discovery of PDE5 inhibitors continues for their potential new applications. Reported here is discovery of a series of new PDE5 inhibitors by structure-based design, molecular docking, chemical synthesis, and enzymatic characterization. The best compound, 3-(4-hydroxybenzyl)-1-(thiophen-2-yl)chromeno[2,3-c]pyrrol-9(2H)-one (57), has an IC₅₀ of 17 nM against the PDE5 catalytic domain and good selectivity over other PDE families. The crystal structure of the PDE5 catalytic domain in complex with 57 was determined at 2 Å resolution and showed that 57 occupies the same pocket as other PDE5 inhibitors, but has a different binding pattern in detail. On the basis of the binding pattern of 57, a novel scaffold can be proposed as a candidate of PDE inhibitors.     

咪苯嗪酮对花生四烯酸诱导的大鼠脑血栓形成的影响

花生四烯酸(AA)0.25～1 mg·kg~(-1)经颈内动脉注射能诱发大鼠同侧大脑半球脑内血栓形成,明显增加伊文思蓝通过血脑屏障渗入脑实质的量,峰值为205±s 50 mg·kg~1脑组织,相应对照组为10±s 5mg·kg~1,咪苯嗪酮0.25～0.5mg·kg~1 iv能对抗AA引起的大鼠脑血栓形成,显著降低脑实质内伊文思蓝的含量,作用呈剂量依赖性,且强于哒唑氧苯     

呋苄头孢菌素和呋苄青霉素钾的极谱行为研究

本文研究呋苄头孢菌素和呋苄青霉素钾的极谱行为。两样品在-1.1 V附近有一清晰的单扫描极谱波,在一定的浓度范围内蜂电流与样品浓度之间呈良好的线性关系,可用作定量分析。本文还对电极反应机理进行了初步的探讨,并证明此波具有吸附性质。     

Cannabinoids inhibit sodium-dependent, high-affinity excitatory amino acid transport in cultured rat cortical astrocytes

Cannabinoids have been shown to increase the extracellular levels of glutamate in vivo and in vitro, but no studies have evaluated the possible involvement of glial glutamate reuptake system. The present study investigates whether cannabinoids and endocannabinoid, anandamide have an effect on astroglial excitatory amino acid (EAA) transport. The kinetics of glutamate transport was studied in rat cortical astrocytes, using the radiolabeled, non-metabolized amino acid, D-[3H] aspartate in the absence or presence of cannabinoid receptor agonists. The results show that in vehicle controls the uptake of d-aspartate was rapid, sodium-dependent and saturated within the first 5 min, resulting in a K(m) 7.365+/-1.16 micromol/L (n=5) and the maximum velocity (V(max)) 1207+/-51 nmol/mg protein/min. Addition of the synthetic cannabinoid analog R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolol][1,2,3de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone (WIN 55,212-2; 3 micromol/L) increased the K(m) (26.25+/-4.84 micromol/L) without affecting the V(max) (1122+/-77 nmol/mg protein/min), suggesting the inhibition was competitive and reversible. Various other cannabinoid agonists also inhibited D-aspartate uptake in a dose-dependent and stereospecific manner. The cannabinoid inhibition of EAA transport was partially blocked by the cannabinoid type-1 (CB1) receptor antagonist N-(piperidin-1-yl-5(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamidehydrochloride (SR141716A; 100 nmol/L). The inhibitory effects of WIN 55,212-2, or its endogenous counterpart anandamide were reversed by 98,059, an inhibitor of mitogen-activated kinase (MAPK) kinase (MEK). These results suggest that cannabinoids and endocannabinoids may constitute a novel class of inhibitors of astroglial glutamate transport system.     

Effects of D3.49A,R3.50A,and A6.34E mutations on ligand binding and activation of the cannabinoid-2 (CB2) receptor

In several G protein-coupled receptors (GPCRs), the Asp-Arg-Tyr (DRY) motif at the bottom of third transmembrane domain and the amino acid at position 6.34 in the sixth transmembrane domain have been shown to play important roles in signal transduction. In this study, we propose that in the cannabinoid-2 (CB2) receptor, R3.50 in the DRY motif may be crucial for interacting with G proteins, and D3.49 and A6.34 may be important for constraining the receptor in an inactive conformation. To test our hypothesis, R3.50A, D3.49A, and A6.34E mutations of the human CB2 receptor were made by site-directed mutagenesis. These mutant receptors were stably transfected into human embryonic 293 cells, and their ligand binding and signal transduction properties were analyzed. Similar to other GPCRs, R3.50 of the CB2 receptor is crucial for signal transduction. Unlike other GPCRs, D3.49 and A6.34 of the CB2 receptor do not seem to be important for keeping the receptor in an inactive state. Furthermore, D3.49A and A6.34E mutations abolished ligand binding, and all three mutations abolished constitutive activity of the wild-type CB2 receptor.     

Investigations on the synthesis and pharmacological properties of 4-alkoxy-2-[2-hydroxy-3-(4-aryl-1-piperazinyl)propyl]-6-methyl-1H-pyrrolo[3,4-c]pyridine-1,3(2H)-diones

Synthesis of 2-[2-hydroxy-3-(4-aryl-1-piperazinyl)propyl] derivatives of 4-alkoxy-6-methyl-1H-pyrrolo[3,4-c]pyridine-1,3(2H)-diones (8-12) is described. The chlorides used in the above synthesis can exist in two isomeric forms: chain (18-20) and cyclic (19a, 20a). The compounds 8-12 exhibited potent analgesic activity which was superior than that of acetylsalicylic acid in two different tests. Most of the investigated imides suppressed significantly spontaneous locomotor activity in mice.     

Integrity of extracellular loop 1 of the human cannabinoid receptor 1 is critical for high-affinity binding of the ligand CP 55,940 but not SR 141716A

Like other G-protein coupled receptors with hydrophobic ligands, the human cannabinoid receptor 1 (CB1) is thought to bind its ligands within the transmembrane region of the receptor. However, for some of these receptors the extracellular loops (ECs) have also been shown to play a role in ligand recognition and selectivity. We have taken a mutagenesis approach to examine the role of the amino terminus, EC1, and EC3 of CB1 in ligand binding. Eight mutant receptors, each with a dipeptide insertion, were constructed, expressed, and evaluated for binding to the cannabinoid ligands (-)-cis-3[2-hydroxy-4-(1',1'-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP 55,940) and N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR 141716A). Mutants with insertions in the membrane distal region of the amino terminus or EC3 maintained affinity for both ligands. Those with insertions in the membrane proximal region of the amino terminus or EC1 exhibited a loss of affinity for CP 55,940 while retaining wild-type affinity for SR 141716A. Representative mutants were analyzed for agonist-induced inhibition of cyclic AMP accumulation, and the results indicated that G-protein coupling remained intact. Alanine substitution mutants were made to address whether it was the perturbation of the overall structure of the region or the displacement of particular side chains that was responsible for the loss of CP 55,940 binding. We conclude that a structurally intact EC1, but not the comparably short EC3, is essential for high-affinity CP 55,940 binding.     

1-Ethyl-4-(isopropylidenehydrazino)-1H-pyrazolo-(3,4-b)-pyridine-5-carboxylic acid, ethyl ester, hydrochloride (SQ 20009)--a potent new inhibitor of cyclic 3',5'-nucleotide phosphodiesterases

The properties of SQ 20009 [1-ethyl-4-(isopropylidenehydrazino)-1H-pyrazolo-(3,4-b)-pyridine-5-carboxylic acid, ethyl ester, HCl] as a cyclic nucleotide phosphodiesterase inhibitor have been investigated. The phosphodiesterase preparations used in this study were ammonium sulfate-fractionated supernatants of homogenates of rat brain, rabbit brain, rat adrenal, rat lipocyte and cat heart; commercially available beef heart phosphodiesterase was also studied. The concentrations of SQ 200009 required to inhibit these phosphodiesterase activities 50 per cent were 2·0, 4·8, 20, 21, 27 and 60 μM, respectively, using 1·6 × 10⁻⁷ M cyclic AMP as substrate. SQ 20006 (the parent of SQ 20009 lacking the 4-isopropylidene moiety), theophylline and caffeine were also tested against all six enzyme preparations. Whereas SQ 20009 was more potent than SQ 20006 using the phosphodiesterase prepared from rat adrenal, the potencies were reversed when the lipocyte enzyme was used. SQ 20009 was approximately 60 and 75 times as potent an inhibitor of rat brain cyclic AMP phosphodiesterase as were theophylline and caffeine respectively. The kinetic properties of the phosphodiesterases prepared from rat brain, cat heart and beef heart were also investigated. Using the rat brain enzyme, two K_m values for cyclic AMP, 4·0 × 10⁻⁶ and 1·2× 10⁻⁴ M and a single K_m, 2·0 × 10⁻⁵ M, for cyclic GMP were confirmed. The K_i of SQ 20009 against the low K_m cyclic AMP phosphodiesterase was 2·0 × 10⁻⁶ M and that for cyclic GMP hydrolysis was 2·4 × 10⁻⁵ M. The inhibition by SQ 20009 of the hydrolysis of both cyclic nucleotides by both the rat brain and beef heart phosphodiesterases was competitive. The cat heart cyclic nucleotide phosphodiesterase was inhibited non-competitively by SQ 20009; the K_i for cyclic AMP hydrolysis was 6·4 × 10⁻⁵ M, and the K_i for cyclic GMP hydrolysis was 3·0 × 10⁻⁵ M. The inhibition by SQ 20009 of cyclic AMP hydrolysis by both the rat brain and cat heart preparations was reversible.     

咪苯嗪酮对血小板聚集、血栓形成和血小板环磷酸腺苷含量的影响

本文观察了新型强心剂咪苯嗪酮(Cl-914)对血小板聚集、血栓形成和血小板cAMP含量的影响。用比浊法测定Cl-914体外抑制AA,ADP和胶原诱导兔血小板聚集的IC_(50),分别为2.6,8.9和15.8μM;大鼠iv Cl-914 1.25mg/kg能抑制实验性血栓形成,20 mg/kg能抑制上述三种诱导剂引起的血小板聚集。在体外,用竞争性蛋白结合法测定,CI-914可使洗涤兔血小板cAMP含量明显升高。CI-914能以剂量依赖方式协同PGE_1抑制血小板聚集和升高血小板cAMP的含量。提示CI-914升高血小板cAMP含量可能是其抑制血小板聚集和抗血栓形成的主要机理。     

Binding thermodynamics at the human cannabinoid CB1 and CB2 receptors

The thermodynamic parameters ΔG°, ΔH° and ΔS° of the binding equilibrium of agonists and antagonists at cannabinoid CB₁ and CB₂ receptors were determined by means of affinity measurements at different temperatures and van’t Hoff plots were constructed. Affinity constants were measured on CHO cells transfected with the human CB₁ and CB₂ receptors by inhibition assays of the binding of the cannabinoid receptor agonist [³H]-CP-55,940. van’t Hoff plots were linear for agonists and antagonists in the temperature range 0-30 °C. The thermodynamic parameters for CB₁ receptors fall in the ranges 17 ≤ ΔH° ≤ 59 kJ/mol and 213 ≤ ΔS° ≤ 361 kJ/mol for agonists and −52 ≤ ΔH° ≤ −26 kJ/mol and −12 ≤ ΔS° ≤ 38 kJ/mol for antagonists. The thermodynamic parameters for CB₂ receptors fall in the ranges 27 ≤ ΔH° ≤ 48 kJ/mol and 234 ≤ ΔS° ≤ 300 kJ/mol for agonists and −19 ≤ ΔH° ≤ −17 kJ/mol and 43 ≤ ΔS° ≤ 74 kJ/mol for antagonists. Collectively, these data show that agonist binding is always totally entropy-driven while antagonist binding is enthalpy and entropy-driven, indicating that CB₁ and CB₂ receptors are thermodynamically discriminated. These data could give new details on the nature of the forces driving the CB₁ and CB₂ binding at a molecular level. Enthalpy, entropy, free energy and binding affinity for each ligand to its receptor can all be assessed and therefore the optimal binding profile discovered. Carrying out these binding investigations as early as possible in the discovery process increases the probability that a lead compound will become a successful pharmaceutical compound.     

NMDA receptor heterogeneity in mammalian tissues: focus on two agonists, (2S,3R,4S) cyclopropylglutamate and the sulfate ester of 4-hydroxy-(S)-pipecolic acid

Several potent and selective agonists of the glutamate (L-GLU) receptors of N-methyl-D-aspartate (NMDA) type have been tested on the L-[³H]GLU binding to rat cortical membranes, on the depolarization of mouse cortical wedges and on the contraction of guinea pig longitudinal muscle myenteric plexus preparations with the aim of comparing the NMDA receptors present in the cortex and those present in the gut.When the depolarization of the cortical wedges was evaluated, the EC₅₀ values of the agonists were (M): (R,S)-(tetrazol-5-yl)-glycine (TG) 0.3; trans-4-hydroxy (S)-pipecolic acid-4-sulfate (t-HPIS) 0.7; 1-aminocyclobutane-cis-1,3-dicarboxylic acid (ACBD) 0.8; NMDA 8; (2S,3R,4S) cyclopropylglutamate (L-CGA C) 12; quinolinic acid (QUIN) 400. When the contraction of the longitudinal muscle myenteric plexus was evaluated, the EC₅₀ values were (M): L-CGA C 1; TG 8; ACBD 50; t-HPIS 100; QUIN 500 and NMDA 680. When the displacement of NMDA specific L-[³H]GLU binding from rat cortical membranes was evaluated, the IC₅₀ values were (NM): L-CGA C 0.003; TG 0.005; ACBD 0.044; t-HPIS 0.062; NMDA 0.31 and QUIN 15. No significant correlation was found when the EC₅₀ values obtained in the ileum were plotted against the EC₅₀ values obtained in the cortex (r = 0.47). In particular it was noted that L-CGA C was approximately three orders of magnitude more potent than NMDA when tested in the ileum but had a potency not significantly different from that of NMDA when tested in the cortex. On the contrary, t-HPIS was particularly potent in cortical wedges.The results of these experiments suggest that different populations of NMDA receptors are present in the tissues of the three mammalian species investigated. In particular, the receptors present in the mouse cortical wedges are selectively stimulated by t-HPIS while those present in the guinea pig myenteric plexus are preferentially stimulated by L-CGA C. Thus at least two functional subtypes of NMDA receptors may be identified in mammalian tissues by using the order of potency of selective agonists.     

The role of adenosine A2A and A2B receptors in the regulation of TNF-alpha production by human monocytes

Adenosine is an endogenous nucleoside that regulates many physiological processes through the activation of its four receptors: A(1), A(2A), A(2B) and A(3). Previous studies have identified the involvement of A(2) receptors in the inhibitory activity of adenosine analogues on tumor necrosis factor-alpha (TNF-alpha) production by lipopolysaccharide (LPS) activated monocytes, but the relative contributions of A(2A) versus A(2B) receptors have not been determined in human primary monocytes. Nor has the role of A(1) and A(3) been clearly identified in the system. The lack of such information impacts on the selection of adenosine receptor agonists for disease intervention. Using LPS-stimulated human primary monocytes, we found that the adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA) or the A(2A) receptor agonist, 4-[2-[[6-amino-9-(N-ethyl-b-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride (CGS21680) produced a concentration-dependent inhibition of TNF-alpha production, with IC(50)s of 58.4nM (32.7-104.5nM, 95% confidence interval) and 49.2nM (22.7-105.9nM, 95% confidence interval), respectively. The selective A(2A) receptor blocker, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylaminso]ethyl)phenol (ZM241385, 30nM), antagonized the effects of NECA and CGS21680 (pK(B) estimates were 8.7+/-0.1 and 8.9+/-0.1, respectively), while the selective A(2B) antagonist, N-(4-cyano-phenyl)-2-[4-(2,6-dioxo-1,3-dipropyl-2,3,4,5,6,7-hexahydro-1H-purin-8-yl)-phenoxy]-acetamide (MRS1754, 100nM), failed to antagonize the effects of either agonist. Furthermore, neither the A(1) receptor agonist, 2-chloro-N(6)-cyclopentyladenosine (CCPA) nor the A(3) receptor agonist, 1-[2-chloro-6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-b-d-ribofuranuronamide (2-Cl-IB-MECA) showed significant inhibitory activity at concentrations that effectively bind to their respective receptors. We conclude that A(2A) receptor activation is predominantly responsible for the inhibitory effects of adenosine receptor agonists on TNF-alpha production from LPS-stimulated monocytes.     

Mass spectrometric analysis of 4-hydroxy-1-(3-pyridyl)-1-butanone-releasing DNA adducts in human lung

An improved analytical method was developed for the analysis of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB)-releasing DNA adducts in lung samples of patients undergoing surgery for lung cancer. HPB-releasing adducts can be formed by metabolic activation of the tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N'-nitrosonornicotine, and have been reported to play an important role in tobacco carcinogenesis. [2,2,3,3-D(4)]HPB (D(4)-HPB) was used as an internal standard, and HPB released by acid hydrolysis of DNA was determined by gas chromatography/mass spectrometry in the negative ion chemical ionisation mode. The method is sensitive with a limit of detection of 5.9 fmol HPB and a limit of quantification of 15.2 fmol HBP/mg DNA. The recovery of HPB was 82+/-17% and the background response was 10.1+/-1.8 fmol HPB/sample. The concentration of HPB-releasing lung DNA adducts was significantly higher (p<0.0001) in 21 self-reported smokers compared to in 11 self-reported nonsmokers (404+/-258 fmol versus 59+/-56 fmol HPB/mg DNA, respectively). HPB-releasing hemoglobin adduct concentrations were only marginally higher in a subset of 12 smokers compared to in 7 nonsmokers (63+/-53 fmol versus 42+/-34 fmol HPB/g hemoglobin; p=0.36). No correlation was found between HPB-releasing adducts in DNA and hemoglobin (p=0.074).     

Novel Alexa Fluor-488 labeled antagonist of the A2A adenosine receptor: Application to a fluorescence polarization-based receptor binding assay

Fluorescence polarization (FP) assay has many advantages over the traditional radioreceptor binding studies. We developed an A_2A adenosine receptor (AR) FP assay using a newly synthesized fluorescent antagonist of the A_2AAR (MRS5346), a pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine derivative conjugated to the fluorescent dye Alexa Fluor-488. MRS5346 displayed a K_i value of 111 ± 16 nM in radioligand binding using [³H]CGS21680 and membranes prepared from HEK293 cells stably expressing the human A_2AAR. In a cyclic AMP functional assay, MRS5346 was shown to be an A_2AAR antagonist. MRS5346 did not show any effect on A₁ and A₃ ARs in binding or the A_2BAR in a cyclic AMP assay at 10 μM. Its suitability as a fluorescent tracer was indicated in an initial observation of an FP signal following A_2AAR binding. The FP signal was optimal with 20 nM MRS5346 and 150 μg protein/mL HEK293 membranes. The association and dissociation kinetic parameters were readily determined using this FP assay. The K_d value of MRS5346 calculated from kinetic parameters was 16.5 ± 4.7 nM. In FP competition binding experiments using MRS5346 as a tracer, K_i values of known AR agonists and antagonists consistently agreed with K_i values from radioligand binding. Thus, this FP assay, which eliminates using radioisotopes, appears to be appropriate for both routine receptor binding and high-throughput screening with respect to speed of analysis, displaceable signal and precision. The approach used in the present study could be generally applicable to other GPCRs.     

Noble 2-[3-(cyclopentyloxy)-4-methoxyphenyl]-1 -isoindolinone derivatives. Part I: synthesis and SAR studies for the inhibition of TNF-α production

This study describes the synthesis and in vitro evaluation of noble 2-[3-(cyclopentyloxy)-4-methoxyphenyl]-1-isoindolinone derivatives for the inhibition of TNF-alpha production. Among these compounds, 2-[3-(cyclopentyloxy)-4-methoxyphenyll-3-methyl-1-isoindolinone (5) was the most potent in inhibitory activity of TNF-alpha production in LPS-stimulated RAW264.7 cells.