Developmental expression of galanin-like immunoreactivity by members of the avian sympathoadrenal cell lineage

So far, no clear correlation has been found between the effects of dopamine D1 receptor agonists on motor behavior in primate models of Parkinson's disease and their ability to stimulate adenylate cyclase in rats, the benzazepine SKF 83959 (3-methyl-6-chloro-7,8-hydroxy-1-[3-methylphenyl]-2,3,4,5-tetrahydro-]H- 3-benzazepine) being the most striking example. Since this discrepancy might be attributed to: (A) the different species used to study these effects or (B) the interaction of SKF 83959 with other catecholamine receptors, the aims of this study were: (1) to study the ability of SKF 83959 to stimulate adenylate cyclase in cultured human and monkey glial cells equipped with dopamine D1 receptors and (2) to evaluate the affinity for and the functional interaction of SKF 83959 with other catecholamine receptors. Binding studies revealed that SKF 83959 displayed the highest affinity for the dopamine D1 receptor (pKi=6.72) and the alpha2-adrenoceptor (pKi=6.41) and moderate affinity for the dopamine D2 receptor and the noradrenaline transporter. In monkey and human cells, SKF 83959 did not stimulate cyclic adenosine monophosphate (cAMP) formation to a significant extent, but antagonized very potently the dopamine-induced stimulation of cAMP formation in both cell types. The compound stimulated basal dopamine outflow and inhibited depolarization-induced acetylcholine release only at concentrations > 10 microM. Finally, SKF 83959 concentration dependently increased electrically evoked noradrenaline release, indicating that it had alpha2-adrenoceptor blocking activity and interfered with the noradrenaline transporter. In conclusion, SKF 83959 is a potent dopamine D1 receptor and alpha2-adrenoceptor antagonist. Thus, the anti-parkinsonian effects of SKF 83959 in primates are not mediated by striatal dopamine D1 receptors coupled to adenylate cyclase in a stimulatory way.     

Functional assessment of alpha 1-adrenoceptor subtypes in porcine coronary artery

1. alpha 1-Adrenoceptors are known to play an important role in vasoconstriction in response to adrenergic stimulation. However, the functional importance of alpha 1-adrenoceptor subtypes at the epicardial coronary artery remains unclear. We examined alpha 1-adrenoceptor subtypes by comparing functional affinities for alpha-adrenoceptor antagonists on noradrenaline (NA)-induced vasoconstriction in porcine denuded right coronary arteries. 2. Noradrenaline induced a dose-dependent vasoconstriction in incubated vessel rings. Prazosin and phentolamine were potent and competitive antagonists for NA-induced contraction (pA2 10.27 and 9.03, respectively). In contrast, the selective alpha 2-adrenoceptor antagonist yohimbine had a low affinity (pA2 6.13). Two selective alpha 1A-adrenoceptor antagonists, WB 4101 and 5-methyl urapidil, were potent and competitive antagonists of alpha 1-adrenoceptor-induced contraction (pA2 10.67 and 8.90, respectively) and the selective alpha 1D-adrenoceptor antagonist BMY 7378 had a low affinity (pA2 6.06). Noradrenaline-induced contraction was insensitive to the alkylating effects of chlorethylclonidine. These observations indicate that the vasoconstriction is predominantly mediated by the alpha 1A-adrenoceptor subtype. This was also supported by a good correlation between pA2 values from the present study and reported binding affinities (pKi) of various alpha-adrenoceptor antagonists with cloned human alpha 1A-adrenoceptors (r = 0.98), but not for alpha 1B- or alpha 1D-adrenoceptor subtypes (r = 0.77 and 0.41, respectively). 3. Our results indicate that the alpha 1A-adrenoceptor is the main functional receptor subtype in porcine denuded coronary arteries.     

Three-dimensional quantitative structure-activity relationship study of nonsteroidal estrogen receptor ligands using the comparative molecular field analysis/cross-validated r2-guided region selection approach

A newly developed comparative molecular field analysis (CoMFA) technique, the cross-validated r2-guided region selection (CoMFA/q2-GRS) method, has been used to build a quantitative structure-activity relationship (3D-QSAR) for nonsteroidal estrogen receptor (ER) ligands. Ligands included in this study belong to a series of diethylstilbestrol (DES) and indenestrol analogues whose affinities for the mouse ER (mER) have been determined in our laboratory. The final model utilized 30 compounds and yielded a q2GRS (cross-validated r2, guided region selection) of 0.796, as compared to a q2 of 0.720 for conventional CoMFA, with a standard error of prediction of 0.594 at 3 principal components. This model was used to visualize steric and electrostatic features of the ligands that correspond with ER binding affinity. Results obtained from the CoMFA steric and electrostatic plots of this model have also been compared to information from the ER binding affinities of substituted estradiol analogues. This is in an effort to determine structural features of compounds in the CoMFA analysis that may correspond to those of the estradiol analogues and to further clarify the mode of binding of nonsteroidal ER ligands.     

Level and instability of day-to-day psychological well-being and risk for depression

Human alpha1-acid glycoprotein (AAG) is a mixture of at least two genetic variants: the A variant and the F1 and/or S variant or variants, which are encoded by two different genes. In a continuation of previous studies indicating specific drug transport roles for each AAG variant according to its separate genetic origin, this work was designed to (1) determine the affinities of the two main gene products of AAG (i.e., the A variant and a mixture of the F1 and S variants) for 35 chemically diverse drugs and (2) to obtain meaningful 3D-QSARs for each binding site. Affinities were obtained by displacement experiments, leading to qualitative indications about binding site characteristics. In particular, drugs binding selectively to the A variant displayed some common structural features, but this was not seen for the F1*S variants. Three-dimensional QSAR analyses using the CoMFA method yielded a steric model for binding to the A variant, from which a simplified haptophoric model was derived. In contrast, no statistically sound model was found for the F1*S variants, possibly due (among other reasons) to an insufficient number of high affinity ligands in the set.     

Structural analysis by the comparative molecular field analysis method of the affinity of beta-adrenoreceptor blocking agents for 5-HT1A and 5-HT1B receptors

The affinities of 17 beta-adrenoreceptor antagonists for 5-HT1A and 5-HT1B receptors were evaluated in binding assays. A large range of Ki values (2-10,000 nM) was observed and ortho or meta substitution of the aromatic ring carrying the amino chain was implicated in the high affinity Ki values, whereas para substitution elicited a dramatic drop in activity. These variations were analyzed with two molecular design tools: the active analogue approach (AAA) and the new 3D-QSAR (quantitative structure activity relationship) method, comparative molecular field analysis (CoMFA). The AAA method emphasized, by superimposition of selected conformations of the molecules, the favorable and unfavorable volumes implicated in the receptor recognition. CoMFA generated a linear expression between the biological data and the different values of electrostatic and steric fields surrounding the molecules. It predicted the values of selected molecules but also those of new molecules not included in the study. The excellent accuracy of the prediction revealed the potential of the method for the design of new compounds. CoMFA demonstrated the important contribution of steric parameters, evaluated at 92%, compared to the electrostatic field (evaluated at 8%) to explain the affinity for 5-HT1A and 5-HT1B receptors. This study emphasizes also the importance of the occupancy of a hydrophobic pocket in the receptor site located near the area interacting with the aromatic moiety, and subsequently its use for the design of new, potent, specific antagonists of 5-HT1A and 5-HT1B receptors.     

Characterization of alpha1-adrenoceptor subtypes mediating contractions to phenylephrine in rat thoracic aorta, mesenteric artery and pulmonary artery

1. The subtype of alpha1-adrenoceptor mediating contractions to phenylephrine of the rat thoracic aorta, mesenteric artery and pulmonary artery were investigated by use of antagonists which show selectivity between the cloned alpha1-adrenoceptor subtypes in binding studies. 2. Cumulative concentration-contraction curves for phenylephrine were competitively antagonized in the rat thoracic aorta by prazosin (pA2 9.9), WB4101 (pA2 9.6), 5-methylurapidil (pA2 8.1), benoxathian (pA2 9.2) and indoramin (pA2 7.4). These compounds were also competitive antagonists in the mesenteric and pulmonary arteries (except for 5-methylurapidil in the pulmonary artery), (prazosin pA2 9.9 and 9.7; WB4101 pA2 9.8 and 9.6; 5-methylurapidil pA2 7.9 and pK(B) estimate 8.0; benoxathian pA2 8.8 and 9.3; indoramin pA2 7.2 and 7.5, respectively). 3. RS 17053 was not a competitive antagonist in any blood vessel as Schild plot slopes were greater than unity. The pK(B) estimates for RS 17053 were 7.1 in aorta, 7.0 in the mesenteric artery and 7.7 in the pulmonary artery. 4. The alpha1D-subtype selective antagonist BMY 7378 appeared to be non-competitive with shallow Schild plot slopes. The data were better fitted with two lines in all tissues, with Schild plot slopes that were no longer different from unity, except in the pulmonary artery. The higher affinity site for BMY 7378 in the aorta had a pA2 of 9.0, while it was 8.8 and 8.9 in the mesenteric and pulmonary arteries, respectively. 5. MDL73005EF acted in a non-competitive manner in all three blood vessels, with shallow Schild plot slopes. The pK(B) estimates for MDL73005EF were 8.4 in aorta, 7.5 in the mesenteric artery and 8.0 in the pulmonary artery. 6. In all three blood vessels the functionally determined antagonist affinity estimates correlated best with published pKi values for their displacement of [3H]-prazosin binding on membranes expressing cloned alpha1d-adrenoceptors compared with alpha1a- or alpha1b-adrenoceptors. The antagonist affinity estimates in the aorta, mesenteric and pulmonary arteries correlated highly with their previously published pA2 values in rat aorta (alpha1D) and less well with those for alpha1A- and alpha1B-adrenoceptors mediating contraction of the rat epididymal vas deferens and rat spleen, respectively. 7. The results of this study suggest that the contraction to phenylephrine of the rat thoracic aorta, mesenteric artery and pulmonary artery are mediated in part via the alpha1D-subtype of adrenoceptor. The data for both BMY 7378 and MDL73005EF in all three blood vessels are consistent with receptor heterogeneity. However, the identity of the second site is unclear.     

Structure-cytotoxic activity relationship for the toad poison bufadienolides

The toad poison bufadienolides including natural and derivatized compounds were tested for their cytotoxic effects on primary liver carcinoma cells PLC/PRF/5 and their structure-cytotoxic activity relationships were studied. For this study, a ligand-binding model was developed by using a pharmacophore mapping program, Distance Comparisons (DISCO). The structural features that are common to the 3D structures of active bufadienolides were identified to provide approach to a 3D QSAR method by using Comparative Molecular Field Analysis (CoMFA) study and to correlate the steric and electrostatic fields of the molecules to their activities. A valuable model which enables prediction of their activities was obtained from the CoMFA analysis, which may be employed for the drug designs of new bufadienolide analogues.     

Evaluation of the effects of a specific alpha 2-adrenoceptor antagonist, atipamezole, on alpha 1- and alpha 2-adrenoceptor subtype binding, brain neurochemistry and behaviour in comparison with yohimbine

In the present study we evaluated the alpha 1- and alpha 2-adrenoceptor subtype binding, central alpha 2-adrenoceptor antagonist potency, as well as effects on brain neurochemistry and behavioural pharmacology of two alpha 2-adrenoceptor antagonists, atipamezole and yohimbine. Atipamezole had higher selectivity for alpha 2- vs. alpha 1-adrenoceptors than yohimbine regardless of the subtypes studied. Both compounds had comparable affinity for the alpha 2A-, alpha 2C- and alpha 2B-adrenoceptors, but yohimbine had significantly lower affinity for the alpha 2D-subtype. This may account for the fact that significantly higher doses of yohimbine than atipamezole were needed for reversal of alpha 2-agonist (medetomidine)-induced effects in rats (mydriasis) and mice (sedation and hypothermia). The effect on central monoaminergic activity was estimated by measuring the concentrations of transmitters and their main metabolites in whole brain homogenate. At equally effective alpha 2-antagonising doses in the rat mydriasis model, both drugs stimulated central noradrenaline turnover (as reflected by increase in metabolite levels) to the same extent. Atipamezole increased dopaminergic activity only slightly, whereas yohimbine elevated central dopamine but decreased central 5-hydroxytryptamine turnover rates. In behavioural tests, atipamezole (0.1-10 mg/kg) did not affect motor activity but stimulated food rewarded operant (FR-10) responding (0.03-3 mg/kg) whereas yohimbine both stimulated (1 mg/kg) and decreased (> or = 3 mg/kg) behaviour in a narrow dose range in these tests. In the staircase test, both antagonists increased neophobia, but in the two compartment test only yohimbine (> or = 3 mg/kg) decreased exploratory behaviour. The dissimilar effects of the antagonists on neurochemistry and behaviour are thought to be caused by non alpha 2-adrenoceptor properties of yohimbine. In conclusion, the alpha 2-antagonist atipamezole blocked all alpha 2-adrenoceptor subtypes at low doses, stimulated central noradrenergic activity and had only slight effects on behaviour under familiar conditions, but increased neophobia. The low affinity for the alpha 2D-adrenoceptor combined with its unspecific effects complicates the use of yohimbine as pharmacological tool to study alpha 2-adrenoceptor physiology and pharmacology.     

On the Rotational Analysis of the Ground Vibrational State of CH3D Molecule

Three-dimensional quantitative structure-activity relationship (3D-QSAR) models have been developed using comparative molecular field analysis (CoMFA) on a large data set (118 compounds) of diverse cyclic urea derivatives as protease inhibitors against the human immunodeficiency virus type 1 (HIV-1). X-ray crystal structures of HIV-1 protease bound with this class of inhibitors were used to derive the most probable bioactive conformations of the inhibitors. The enzyme active site was used as a constraint to limit the number of possible conformations that are sterically accessible. The test sets have been created keeping in mind structural diversity as well as the uniform simple statistical criteria (mean, standard deviation, high and low values) of the protease inhibitory activities of the molecules compared to the training sets. Multiple predictive models have been developed with the training sets (93 compounds in each set) and validated with the corresponding test sets (25 compounds in each set). All the models yielded high predictive correlation coefficients (q2 from 0.699 to 0.727), substantially high fitted correlation coefficients (r2 from 0.965 to 0.973), and reasonably low standard errors of estimates (S from 0. 239 to 0.265). The steric and electrostatic effects have approximately equal contributions, 45% and 55% (approximately), respectively, toward explaining protease inhibitory activities. This analysis yielded models with significant information on steric and electrostatic interactions clearly discerned by the respective coefficient contour plots when overlapped on the X-ray structure of the HIV-1 protease. The HINT CoMFA study revealed significant contribution of hydrophobicity toward protease inhibitory activity. The 3D visualization technique utilizing these contour plots as well as the receptor site geometry may significantly improve our understanding of the inhibitor-protease (HIV-1) interactions and help in designing compounds with improved activity.     

Comparative molecular field analysis-based prediction of drug affinities at recombinant D1A dopamine receptors

Determination of quantitative structure-activity relationship (QSAR) for affinity at particular dopamine (DA) receptors has become an even greater priority with the cloning of five DA receptor subtypes. The use of agonist affinity at recombinant receptors selectively expressed in clonal cells as the dependent variable in QSAR presents a unique opportunity for accuracy and precision in measurement of biological values. Bound conformations of 11 agonists (for which both affinity data at the recombinant D1A DA receptor and stereochemical configurations were available) were determined by alignment with a template compound, SKF38393, which shows high affinity and selectivity for D1A receptors and is fairly rigid in structure. These aligned structures suggested a 3-point pharmacophore map (one cationic nitrogen and two electronegative centers) of the D1A DA receptor. This map shows both similarities and differences when compared with a previously reported D2 DA receptor pharmacophore map based on biological data from rat brain and with a recently published map of the native D1 DA receptor using several semirigid compounds. Log(1/K(d)) values at recombinant D1A DA receptors were used as the target property for a CoMFA (comparative molecular field analysis) of the 11 aligned structures. The resulting CoMFA model yielded a cross-validated r(2)(q(2)) value of 0.829 and a simple r(2) = 0.96. In contrast, when a CoMFA model was developed for 10 of these compounds using striatal D1 K(d) values, the q(2) value was reduced to 0.178. These results are consistent with the idea that drug affinity data obtained from clonal cells expressing recombinant receptors may be superior to that obtained using heterogeneous mixtures of native receptors prepared from brain membranes. The predictive utility of the CoMFA model was evaluated using several high-affinity dopamine agonists and m- and p-tyramine, two compounds with a single hydroxyl group on the aromatic ring. Predictions were fairly accurate for all compounds but the two tyramines.     

Reversible and non-competitive antagonist profile of CPCCOEt at the human type 1alpha metabotropic glutamate receptor

In transfected CHO cells expressing the human metabotropic glutamate receptor mGlu1alpha, 7-(hydroxyimino)cyclopropan[b]-chromen-1a-carboxylic acid ethylester (CPCCOEt) was found to antagonize L-quisqualate-induced phosphoinositide hydrolysis in a non-competitive and reversible manner (apparent pKi value, 4.76+/-0.18; n=3). This suggests that CPCCOEt antagonizes type 1alpha metabotropic glutamate receptor activation by interacting with a site distinct from the agonist binding site.     

Synthesis and comparative molecular field analysis (CoMFA) of antitumor 3-arylisoquinoline derivatives

In this study a series of 3-arylisoquinoline derivatives were synthesized and cytotoxicity against human melanoma tumor cell evaluated, and a three dimensional quantitative structure-activity relationship was investigated using the comparative molecular field analysis (CoMFA). The results suggested that the electrostatic, steric and hydrophobic factors of 3-arylisoquinolines were strongly correlated with the antitumor activity. Considerable predictive ability (cross-validated r2 as high as 0.721) was obtained through CoMFA.     

Subtype selectivity of a new alpha 1-adrenoceptor antagonist, JTH-601: comparison with prazosin

The existence of alpha 1-adrenoceptors with low affinity for prazosin (alpha 1L group: alpha 1L and alpha 1N subtypes) has been proposed in addition to alpha 1-adrenoceptor subtypes with high affinity for prazosin (alpha 1H group: alpha 1A, alpha 1B and alpha 1D subtypes). A newly synthesized alpha 1-adrenoceptor antagonist, JTH-601 (N-(3-hydroxy-6-methoxy-2,4,5-trimethylbenzyl)-N-methyl-2-(4-hydro xy-2-isopropyl-5-methyl-phenoxy) ethylamine hemifumarate) showed approximately a 10 times higher affinity for the alpha 1L group, a similar affinity for the alpha 1A subtype, but a more than 10 times lower affinity for the alpha 1B and alpha 1D subtypes when compared with prazosin. These results provide a further pharmacological evidence that alpha 1-adrenoceptors with low affinity for prazosin exist in addition to those with high affinity for prazosin, suggesting that JTH-601 may be useful for characterising the alpha 1-adrenoceptor subtypes.     

Overcoming apoptosis: new methods for improving protein-expression systems

This study was designed to further discriminate alpha1-adrenoceptor subtypes in rat aorta and prostate using functional experiments. Responses induced by phenylephrine were equilibrated in both tissues. The pA2 values and slope factors of several alpha1-antagonists were assessed using concentration-response curves. The antagonists used were prazosin, WB-4101, 5-methylurapidil (5-MU), HV-723, and tamsulosin. In addition, the effects of chloroethylclonidine (CEC) and nifedipine on phenylephrine-induced contractions were investigated. A high pA2 value for prazosin was observed in both tissues (aorta 9.84, prostate 9.19) and the ranking of each drug's pA2 value is as follows: tamsulosin > prazosin > WB-4101 > HV-723 > 5-MU in the aorta, and tamsulosin > prazosin > 5-MU > WB-4101 = HV-723 in the prostate. A significant difference between the pA2 value of each drug except for tamsulosin in the aorta and in prostate was observed (p < 0.01). Inhibition of contraction by pretreatment with CEC was 83.9 +/- 2.42% in the aorta, and 6.17 +/- 0.94% in the prostate. On the other hand, inhibition of maximal response by pretreatment with nifedipine (1 micromol/l) was 35.1 +/- 2.2% in the aorta and 24.5 +/- 3.1% in the prostate. A good correlation between these pA2 values and pKi values for recombinant human alpha1b-adrenoceptor expressed in CHO cells (aorta) and alpha1a-subtypes of CEC pretreated rat hippocampus (prostate) were observed. In conclusion, these results suggest that: (1) the contraction of these two tissues is mediated by alpha1H-adrenoceptor with a high affinity for prazosin; (2) alpha1H-adrenoceptors correspond to alpha1b-(aorta) and alpha1a-subtypes (prostate), and (3) each alpha1-adrenoceptor subtype in the aorta and prostate may be alpha1b-(aorta) and alpha1a-subtypes (prostate), respectively.