Synthesis and biological activity of 2-alkylbenzimidazoles bearing a N-phenylpyrrole moiety as novel angiotensin II AT1 receptor antagonists

A series of 2-alkylbenzimidazoles bearing a N-phenylpyrrole moiety were synthesized and evaluated as a novel class of AT(1) receptor antagonists. Among them, compounds 10a and 10g inhibited [(125)I] AngII-binding affinity to AT(1) receptor at nanomolar level and potently inhibited the Ang II-induced pressor response by oral administration. Moreover, evaluation in spontaneously hypertensive rats showed that 10a is an orally active AT(1) receptor antagonist.     

Synthesis and biological evaluation of some 6-arylamidomorphines as analogues of morphine-6-glucuronide

A series of 6-beta-arylamidomorphines was synthesized and biologically evaluated. Various aryl substituents were introduced into the arylamidomorphines to examine substituent structure-activity relationships. Competition binding assays showed that compounds 10a-h bound to the mu opioid receptor with high affinity (0.2-0.6 nM). Functional assays showed that compounds 10a-h acted as full mu opioid receptor agonists. The ED(50) of compound 10e.HCl as an analgesic was 12.6 mg/kg in the tail flick latency test in the rat.     

Naphthofuroquinone derivatives: inhibition of receptor tyrosine kinases

A series of dinaphtho[1,2-b;2',3'-d]furan-7,12-dione derivatives were synthesized and evaluated for inhibitory activities against receptor tyrosine kinases. The naphthofuroquinone compounds with dialkylaminoethoxy group at C(5)-position (7, 8, 10, and 11) manifested strong inhibitory activities against epidermal growth factor receptor and vascular endothelial growth factor receptor. Docking study of 11 with EGFR was also performed.     

MIF-1 and Tyr-MIF-1 augment GABA-stimulated benzodiazepine receptor binding

Behavioral evidence in laboratory animals and human beings indicates possible links between the endogenous opiate and gamma-aminobutyric acid (GABA)-benzodiazepine receptor systems, especially with regard to antagonistic properties. To assess possible interactions between endogenous opiate antagonists and benzodiazepine receptor binding, we evaluated the effects of the peptides MIF-1 and Tyr-MIF-1 on benzodiazepine receptor binding in mouse brain membranes. Neither peptide affected receptor binding in cortex over a broad dose range, but both peptides significantly augmented GABA-stimulated benzodiazepine receptor binding at GABA concentrations of 10(-8) and 10(-7) M. Rosenthal-Scatchard analysis indicated that the increase in binding was largely due to increased apparent affinity. Both peptides augmented GABA-enhanced binding at low doses (MIF-1 10(-11) M, Tyr-MIF-1 10(-13) M) with decreased effects at higher doses. In cerebellum and brainstem, MIF-1 tended to enhance GABA-stimulated binding but Tyr-MIF-1 was inactive. These results indicate benzodiazepine-opiate and benzodiazepine-peptide interactions.     

Selection, synthesis, and structure-activity relationship of tetrahydropyrido[4,3-d]pyrimidine-2,4-diones as human GnRH receptor antagonists

The present article describes a selection of a new class of small molecule antagonists for the h-GnRH receptor, their preparation, and evaluation in vitro. Three computational methods were combined into a consensus score, to rank order virtual templates. The top 5% of templates were further evaluated in silico and assessed for novelty and synthetic accessibility. The tetrahydropyrido[4,3-d]pyrimidine-2,4-dione core was selected for synthesis and evaluated in vitro. Using an array approach for analog design and synthesis, we were able to drive the binding below 10nM for the h-GnRH receptor after two rounds of optimization.     

Opioid agonist/antagonist effect of naloxone in modulating rabbit jejunum contractility in vitro.

Opioid peptides are the most effective drugs in controlling pain; their action is elicited by binding to specific membrane receptors. The gastrointestinal tract represents, after the nervous system, the site in which the opioid receptors are expressed at high levels. The opioid agonist morphine has a significant inhibitory effect on intestinal motility, this action is blocked by naloxone an opioid antagonist mainly active at mu and kappa receptors. In this study the presence of mu opioid receptor on rabbit jejunum was investigated by western blot. The effects of beta-endorphin, the endogenous opioid peptide with the highest affinity to the mu opioid receptor and those of naloxone on spontaneous rabbit jejunum contractions were evaluated. Beta-endorphin (10(-6) M) showed a relaxant effect on jejunum contractility while naloxone showed a dual effect inducing an increase of spontaneous contractility at low concentrations (10(-6) M, 10(-7) M, 10(-8) M) and a decrease when high concentrations (10(-3) M, 10(-4) M, 10(-5) M) were utilized. The obtained results demonstrate that mu opioid receptor is expressed in rabbit jejunum and suggest that this receptor may be involved in mediating the effects of both opioid agonist and antagonist on jejunum contractions.     

Low molecular weight hyaluronan shielding of DNA/PEI polyplexes facilitates CD44 receptor mediated uptake in human corneal epithelial cells

AIM: It was the aim of this study to prepare purified DNA/PEI polyplexes, which are coated with hyaluronan to facilitate CD44 receptor mediated uptake of the DNA/PEI polyplex and to reduce unspecific interactions of the complex with negatively charged extracellular matrix components on the ocular surface. METHODS: Hyaluronans of different molecular weights (<10 kDa, 10-30 kDa and 30-50 kDa) were isolated after enzymatic degradation of high molecular weight hyaluronan via ultrafiltration by centrifugation. The influence of the different hyaluronans used for coating on the stability and transfection efficiency of the complexes was evaluated in vitro. Transfection and uptake studies were performed in human corneal epithelial (HCE) cells. CD44 receptor expression of this cell model was evaluated by immunohistochemistry. RESULTS: Coating of purified DNA/PEI polyplexes with low molecular weight hyaluronan (<10 kDa) facilitated receptor-mediated uptake via the CD44 receptor in HCE cells, increased complex stability in vitro, and effectively shielded the positive surface charges of the polyplex without decreasing its transfection efficiency. Higher molecular weights and larger amounts of hyaluronan in the complexes resulted in lesser improvements in the stability and transfection efficacy of the complexes. CONCLUSIONS: Coating of polyplexes with low molecular weight hyaluronan is a promising strategy for gene delivery to the ocular surface, where CD44 receptor mediated uptake decreased cytotoxicity and reduced non-specific interactions with the negatively charged extracellular matrix components are considered beneficial for increased transfection efficiency of non-viral vectors.     

Modeling of neuropeptide receptors Y1, Y4, Y5, and docking studies with neuropeptide antagonist

Neuropeptide Y (NPY), receptors belong to the G-protein coupled receptor superfamily. NPY mediates several physiological responses, such as blood pressure, food intake, sedation. These actions of NPY are mediated by six receptor subtypes denoted as Y1-Y5 and y6. Modeling of receptor subtypes and binding site identification is an important step in developing new therapeutic agents. We have attempted to model the three NPY receptor types, Y1, Y4, and Y5 using homology modeling and threading methods. The models are consistent with previously reported experimental evidence. To understand the interaction and selectivity of NPY analogues with different neuropeptide receptors, docking studies of two neuropeptide analogues (BVD10 and BVD15) with receptors Y1 and Y4 were carried out. Results of the docking studies indicated that the interaction of ligands BVD10 and BVD15 with Y1 and Y4 receptors are different. These results were evaluated for selectivity of peptide analogues BVD10 and BVD15 towards the receptors.     

Modeling of Neuropeptide Receptors Y1, Y4, Y5, and Docking Studies with Neuropeptide Antagonist Analogues: Implications for Selectivity

Abstract Neuropeptide Y (NPY), receptors belong to the G-protein coupled receptor superfamily. NPY mediates several physiological responses, such as blood pressure, food intake, sedation. These actions of NPY are mediated by six receptor subtypes denoted as Y(1)-Y(5) and y(6). Modeling of receptor subtypes and binding site identification is an important step in developing new therapeutic agents. We have attempted to model the three NPY receptor types, Y1, Y4, and Y5 using homology modeling and threading methods. The models are consistent with previously reported experimental evidence. To understand the interaction and selectivity of NPY analogues with different neuropeptide receptors, docking studies of two neuropeptide analogues (BVD10 and BVD15) with receptors Y1 and Y4 were carried out. Results of the docking studies indicated that the interaction of ligands BVD10 and BVD15 with Y1 and Y4 receptors are different. These results were evaluated for selectivity of peptide analogues BVD10 and BVD15 towards the receptors.     

Tetrahydroprotoberberine alkaloids with dopamine and σ receptor affinity

Two series of analogues of the tetrahydroprotoberberine (THPB) alkaloid (±)-stepholidine that (a) contain various alkoxy substituents at the C10 position and, (b) were de-rigidified with respect to (±)-stepholidine, were synthesized and evaluated for affinity at dopamine and σ receptors in order to evaluate effects on D3 and σ2 receptor affinity and selectivity. Small n-alkoxy groups are best tolerated by D3 and σ2 receptors. Among all compounds tested, C10 methoxy and ethoxy analogues (10 and 11 respectively) displayed the highest affinity for σ2 receptors as well as σ2 versus σ1 selectivity and also showed the highest D3 receptor affinity. De-rigidification of stepholidine resulted in decreased affinity at all receptors evaluated; thus the tetracyclic THPB framework is advantageous for affinity at dopamine and σ receptors. Docking of the C10 analogues at the D3 receptor, suggest that an ionic interaction between the protonated nitrogen atom and Asp110, a H-bond interaction between the C2 phenol and Ser192, a H-bond interaction between the C10 phenol and Cys181 as well as hydrophobic interactions of the aryl rings to Phe106 and Phe345, are critical for high affinity of the compounds.     

New thiazole derivatives as potent and selective 5-hydroxytriptamine 3 (5-HT3) receptor agonists for the treatment of constipation

The syntheses and biological evaluation of a series of novel indeno[1,2-d]thiazole derivatives are described. Several groups reported 5-HT(3) receptor agonists which were mainly evaluated for their activities on the von Bezold-Jarisch reflex (B-J reflex). We discovered that tetrahydrothiazolopyridine derivative 1b had a contractile effect on the isolated guinea pig colon with weak B-J reflex. Our efforts to find a new type of 5-HT(3) receptor agonists on the isolated guinea pig colon focused on the synthesis of a fused thiazole derivative 1d modified from 1b and reverse-fused thiazole derivatives (7-10). In this series, 10f (YM-31636) showed high affinity and selectivity for the cloned human 5-HT(3) receptor; furthermore, it showed potent and selective 5-HT(3) receptor agonistic activity. YM-31636 was examined for its effects on defecation in animals, thus evaluating the compound as an agent against constipation.     

Ligand-mediated internalization, recycling, and downregulation of the epidermal growth factor receptor in vivo [published erratum appears in J Cell Biol 1990 Jan;110(1):following 227]

EGF receptor internalization, recycling,a nd downregulation were evaluated in liver parenchyma as a function of increasing doses of injected EGF. The effect of ligand occupancy in vivo on the kinetics and extent of internalization was studied with changes in the receptor content of isolated plasmalemma and endosome fractions evaluated by direct binding, Scatchard analysis, and Western blotting. For all doses of injected EGF, receptor was lost from the plasmalemma and accumulated in endosomes in a time- and dose-dependent fashion. However, at doses of injected EGF equivalent to less than or equal to 50% surface receptor occupancy (i.e., less than or equal to 1 microgram/100 g body weight), receptor levels returned by 120 min to initial values. This return was resistant to cycloheximide and therefore did not represent newly synthesized receptor. Neither was the return due to replenishment by an intracellular pool of low-affinity receptors as such a pool could not be detected by Scatchard analysis or Western blotting. Therefore, receptor return was due to the recycling of previously internalized receptor. At doses of injected EGF greater than 50% receptor occupancy, net receptor loss-i.e., downregulation-was observed by evaluating the receptor content of total particulate fractions of liver homogenates. At the higher saturating doses of injected EGF (5 and 10 micrograms/100 g body weight), the majority of surface receptor content was lost by 15 min and remained low for at least an additional 105 min. As the kinetics of ligand clearance from the circulation and liver parenchyma were similar for all doses of EGF injected, then the ligand-mediated regulation of surface receptor content and downregulation were not a result of a prolonged temporal interaction of ligand with receptor. Rather, the phenomena must be a consequence of the absolute concentrations of EGF interacting with receptor at the cell surface and/or in endosomes.     

Leptin regulation of prepro-orexin and orexin receptor mRNA levels in the hypothalamus

The aim of this study was to determine the effects of leptin treatment on prepro-orexin and orexin receptor expression in the rat hypothalamus. Adult male rats, food-deprived for 48 and 72 h, were treated one time with vehicle or leptin (10 microg, icv). Prepro-orexin mRNA content was measured by semiquantitative RT-PCR, Northern blot, and in situ hybridization; orexin receptor 1 and 2 mRNA content was quantified by Northern blot and/or semiquantitative RT-PCR. Our results indicate that leptin inhibits a fasting-induced increase in prepro-orexin mRNA and orexin receptor 1 mRNA levels in the rat hypothalamus, while orexin receptor 2 mRNA levels were unchanged in all situations evaluated. These data provide direct evidence for an additional mechanism of adaptation of the hypothalamus to food deprivation and for a new effect of leptin in the regulation of food intake.     

Establishing a cellular FRET-based fluorescence plate reader assay to monitor proNGF-induced cross-linking of sortilin and the neurotrophin receptor p75NTR

Whereas the proform of the nerve growth factor (proNGF) is crucial for eliminating superfluous cells during neuronal development it also promotes apoptosis following brain trauma and neuronal injury. The apoptotic signal is elicited upon formation of a trimeric receptor complex also containing the vps10p domain receptor sortilin and the neurotrophin receptor p75^NTR. However, proNGF-induced receptor complex formation has been difficult to directly assess other than by western blotting. We here describe a fluorescence resonance energy transfer (FRET) based fluorescence plate reader assay to monitor the interaction between fluorescently tagged sortilin and p75^NTR in live cells. The method is based on a standard fluorescent plate reader found in many biochemical laboratories and the results are evaluated using a microscopy-based quantified sensitized acceptor emission FRET approach making use of a pair of FRET standard constructs. As a result, the effect of proNGF on the interaction between sortilin and p75^NTR can be evaluated in live cells allowing for screening and selection of therapeutic compounds interfering with proNGF-induced cell death.     

The effect of angiotensin II on intracellular pH is mediated by AT(1) receptor translocation

The effect of ANG II on intracellular pH (pH(i)) recovery rate and AT(1) receptor translocation was investigated in transfected MDCK cells. The pH(i) recovery rate was evaluated by fluorescence microscopy using the fluorescent probe BCECF-AM. The human angiotensin II receptor isoform 1 (hAT(1)) translocation was analyzed by immunofluorescence and confocal microscope. Our data show that transfected cells in control situation have a pH(i) recovery rate of 0.219 +/- 0.017 pH U/min (n = 11). This value was similar to nontransfected cells [0.211 +/- 0.009 pH U/min (n = 12)]. Both values were significantly increased with ANG II (10(-9) M) but not with ANG II (10(-6) M). Losartan (10(-7) M) and dimethyl-BAPTA-AM (10(-7) M) decreased significantly the stimulatory effect of ANG II (10(-9) M) and induced an increase in Na(+)/H(+) exchanger 1 (NHE-1) activity with ANG II (10(-6) M). Immunofluorescence studies indicated that in control situation, the hAT(1) receptor was predominantly expressed in cytosol. However, it was translocated to plasma membrane with ANG II (10(-9) M) and internalized with ANG II (10(-6) M). Losartan (10(-7) M) induced hAT(1) translocation to plasma membrane in all studied groups. Dimethyl-BAPTA-AM (10(-7) M) did not change the effect of ANG II (10(-9) M) on the hAT(1) receptor distribution but induced its accumulation at plasma membrane in cells treated with ANG II (10(-6) M). With ionomycin (10(-6) M), the receptor was accumulated in cytosol. The results indicate that, in MDCK cells, the effect of ANG II on NHE-1 activity is associated with ligand binding to AT(1) receptor and intracellular signaling events related to AT(1) translocation.     

Tumor-promoting phorbol diesters mediate phosphorylation of the epidermal growth factor receptor

4 beta-Phorbol 12 beta-myristate 13 alpha-acetate (PMA) markedly inhibited the binding of low concentrations (less than 10(-9 m) of 125I-epidermal growth factor (EGF) to A431 human epidermoid carcinoma cells. However, very little change in the binding of 125-I-EGF at high concentrations (greater than 10(-8) M) was observed in response to PMA. Affinity labeling of the 170,000-dalton EGF receptor with 125I-EGF and disuccinimidyl suberate was also decreased by the tumor promoter at low, but not high, concentrations of 125I-EGF. In order to examine this action of PMA on the EGF receptor, the receptor phosphorylation state was evaluated in A431 cells that had been incubated with [32P]phosphate for 3 h prior to the addition of PMA. The 32P content of the EGF receptor purified with EGF-Sepharose was increased by 38% compared with the same amount of receptor isolated from control cells. The increase in EGF receptor phosphorylation was dose-dependent with a half-maximal effect between 0.1 and 1 nM PMA and was specific for tumor promoting analogues of phorbol diesters. Phosphoamino acid analysis indicated that the increase in the 32P content of the EGF receptor was mainly due to phosphoserine. These results demonstrate that the EGF receptor is a target for PMA action and suggest that the mechanism of PMA action on the response of cells to epidermal growth factor may be mediated in part by phosphorylation of the EGF receptor.     

Glucose-induced islet blood flow increase in rats: interaction between nervous and metabolic mediators

This study investigated the mechanisms for glucose-induced islet blood flow increase in rats. The effects of adenosine, adenosine receptor antagonists, and vagotomy on islet blood flow were evaluated with a microsphere technique. Vagotomy prevented the islet blood flow increase expected 3, 10, and 20 min after injection of glucose, whereas theophylline (a nonspecific adenosine receptor antagonist) prevented the islet blood flow increase from occurring 10 and 20 min after glucose administration. Administration of selective adenosine receptor antagonists suggested that the response to theophylline was mediated by A1 receptors. Exogenous administration of adenosine did not affect islet blood flow, but local accumulation of adenosine, induced by the adenosine uptake inhibitor dipyridamole, caused a doubling of islet blood flow. In conclusion, the increased islet blood flow seen 3 min after induction of hyperglycemia is caused by the vagal nerve, whereas the increase in islet blood perfusion seen at 10 and 20 min after glucose administration is caused by both the vagal nerve and adenosine.     

Analysis of receptor reserves in canine tracheal smooth muscle

The receptor reserves for acetylcholine, 5-hydroxytryptamine, and histamine in canine tracheal muscle were evaluated. Muscle strips were dissected free of epithelial and connective tissue and suspended for isometric tension recording in modified Krebs-Ringer solution. Dissociation constants for all three agonists were determined by analysis of their concentration-response curves under control conditions and after partial inactivation of receptors by phenoxybenzamine dihydrochloride. The values of KA for acetylcholine, 5-hydroxytryptamine, and histamine were 1.8 X 10(-5) M, 1.35 X 10(-6) M, and 5.0 X 10(-5) M, respectively. Dissociation constants were used to determine receptor occupancy-response relationships. Maximal responses to acetylcholine were obtained by activation of only 4.0 +/- 1.0% of receptors, indicating the presence of a very large receptor-reserve. In contrast, a maximal response to 5-hydroxytryptamine and histamine required activation of 78.0 +/- 11.0 and 87.7 +/- 1.6% of the receptors, respectively, indicating very modest receptor reserves. The differences in receptor-reserve characteristics for these agonists in airway muscle might contribute to the differential effects of inhibitory and facilitory influences on contractions elicited by them.