Mutations of aspartate 103 in the Hm2 receptor and alterations in receptor binding properties of muscarinic agonists

Aspartate 103 (D103) in the third transmembrane domain of the Hm2 receptor was mutated to glutamate (D103E), asparagine (D103N), or alanine (D103A). As measured by [3H]-NMS, no significant binding was observed in D103A, while a 2-fold decrease in ligand affinity was seen in D103E and a 32-fold decrease in affinity was found in the D103N mutant. Examination of reference agonists showed greater loss of affinity in D103N than in D103E with the rank order of change being: L-607,207>carbachol>arecoline>pilocarpine>oxotremorine>McN-A-343. Of the novel 1-azabicyclo[2.2.1]-heptan-3-one oxime agonists examined, arylacetylene oximes showed little alteration in binding in either the D103E or D103N mutants, while the geometric isomers of several bicyclic aryl-ene-yne oximes showed significant changes in affinity, especially in the D103N mutant. Thus, overall size of the agonist and/or spatial orientation of the molecule within the binding pocket contribute to changes measured in binding.     

Classification of kinin receptors

This minireview is divided into three parts: the first part refers to the characterization and classification of kinin receptors using agonists and antagonists in isolated tissues (classical pharmacology). Two kinin receptors have been considered on the basis of their distinct pharmacology, namely the B1 receptor of the rabbit aorta (rank order of potency of agonists: LysdesArg9BK > desArg9BK > or = LysBK > BK; apparent affinities of antagonists Lys[Leu8]desArg9BK (pIC50 8.4) > [Leu8]desArg9BK (pIC50 7.4) > HOE 140, a B2 receptor antagonist, pIC50<5.0), and the B2 receptor of the rabbit jugular vein (potency of agonists: LysBK = BK > LysdesArg9BK = desArg9BK and HOE 140 (pIC50 9.0) > Lys[Leu8]desArg9BK, pIC50<5.0). The second part describes species-related B1 receptor subtypes, demonstrated by different pharmacological profiles of agonists and antagonists: human, rabbit and pig subtypes (LysdesArg9BK > desArg9BK and Lys[Leu8]desArg9BK > [Leu8]desArg9BK) and dog, rat, mouse and hamster B1 receptors (desArg9BK = LysdesArg9BK and [Leus]desArg9BK = Lys[Leu8]desArg9BK). Affinities of agonists and antagonists in some species (man, rabbit, pig) are significantly increased (at least 10-fold) by the presence of a Lys at their N-terminus. The last part describes species-related B2 receptor subtypes supported by results obtained with non-peptide receptor agonists (FR 190997) and antagonists (FR 173657). While BK acts as a full agonist in man, rabbit and pig, FR 190997 behaves as a full agonist on human, as partial agonist on rabbit, and as pure antagonist on pig B2 receptors. Various hypotheses are considered to interpret these findings.     

D-amino acid and alanine scans of the bioactive portion of porcine motilin.

A recent systematic study of porcine motilin fragments has clearly shown that biological activity resides in the amino-terminal end. The amino-terminal tetradecapeptide retains more than 90% of the potency of the full molecule. We now examined the effect of replacement of residues 1 through 11 by either their D-isomer or by alanine in [Leu13]pMOT(1-14). Peptides were synthesized using Fmoc solid phase methodology, purified by HPLC, and assayed for their ability to displace bound motilin (rabbit antral smooth muscle homogenate) and to induce contractions (isolated rabbit duodenal segments). The negative logarithm of the concentration displacing 50% of the tracer (pIC50), or producing 50% of the maximal contractile response (pEC50), was determined. All compounds were still full agonists. A reduction in potency of more than two log units was seen for the compounds in which residues 1 (Phe), 4 (Ile), and 7 (Tyr) were replaced by Ala and residues 3 (Pro), 4 (Ile), and 6 (Thr) by their D-isomer. The largest drop was noted for the analogs substituted at position 4. For all compounds there was an almost perfect correlation between the pIC50 and the pEC50 values (r = 0.96), although the pEC50 was consistently smaller. These results show that the biological activity of motilin is mainly determined by the first seven residues. The pharmacophore consists of the aromatic rings from Phe1 and Tyr7 and the aliphatic side chains from Val2 and Ile4. Pro3, Phe5, and Thr6 may stabilize the bioactive conformation.     

Potent vasodilator responses to human urotensin-II in human pulmonary and abdominal resistance arteries

The peptide human urotensin-II (hUT-II) and its receptor have recently been cloned. The vascular function of this peptide in humans, however, has yet to be determined. Vasoconstrictor and vasodilator responses to hUT-II were investigated in human small muscular pulmonary arteries [approximately 70 microm internal diameter (ID)] and human abdominal resistance arteries (approximately 200 microm ID). Vasodilator responses were investigated in endothelin-1 (3 nM) precontracted vessels and, in the small pulmonary vessels, compared with the known vasodilators adrenomedullin, sodium nitroprusside, and acetylcholine. In human small pulmonary arteries, hUT-II did not induce vasoconstriction but was a potent vasodilator [-log M concentration causing 50% of the maximum vasodilator effect (pIC(50)) 10.4 +/- 0.5; percentage of reduction in tone (E(max)) 81 +/- 8% (vs. 23 +/- 11% in time controls), n = 5]. The order of potency for vasodilation was human urotensin-II = adrenomedullin (pIC(50) 10.1 +/- 0.4, n = 6) > sodium nitroprusside (pIC(50) 7.4 +/- 0.2, n = 6) = acetylcholine (pIC(50) 6.8 +/- 0.3, n = 6). In human abdominal arteries, hUT-II did not induce vasoconstriction but was a potent vasodilator [pIC(50) 10.3 +/- 0.7; E(max) 96 +/- 8% (vs. 43 +/- 16% in time controls), n = 4]. This is the first report that hUT-II is a potent vasodilator but not a vasoconstrictor of human small pulmonary arteries and systemic resistance arteries.     

Synthesis and contractile activity of new pseudopeptido and thioaromatic analogues of leukotriene D4.

Seven new pseudopeptido and thioaromatic leukotriene analogues were synthesized and their agonist-antagonist and binding activities investigated. The synthesis led to the pleasing observation that the analogue in which the cysteinyl-glycine moiety was replaced by a 6-mercapto-3-(E)-hexenoic acid, not only exhibited potent affinity (guinea-pig lung parenchyma, IC50: 5 x 10(-9) M) but also showed 30% of the LTD4 agonist activity (guinea-pig ileum, ED 50: 2.7 x 10(-9)) giving very important key information on LTD4 geometry to the receptor. This compound was the first stable new pseudopeptido-leukotriene with such agonist activity and should contribute to the understanding of the metabolism of leukotriene D4. In addition, inversion of chirality at C5 and C6 carbon atoms of the leukotriene chain or replacement of the cysteinyl-glycine moiety by a thioaromatic acid led to new weak antagonists of the LTD4.     

Inhibition of [3H]ponasterone A binding by ecdysone agonists in the intact Kc cell line.

Inhibition of the binding of [3H]ponasterone A ([3H]PoA) by ecdysone agonists including diacylhydrazines such as RH-5849, tebufenozide (RH-5992) and methoxyfenozide (RH-2485) was examined in intact Drosophila Kc cells. The reciprocal logarithm of the concentration at which there is 50% inhibition of [3H]PoA binding, pIC(50) (M), was determined as the binding activity for all compounds from each concentration-response curve. The order of the activity was PoA>20-hydroxyecdysone>cyasterone>inokosterone>or=makisterone A>methoxyfenozide>or=tebufenozide>ecdysone>RH-5849. The ranking of steroidal ecdysone analogs is consistent with that obtained against Spodoptera Sf-9 cells. Furthermore, in terms of pIC(50), all binding activity for ecdysone analogs, except ecdysone, estimated in the Kc cell line system was significantly higher than that for the Sf-9 cell line system. However, the activity of ecdysone was comparable between Kc and Sf-9 cells. The activity of diacylhydrazine analogs against Kc cells was significantly low compared with that against Sf-9 cells. The potency of methoxyfenozide was 1/200 that of PoA, which showed the highest activity in the Kc cell line system among all compounds tested. The activity of tebufenozide analogs having an n-pentyl or n-hexyl group instead of a 4-ethylphenyl group was similar to that of RH-5849.     

Synthesis of ponasterone A derivatives with various steroid skeleton moieties and evaluation of their binding to the ecdysone receptor of Kc cells

A series of ponasterone A (PNA) derivatives with various steroid moieties were synthesized to measure their binding activity to the ecdysone receptors of Drosophila Kc cells. The activity of compounds was evaluated by determining the concentration required to give the 50% inhibition (IC(50) in M) of the incorporation of [(3)H]PNA to Drosophila Kc cells. Compounds with no functional groups such as OH and CO group in the steroid skeleton moiety were inactive. By the introduction of functional groups such as the OH and the CO group in the steroidal structure, these compounds became active. Some compounds containing the A/B-trans ring fusion, which is different from that (A/B-cis) of ecdysteroids were also active. The oxidation of CH(2) at 6-position to CO, enhanced the activity 19 times, but the activity was erased by the reduction of oxo to OH group at 6-position. The activity was enhanced about 250 times by the conversion of A/B ring configuration from trans [(20R,22R)-2beta,3beta,20,22-tetrahydroxy-5alpha-cholestan-6-one: pIC(50)=4.84] to cis [(20R,22R)-2beta,3beta,20,22-tetrahydroxy-5beta-cholestan-6-one: pIC(50)=7.23]. The latter cis-type compound which is the most potent among compounds synthesized in this study was equipotent to the natural molting hormone, 20-hydroxyecdysone, even though it is 1/50 of PNA.     

The first X-ray crystal structure of the glucocorticoid receptor bound to a non-steroidal agonist

The amino-pyrazole 2,6-dichloro-N-ethyl benzamide 1 is a selective GR agonist with dexamethasone-like in vitro potency. Its X-ray crystal structure in the GR LBD (Glucocorticoid ligand-binding domain) is described and compared to other reported structures of steroidal GR agonists in the GR LBD (3E7C).     

Effect of novel N-aryl sulfonamide substituted 3-morpholino arecoline derivatives as muscarinic receptor 1 agonists in Alzheimer's dementia models

A series of novel, potent, and selective muscarinic receptor 1 agonists (M1 receptor agonists) that employ a key N-substituted morpholine Arecoline moiety has been synthesized as part of research effort for the therapy of Alzheimer’s diseases. The ester group of arecoline (which is reported as muscarinic agonist) has been replaced by N-substituted morpholine ring. The structure–activity relationship reveals that the electron donating 4-substituted sulfonyl derivatives (9a, 9b, 9c, and 9e) on the nitrogen atom of the morpholine ring increases the affinity of M1 receptor binding 50- to 80-fold greater than the corresponding arecoline. Other derivatives also showed considerable M1 receptor binding affinity.     

Polyclonal antibodies to EB1089 (seocalcitol), an analog of 1alpha,25-dihydroxyvitamin D(3)*

A hapten derivative of EB1089 [1(R),3(S),25-trihydroxy-26,27-dimethyl-9,10-seco-24-homocholesta-5(Z),7(E),10(19),22(E),24(E)-pentaene], a side-chain analog of 1alpha,25-dihydroxyvitamin D(3), was synthesized for raising antibodies with a high specificity for EB1089. The A-ring moiety of EB1089 was replaced in the hapten by a linker for conjugation to a protein. Three polyclonal antibodies were obtained by immunizing rabbits with a BSA-conjugate of the hapten. The antibodies were characterized for titer, avidity and specificity using an enzyme immunoassay with covalently bound EB1089. The three antibodies had similar binding profiles and were highly selective for EB1089 and its metabolites over the naturally occurring vitamin D metabolites. Cross-reactivities with 25-hydroxyvitamin D(3), the most abundant vitamin D metabolite in serum, were in the range 0.01-0.2% relative to EB1089.     

A novel hypothesis for the binding mode of HERG channel blockers

We present a new docking model for HERG channel blockade. Our new model suggests three key interactions such that (1) a protonated nitrogen of the channel blocker forms a hydrogen bond with the carbonyl oxygen of HERG residue T623; (2) an aromatic moiety of the channel blocker makes a pi-pi interaction with the aromatic ring of HERG residue Y652; and (3) a hydrophobic group of the channel blocker forms a hydrophobic interaction with the benzene ring of HERG residue F656. The previous model assumes two interactions such that (1) a protonated nitrogen of the channel blocker forms a cation-pi interaction with the aromatic ring of HERG residue Y652; and (2) a hydrophobic group of the channel blocker forms a hydrophobic interaction with the benzene ring of HERG residue F656. To test these models, we classified 69 known HERG channel blockers into eight binding types based on their plausible binding modes, and further categorized them into two groups based on the number of interactions our model would predict with the HERG channel (two or three). We then compared the pIC(50) value distributions between these two groups. If the old hypothesis is correct, the distributions should not differ between the two groups (i.e., both groups show only two binding interactions). If our novel hypothesis is correct, the distributions should differ between Groups 1 and 2. Consistent with our hypothesis, the two groups differed with regard to pIC(50), and the group having more predicted interactions with the HERG channel had a higher mean pIC(50) value. Although additional work will be required to further validate our hypothesis, this improved understanding of the HERG channel blocker binding mode may help promote the development of in silico predictions methods for identifying potential HERG channel blockers.     

The metabotropic glutamate receptor (MGR): pharmacology and subcellular location.

A pharmacological characterization of the metabotropic glutamate receptor (MGR) was performed in striatal neurons. Among the excitatory amino acid receptor antagonists tested, only D, L-2-amino-3-phosphonopropionate (D, L-AP3) inhibited QA-induced inositol phosphate (InsP) formation in a competitive manner (mean pKi = 4.45 +/- 0.43, n = 4). However, this drug was a partial agonist of MGR since it stimulated the inositol-phosphate formation. We found that D, L-AP3 also inhibited NMDA-induced calcium increase, in a competitive manner (mean pIC50 = 4.34 +/- 0.22, n = 8, and mean pKi = 3.7 +/- 0.11 n = 5). 1 mM of the ionotropic agonists alpha-amino-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate (KA) or domoate (DO) (100 microM or higher) induced a significant InsP formation in striatal neurons. The InsP responses induced by all these agonists were totally blocked by the phorbol ester phorbol-12,13-dibutyrate (PdBu), but not by atropine or prazosin. Agonist-induced increases of intracellular calcium concentrations ([Ca2+]i) were insensitive to PdBu, suggesting that all these substances were able to stimulate the MGR in striatal neurons. Trans-1-amino-cyclopentyl-1,3-dicarboxylate (trans-ACPD) evoked dose-dependent inositol phosphate formations with an EC50 of 29 microM but had no significant effect on NMDA or AMPA receptors, as measured by the patch clamp technique. In the presence of 30 microM of AMPA, trans-ACPD induced a significant release of arachidonic acid (AA) in striatal neurons. No important AA release was observed by any of these agonists alone. 56 mM K+ did not mimic AMPA in this associative ionotropic/metabotropic effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Down-regulation by nuclear factor kappaB of human 25-hydroxyvitamin D3 1alpha-hydroxylase promoter

1,25-(OH)(2) vitamin D(3) is important for calcium homeostasis and cell differentiation. The key enzyme for the activation of liver-derived 25(OH) vitamin D(3) is 25-hydroxyvitamin D(3) 1alpha-hydroxylase. It is expressed mainly in the kidney but also in peripheral tissues. A 1413-bp fragment of the 1alpha-hydroxylase promoter was cloned into luciferase vectors pGL2basic and pGL3basic. Sequence analyses revealed four base exchanges and three base deletions compared with the published sequence which were identically found in five control persons. In silico promoter analyses revealed 17 putative nuclear factor (NF)kappaB sites, 10 of which were found to bind NFkappaB in EMSA experiments. Cotransfection of NFkappaB p50 and p65 subunits resulted in dramatic reduction of the promoter activity of the full-length construct as well as a series of 5'-deletion constructs. Deletion of the farmost 3'-situated NFkappaB-responsive element almost abolished NFkappaB responsiveness. Treatment of human embryonic kidney 293 cells with sulfasalazine, a NFkappaB inhibitor, resulted in enhanced 1alpha-hydroxylase mRNA production. Down-regulation of 1alpha-hydroxylase promoter through NFkappaB signaling may contribute to the pathogenesis of inflammation-associated osteopenia/osteoporosis.     

2-Aryl-N-acyl indole derivatives as liver X receptor (LXR) agonists

Structure-activity relationship studies on a series of Boc-indole derivatives as LXR agonists are described. Compound 1 was identified as an LXR agonist through structure-based virtual screening followed by high-throughput gene profiling. Replacement of the indan linker portion in 1 with an open-chain linker resulted in compounds with similar or improved in vitro potency and cellular functional activity. The Boc group at the N-1 position of the indole moiety can be replaced with a benzoyl group. The SAR studies led to the identification of compound 8, a potent LXRbeta agonist with an EC50 of 12 nM in the cofactor recruitment assay.     

人源CDC50基因的分子克隆及在E.coli中的表达

糖皮质激素受体 (GR)为一种特异性配体诱导的转录因子在机体发育、生理及行为等过程中发挥重要作用 .这一过程往往受到转录协同调节因子的控制 ,包括协同抑制因子或协同激活因子的作用 .克隆了人源双跨膜受体分子 ,并命名为hCDC5 0 .该分子编码区有 10 5 6bp ,与鼠源基因mCDC5 0在核苷酸水平有 90 %一致性 .将hCDC5 0蛋白的 16 4～ 317编码序列与GST融合构建原核表达质粒进行重组蛋白的表达与纯化并制备多抗 .用二维双向扩散检测抗体为阳性 ,抗原的滴度为12 5 μg/ml.体外翻译研究显示该基因编码蛋白约为 4 0kD ,与Western检测在动物细胞中的表达一致 .用hCDC5 0真核表达质粒与GR萤光报告系统共转染COS 7细胞显示 ,hCDC5 0对GR信号转导有明显的抑制作用 ,其对GR信号通路的抑制作用达 3～ 4倍以上 .hCDC5 0可能为一个新的协同抑制因子 ,在抑制GR的转录激活过程中起作用     

Antiplasmodial activity of nitroaromatic and quinoidal compounds: redox potential vs. inhibition of erythrocyte glutathione reductase

Prooxidant nitroaromatic and quinoidal compounds possess antimalarial activity, which might be attributed either to their formation of reactive oxygen species or to their inhibition of antioxidant enzyme glutathione reductase (GR, EC 1.6.4.2). We have examined the activity in vitro against Plasmodium falciparum of 24 prooxidant compounds of different structure (nitrobenzenes, nitrofurans, quinones, 1,1'-dibenzyl-4,4'-bipyridinium, and methylene blue), which possess a broad range of single-electron reduction potentials (E(1)(7)) and erythrocyte glutathione reductase inhibition constants (K(i(GR))). For a series of homologous derivatives of 2-(5'-nitrofurylvinyl)quinoline-4-carbonic acid, the relationship between compound K(i(GR)) and concentration causing 50% parasite growth inhibition (IC(50)) was absent. For all the compounds examined in this study, the dependence of IC(50) on their K(i(GR)) was insignificant. In contrast, IC(50) decreased with an increase in E(1)(7) and positive electrostatic charge of aromatic part of molecule (Z): log IC(50) (microM) = -(0.9846 +/- 0.3525) - (7.2850 +/- 1.2340) E(1)(7) (V) - (1.1034 +/- 0.1832) Z (r(2) = 0.8015). The redox cycling activity of nitroaromatic and quinoidal compounds in ferredoxin:NADP(+) reductase-catalyzed reaction and the rate of oxyhemoglobin oxidation in lysed erythrocytes increased with an increase in their E(1)(7) value. Our findings imply that the antiplasmodial activity of nitroaromatic and quinoidal compounds is mainly influenced by their ability to form reactive oxygen species, and much less significantly by the GR inhibition.     

Substituted phenyl as a steroid A-ring mimetic: Providing agonist activity to a class of arylsulfonamide nonsteroidal glucocorticoid ligands

A class of arylsulfonamide glucocorticoid receptor agonists that contains a substituted phenyl group as a steroid A-ring mimetic is reported. The structural design and SAR that provide the functional switching of a GR antagonist to an agonist is described. A combination of specific hydrogen bonding and lipophilic elements on the A-ring moiety is required to achieve potent GR agonist activity. This study culminated in the identification of compound 23 as a potent GR agonist with selectivity over the PR and MR nuclear hormone receptors.