Complexing properties of N-methylpyridium alpha-amidooxime iodide (MAI) and spectrophotometric investigations on MAI aqueous solutions and its protolitic equilibria

The effect of pH on electronic spectra of MAI was investigated. The N-methylpyridium alpha-amidooxime iodide (MAI) in water solution is hydrolized to weak base with log K2H = 10.14. At low concentration in water solution the geometrical isomers are likely to exist, yielding 4 isosbestic points in UV spectrum. In syn isomer the lone electron pair of -NH2 group is probably blocked through intramolecular hydrogen bonding with oxime group. The effect of hydrogen ions concentration on spectra of watersolution of MAI was examined within range Ho = -3.87 to pH 12.97. Determined protolitic constants are as follows: log K1H(pKa) = 12.14 dissociation of oxime proton, log K3BH = 4.02 - protonation of -NH2 group in anti isomer, log K3AH = 1.62 protonation of -NH2 group in syn isomer, log K4H = -2.42 - protonation of oxime nitrogen or oxygen.     

Aluminum impairs glucose utilization and cholinergic activity in rat brain in vitro

The effects of AlCl3 on the production of 14CO2 from [U-14C]glucose and high affinity choline transport in rat brain synaptosomes, and on carbachol-stimulated hydrolysis of phosphoinositides in cortical slices were studied. In buffer containing either high K+ (50 mM) or low K+ (4.9 mM), 1 mM AlCl3 significantly depressed the synaptosomal production of 14CO2 from [U-14C]glucose to 54% and 44% of control rates, respectively. At a concentration of 0.1 mM, AlCl3 depressed the evolution of 14CO2 from [U-14C]glucose from synaptosomes incubated in the high K+ buffer, but did not significantly change 14CO2 production from synaptosomes in the low K+ buffer. Aluminum chloride also inhibited high affinity choline transport in synaptosomes prepared from rat cortex and from hippocampus with an IC50 of approximately 0.5 mM. In brain slices the carbachol-stimulated hydrolysis of phosphoinositides was inhibited by AlCl3 in a dose-dependent manner. One millimolar, 0.5 mM and 0.1 mM AlCl3 inhibited the carbachol-stimulated release of inositol phosphates by 75%, 44% and 33%, respectively. These same concentrations of AlCl3 inhibited the incorporation of [3H]inositol into phospholipids. This inhibitory effect was not dose-dependent as all 3 concentrations of AlCl3 inhibited phospholipid labelling to the same extent (27-37%). These results are discussed in relation to the in vivo neurotoxicity of aluminum.     

The characteristics of low and high affinity [3H]-prazosin binding to membranes from rat renal cortex

The characteristics of [3H]-prazosin binding in renal cortical membranes of the rat have been assessed under a variety of buffer conditions. At 37 degrees, in Krebs' phosphate and Tris buffer, [3H]-prazosin bound to two sites, a small population of high affinity sites with properties of alpha1-adrenoceptors and a much larger population of low affinity sites with different characteristics. High affinity [3H]-prazosin binding was insensitive to Na+, K+, Ca2+ and Mg2+ ions, but low affinity [3H]-prazosin binding was markedly increased in Krebs' phosphate or sodium phosphate buffer and further enhanced in membranes pretreated with EGTA. Binding was decreased in the presence of Ca2+, the decrease in binding mainly being due to a decrease in the number of low affinity sites labelled by the ligand. Low affinity [3H]-prazosin binding was increased at 37 degrees and relatively insensitive to alpha-adrenoceptor antagonists which were weak competitors while catecholamines failed to compete for low affinity binding. Scatchard plots of [3H]-prazosin binding performed using (-)-noradrenaline (1 mM) to define non-specific binding defined binding only to alpha 1-adrenoceptors. This provides a means of differentiating high and low affinity [3H]-prazosin binding.     

Kir6.2-dependent high-affinity repaglinide binding to beta-cell K(ATP) channels

1. The beta-cell K(ATP) channel is composed of two types of subunit - the inward rectifier K(+) channel (Kir6.2) which forms the channel pore, and the sulphonylurea receptor (SUR1), which serves as a regulatory subunit. The N-terminus of Kir6.2 is involved in transduction of sulphonylurea binding into channel closure, and deletion of the N-terminus (Kir6.2DeltaN14) results in functional uncoupling of the two subunits. In this study, we investigate the interaction of the hypoglycaemic agents repaglinide and glibenclamide with SUR1 and the effect of Kir6.2 on this interaction. We further explore how the binding properties of repaglinide and glibenclamide are affected by functional uncoupling of SUR1 and Kir6.2 in Kir6.2DeltaN14/SUR1 channels. All binding experiments are performed on membranes in ATP-free buffer at 37 degrees C. 2. Repaglinide was found to bind with low affinity (K(D)=59+/-16 nM) to SUR1 alone, but with high affinity (increased approximately 150-fold) when SUR1 was co-expressed with Kir6.2 (K(D)=0.42+/-0.03 nM). Glibenclamide, tolbutamide and nateglinide all bound with marginally lower affinity to SUR1 than to Kir6.2/SUR1. 3. Repaglinide bound with low affinity (K(D)=51+/-23 nM) to SUR1 co-expressed with Kir6.2DeltaN14. In contrast, the affinity for glibenclamide, tolbutamide and nateglinide was only mildly changed as compared to wild-type channels. 4. In whole-cell patch-clamp experiments inhibition of Kir6.2DeltaN14/SUR1 currents by both repaglinide and nateglinde is abolished. 5. The results suggest that Kir6.2 causes a conformational change in SUR1 required for high-affinity repaglinide binding, or that the high-affinity repaglinide-binding site includes contributions from both SUR1 and Kir6.2. Glibenclamide, tolbutamide and nateglinide binding appear to involve only SUR1.     

Interaction of warfarin with human serum albumin. A stoichiometric description

Reversible binding of warfarin to defatted serum albumin was studied by equilibrium dialysis at pH 7.4, in a 66 mM sodium phosphate buffer at 37 degrees. The binding isotherm could be described by two stoichiometric binding constants, K1 in the range 141,000 to 192,000 M-1 and K2 at 39,000 to 57,000 M-1. At least two additional molecules could be bound but gave indeterminate binding constants. The product K3 X K4 was about 4.7 X 10(7) M-2. Different site models were possible, either one high affinity and several low affinity sites, or two high affinity sites, cooperative, independent, or anticooperative, together with two low affinity sites. Binding affinity for the first warfarin molecule did not vary with pH in the interval from 6 to 9. The affinity decreased with increasing concentrations of sodium sulfate, sodium chloride, and calcium chloride, depending upon ionic strength. Specific effects of chloride and calcium ions were not observed. Light absorption spectra indicated that the warfarin anion was bound to albumin. All observations were consistent with a binding process involving albumin and the warfarin anion, without participation of hydrogen ions and not influenced by the N-B conformational transition of albumin.     

Structure-activity relationship studies of CNS agents. Part VIII. Bulk tolerance around the protonation center of 4-substituted 1-(3-chlorophenyl)piperazines at 5-HT1A and 5-HT2 receptors.

The effect of a steric hindrance around the protonation center of the model 4-substituted 1-(3-chlorophenyl)-piperazines 1-9 and 11-14 on their affinity for 5-HT1A and 5-HT2 receptor sites was investigated. Additional evidence for hydrophobic interactions between the N-4 hydrocarbon substituents and 5-HT1A receptors has been presented. However, the hydrophobic forces play a minor role in stabilization of the bioactive complex with 5-HT2 receptors. It has also been found that even bulky substituents around the protonation center of 1-aryl-piperazines are well tolerated at both 5-HT1A and 5-HT2 sites.     

Modulation of dopamine receptors by cations in 7315a, MtTW15 and estradiol-induced pituitary tumors

Modulation of dopamine (DA) receptors by cations (Na+, K+, Mg2+, Ca2+) was compared in 7315a, MtTW15, and estradiol valerate-induced (EV-T) pituitary tumors, and intact adenohypophysis. In 7315a tumors, the affinity of [3H]spiperone binding measured at 25 degrees remained unchanged in the presence of each cation individually or all these cations together (IONS) compared to the affinity obtained using a buffer without ions; the density (Bmax) was not affected by monovalent cations or Mg2+ and was decreased by Ca2+ or IONS. When binding experiments were done at 37 degrees, monovalent cations increased affinity whereas divalent cations or IONS did not modify it, and none of these cations affected Bmax values. In MtTW15 tumors, the affinity of [3H]spiperone binding measured at 25 degrees was not changed by Na+ or IONS and was decreased by K+ or divalent cations; the density was decreased by K+ and unchanged by all the other cations. When binding experiments were done at 37 degrees, Na+ increased the affinity, whereas all the other cations did not affect it: the density was unaffected by all the cations studied. In EV-T assayed at 37 degrees, the affinity was increased by monovalent cations or Mg2+ and was unchanged by Ca2+; monovalent cations did not affect the density of [3H]spiperone binding and divalent cations increased it. In binding experiments performed at 25 degrees and 37 degrees, choline chloride did not change the affinity or the density of [3H]spiperone binding to DA receptors in the three pituitary tumors investigated, suggesting that the effect of cations was specific and not due to differences in ionic strength. In the rat normal anterior pituitary, Na+ increased the affinity of [3H]spiperone for the DA receptors, whereas the affinity was unchanged by Ca2+; the density of [3H]spiperone binding was unaffected by these cations. Our results suggest that DA receptors in 7315a and MtTW15 tumors are regulated abnormally by sodium, potassium, magnesium and calcium. In contrast, DA receptors in EV-T are regulated normally by monovalent cations and abnormally by divalent cations as compared to these receptors in intact pituitary tissue.     

Influence of monovalent cations on the binding of a charged and an uncharged ('carbo'-)muscarinic antagonist to muscarinic receptors.

1. The effect of the buffer concentration on binding of [3H]-N-methylscopolamine to muscarinic receptors M2 was tested in rat heart. Tracer binding was of low affinity in a 20 mM imidazole buffer (pKD 8.3), inhibited by an increase from 10 to 100 mM of the sodium phosphate buffer concentration (pKD 9.92 to 9.22), slightly inhibited by an increase of the Tris/HC1 buffer concentration from 20 to 100 mM (pKD 9.70 to 9.47) and unaffected by an increase of the histidine/HC1 buffer concentration from 20 to 100 mM (pKD 9.90 to 9.82). We chose the last buffer to analyse the effect of ions on antagonists binding to cardiac M2 receptors and to transiently expressed wild-type and (Y533-->F) mutant m3 muscarinic receptors in COS-7 cells. 2. Equilibrium [3H]-N-methylscopolamine binding to cardiac M2 receptors was inhibited, apparently competitively, by monovalent salts (LiCl > or = NaCl > or = KCl). In contrast, binding of the uncharged 3,3-dimethylbutan-1-ol ester of diphenylglycolic acid (BS-6181) was facilitated by addition of monovalent salts (LiCl > or = NaCl > or = KCl) to the binding buffer. This cation binding pattern is consistent with interaction with a large, negative field strength binding site, such as, for instance, a carboxylic acid. 3. In the presence of 100 mM NaCl, [3H]-N-methylscopolamine had a similar affinity for the wild-type m3 receptor (pKD 9.85) and for a (Y533-->F) mutant m3 receptor (pKD 9.68). However, in the absence of added salts, the tracer had a significantly lower affinity for the mutated (pKD 10.19) as compared to the wild-type (pKD 10.70) m3 receptor. BS-6181 had a significantly lower affinity for the (Y533-->F) mutant m3 muscarinic receptor, as compared to the wild-type m3 receptor, both in the absence (pKD 6.19-6.72) in the presence (pKD 6.48-7.40) of 100 mM NaCl. The effects of NaCl on binding of the uncharged ester and of [3H]-N-methylscopolamine to the m3 receptor were decreased by the mutation. 4. Taken together, these results support the hypothesis that monovalent cations from the buffer may interact with the cation binding site of the receptors (an aspartate residue in the third transmembrane helix of muscarinic receptors). Buffer cations may inhibit competitively the binding of (charged) muscarinic ligands having a tertiary amine or ammonium group, while facilitating the receptor recognition by uncharged, isosteric 'carbo-analogues'. Mutation of the (Y533-->F) of the m3 receptor decreased the affinity of the receptor for positive charges, including the sodium ion.     

The effect of pH on the binding of sodium aurothiosulphate to human serum albumin. A possible binding mechanism

The effect of pH on the binding of aurothiosulphate to human serum albumin was studied in unbuffered solutions at 37 degrees and ionic strength 0.15-0.16 M. In the investigated pH range, 6.3-8.4, the effect of pH on the high affinity association constant K1 was very different from that on the lower affinity constants K2-K4. K1 was virtually constant except for a two-fold decrease in the narrow pH range 7.5-7.9, which was explained as a H+ induced local conformation change in the environment of site 1. Contrary to this, K2-K4 decreased monotonically with increasing pH, which could be entirely accounted for by a change in electrostatic interaction. A conceivable binding mechanism consistent with the results might be: that gold binds as Au+ to the high affinity binding site by exchanging a H+ and that this site might be the free sulphydryl group in cysteine or the terminal alpha-amino group; and that gold binds as Au(S2O3)3-(2) to the lower affinity binding sites which might be the protonated basic side chain group, i.e. epsilon-amino groups.     

Correlation of apparent affinity values from H3-receptor binding assays with apparent affinity (pKapp) and intrinsic activity (alpha) from functional bioassays

BACKGROUND AND PURPOSE: Agonist apparent affinities (pK(I)') in histamine H(3)-receptor binding assays were higher than expected from apparent affinity values (pK(app)) estimated in bioassay. Here, we investigate whether the degree of pK(I)' overestimation is related to agonist intrinsic efficacy, by studying the effect of buffer composition on the pK(I)' of ligands with varying intrinsic activity. EXPERIMENTAL APPROACH: In the guinea-pig ileum bioassay, intrinsic activity (alpha) was determined from the maximal inhibition of the contraction produced by increasing agonist concentration. pK(app) values were estimated using the method of Furchgott. The pK(L) of [(3)H]clobenpropit in guinea-pig cerebral cortex was estimated by saturation analysis in 20 mM HEPES-NaOH buffer (buffer B(0,0,0)), or buffer B(0,0,0) containing 70 mM CaCl(2), 100 mM NaCl and 100 mM KCl (buffer B(0.07,0.1,0.1)). PK(I) values were determined in competition studies in both buffers. KEY RESULTS: [(3)H]clobenpropit saturation isotherms had n (H) values of unity in both buffers. In buffer B(0.07,0.1,0.1), agonist pK(I)' values were closer to pK(app) values than in buffer B(0,0,0) but were associated with n (H) values <1. A two-site analysis of agonist data in buffer B(0.07, 0.1, 0.1) provided a better fit than a one-site fit and low affinity values (pK(IL)) were comparable to pK(app). Differences between the pK(I)' in buffer B(0,0,0) and pK(IL) values in buffer B(0.07,0.1,0.1) (DeltapK) were correlated with alpha. CONCLUSIONS AND IMPLICATIONS: H(3)-receptor binding assays conducted in buffer B(0,0,0) and buffer B(0.07,0.1,0.1) can provide a measure of ligand affinity (pK(app)) and intrinsic efficacy. The assay predicts that some ligands previously classified as H(3)-receptor antagonists may possess residual intrinsic efficacy.     

Simple, intuitive calculations of free energy of binding for protein-ligand complexes. 2. Computational titration and pH effects in molecular models of neuraminidase-inhibitor complexes

One factor that can strongly influence predicted free energy of binding is the ionization state of functional groups on the ligands and at the binding site at which calculations are performed. This analysis is seldom performed except in very detailed computational simulations. In this work, we address the issues of (i) modeling the complexity resulting from the different ionization states of ligand and protein residues involved in binding, (ii) if, and how, computational methods can evaluate the pH dependence of ligand inhibition constants, and (iii) how to score the protonation-dependent models. We developed a new and fairly rapid protocol called "computational titration" that enables parallel modeling of multiple ionization ensembles for each distinct protonation level. Models for possible protonation combinations for site/ligand ionizable groups are built, and the free energy of interaction for each of them is quantified by the HINT (Hydropathic INTeractions) software. We applied this procedure to the evaluation of the binding affinity of nine inhibitors (six derived from 2,3-didehydro-2-deoxy-N-acetylneuraminic acid, DANA) of influenza virus neuraminidase (NA), a surface glycoprotein essential for virus replication and thus a pharmaceutically relevant target for the design of anti-influenza drugs. The three-dimensional structures of the NA enzyme-inhibitor complexes indicate considerable complexity as the ligand-protein recognition site contains several ionizable moieties. Each computational titration experiment reveals a peak HINT score as a function of added protons. This maximum HINT score indicates the optimum pH (or the optimum protonation state of each inhibitor-protein binding site) for binding. The pH at which inhibition is measured and/or crystals were grown and analyzed can vary from this optimum. A protonation model is proposed for each ligand that reconciles the experimental complex structure with measured inhibition and the free energy of binding. Computational titration methods allow us to analyze the effect of pH in silico and may be helpful in improving ligand binding free energy prediction when protonation or deprotonation of the residues or ligand functional groups at the binding site might be significant.     

Solid-supported lipid membranes as a tool for determination of membrane affinity: high-throughput screening of a physicochemical parameter

Quantification of membrane affinity is an important early screening step in modern drug design. However, current approaches using different lipid membrane models usually are time-consuming or show severe experimental drawbacks. In this paper we describe the use of solid-supported lipid membranes (TRANSIL) as a new tool for the determination of membrane affinity. Eighteen pharmaceuticals (neutrals, acids, and bases) have been analyzed for their lipophilicity at physiological pH in an automated setup; phase separation of lipid and aqueous phase can be achieved simply by a short low-speed centrifugation or filtration. The membrane affinity is then calculated by quantification of the total drug concentration and the amount of drug remaining in the aqueous phase after incubation with solid-supported lipid membranes. Lipophilicity parameters relying on solid-supported lipid membranes correlate well with octanol-water partition coefficients K(ow) for neutral organic compounds (range of log K(ow) = 1.5-5, n = 7, r = 0.93). Data acquisition with this lipid membrane model system is highly re-producible. Even in the case of ionizable drugs, where K(ow) tends to underestimate membrane affinity, the latter can be correctly quantified using solid-supported lipid membranes: data comparison shows good agreement of the presented approach with established but time-consuming standardized lipid/buffer systems. Solid-supported lipid membranes allow a fast and reliable quantification of membrane affinity, enabling high-throughput screening of this physicochemical parameter.     

Interaction of K(ATP) channel modulators with sulfonylurea receptor SUR2B: implication for tetramer formation and allosteric coupling of subunits.

Sulfonylurea receptors (SURs) are subunits of ATP-sensitive K(+) channels (K(ATP) channels); they mediate the channel-closing effect of sulfonylureas such as glibenclamide and the channel-activating effect of K(ATP) channel openers such as the pinacidil analog P1075. We investigated the inhibition by MgATP and P1075 of glibenclamide binding to SUR2B, the SUR subtype in smooth muscle. To increase specific binding, experiments were also performed using SUR2B(Y1206S), a mutant with higher affinity for glibenclamide than for the wild-type (K(D )= 4 versus 22 nM, respectively) but otherwise exhibiting similar pharmacological properties. In the absence of MgATP, [(3)H]glibenclamide binding to both SURs was homogenous. MgATP inhibited [(3)H]glibenclamide binding to both SURs to 25% by reducing the apparent number of glibenclamide binding sites, leaving the affinity unchanged. In the absence of MgATP, P1075 inhibited [(3)H]glibenclamide binding in a monophasic manner with K(i) approximately 1 microM. In the presence of MgATP (1 mM), inhibition was biphasic with one K(i) value resembling the true affinity of P1075 for SUR2B (2-6 nM) and the other resembling K(i) in the absence of MgATP (approximately 1 microM). The data show that (1) MgATP induces heterogeneity in the glibenclamide sites; (2) the high-affinity glibenclamide sites remaining with MgATP are linked to two classes of P1075 sites; and (3) P1075 interacts specifically with SUR2B also in the absence of MgATP. The data are discussed with the assumption that SUR2B, expressed alone, forms tetramers; that MgATP induces allosteric interactions between the subunits; and that mixed SUR2B-glibenclamide-P1075 complexes can exist at equilibrium.     

1,3-Diamino-6,7-dimethoxyisoquinoline derivatives as potential alpha 1-adrenoceptor antagonists

Treatment of 2-methyl-4,5-dimethoxybenzonitrile (3) with LDA followed by reaction with an N,N-disubstituted cyanamide provided a series of 1,3-diamino-6,7-dimethoxyisoquinolines (2), which were evaluated for alpha-adrenoceptor binding affinity and antihypertensive activity. 1-Amino-3-(dimethylamino)-6,7-dimethoxyisoquinoline (4) showed no significant affinity (Ki much greater than 10(-6) M) for alpha 1-adrenoceptors, while the corresponding 3-(2-furoylpiperazin-1-yl) analogue (8; Ki = 1.6 X 10(-7) M) was some 1000-fold less potent than prazosin. pKa data showed that N-2 protonation (34%) of 4 (pKa = 7.1) would occur at physiological pH, in agreement with X-ray crystallographic analysis of 8.HCl. Comparison of positive charge distribution following protonation of 4 with the corresponding quinoline and quinazoline cations confirmed N-1 protonation is required for these heterocyclic nuclei to bind efficiently to the alpha 1-adrenoceptor. Computer-assisted comparison of the X-ray structures of 8 and prazosin suggested that the 4.0 kcal/mol difference in alpha 1-adrenoceptor binding energies was largely due to salt-bridge formation (ca. 3.0 kcal/mol) between the protonated quinazoline and the receptor protein. None of the isoquinolines (2) proved to be effective antihypertensive agents in rats even when administered at relatively high doses (10 mg/kg). These results support the hypothesis that the antihypertensive activity of prazosin, doxazosin, and related derivatives derives solely from alpha 1-adrenoceptor blockade.     

Stimulation and suppression of renin release from incubations of rat renal cortex by factors affecting calcium flux.

Inhibition of renin secretion from incubations of rat kidney cortex by angiotensin II (AII), ouabain and K+ depletion, depended on the presence of external Ca2+. AII inhibition of isoprenaline-stimulated renin secretion was only partially dependent on external Ca2+. Ouabain and K+ depletion inhibited isoprenaline-stimulated renin release but only in the presence of external Ca2+. Since, in Ca2+-free medium, isoprenaline stimulated renin release when the Na+/K+-ATPase was blocked, isoprenaline probably does not act through the Na+/K+-ATPase. Lanthanum blocked the stimulation of renin release by isoprenaline. Ethylenediamine tetra-acetic acid (EDTA) and ethyleneglycol-bis-(beta-amino-ethyl ether) N,N'-tetra-acetic acid (EGTA) increased renin secretion to a similar degree in Ca2+- and Mg2+-free buffer. When Mg2+ was present the effect of EGTA, but not EDTA, was considerably reduced. Verapamil reduced the fall in basal renin secretion in normal but not Ca2+-free buffer. Verapamil did not block the inhibitory effects of AII or ouabain and did not alter the stimulation of renin secretion by isoprenaline. Bay K 8644 inhibited renin secretion from cortex incubated in medium containing 15 mM K+ and this was dependent on extracellular Ca2+. In normal buffer (5.9 mM K+) Bay K 8644 increased renin secretion.     

Discovery of a novel A2B adenosine receptor antagonist as a clinical candidate for chronic inflammatory airway diseases.

Recently, we have reported a series of new 1,3-symmetrically (R 1 = R 3) substituted xanthines ( 3 and 4) which have high affinity and selectivity for the human adenosine A 2B receptors (hA(2B)-AdoR). Unfortunately, this class of compounds had poor pharmacokinetic properties. This prompted us to investigate the effect of differential alkyl substitution at the N-1 and N-3 positions ( N 1-R not equal to N 3-R) on A(2B)-AdoR affinity and selectivity; we had the dual objectives of enhancing affinity and selectivity for the A(2B)-AdoR, as well as improving oral bioavailability. This effort has led to the discovery of compound 62, that displayed high affinity and selectivity for the hA(2B)-AdoR (K(i) = 22 nM). In addition, compound 62 showed high functional potency in inhibiting the accumulation of cyclic adenosine monophosphate induced by 5'- N-ethylcarboxamidoadenosine in HEK-A(2B)-AdoR and NIH3T3 cells with K(B) values of 6 and 2 nM, respectively. In a single ascending-dose phase I clinical study, compound 62 had no serious adverse events and was well tolerated.     

Angiotensin II pseudopeptides containing 1,3,5-trisubstituted benzene scaffolds with high AT2 receptor affinity

Two 1,3,5-trisubstituted aromatic scaffolds intended to serve as gamma-turn mimetics have been synthesized and incorporated in five pseudopeptide analogues of angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe), replacing Val-Tyr-Ile, Val-Tyr, or Tyr-Ile. All the tested compounds exhibited nanomolar affinity for the AT2 receptor with the best compound (3) having a K(i) of 1.85 nM. Four pseudopeptides were AT2 selective, while one (5) also exhibited good affinity for the AT1 receptor (K(i) = 30.3 nM). This pseudopeptide exerted full agonistic activity in an AT2 receptor induced neurite outgrowth assay but displayed no agonistic effect in an AT1 receptor functional assay. Molecular modeling, using the program DISCOtech, showed that the high-affinity ligands could interact similarly with the AT2 receptor as other ligands with high affinity for this receptor. A tentative agonist model is proposed for AT2 receptor activation by angiotensin II analogues. We conclude that the 1,3,5-trisubstituted benzene rings can be conveniently prepared and are suitable as gamma-turn mimics.     

Characterisation of [(125)I]-apamin binding sites in rat brain membranes with HE293 cells transfected with SK channel subtypes

The pharmacology of [(125)I]-apamin binding sites was examined in rat cortical and hippocampal tissue and compared with membranes prepared from human embryonic kidney (HEK293) cells transfected with SK channel subtypes hSK1, rSK2 and rSK3. The K(D) of [(125)I]-apamin in rat cortex and hippocampus was similar to the apamin-sensitive subtypes, rSK2 and rSK3 (K(D) (pM): 6.4, 7.08, 6.56 and 8.94, respectively). In addition, [(125)I]-apamin had a K(D)=270.4pM for the putatively 'apamin-insensitive' hSK1. Apamin had about a three-fold higher affinity than [(125)I]-apamin in brain tissue and in the cells expressing the different SK channel subtypes. Pancuronium, bicuculline and d-tubocurarine displayed micromolar affinity for all five-membrane preparations, whereas dequalinium and gallamine appear to show some subtype selectivity. Tetraethylammonium (TEA) and 4-aminopyridine (4-AP) had millimolar affinity and linopirdine had no effect. In conclusion, the pharmacology of [(125)I]-apamin binding in the cortex and hippocampus was similar to that in the apamin-sensitive clones, rSK2 and rSK3. In addition, we demonstrated low affinity [(125)I]-apamin binding for hSK1 and identified compounds that show subtype selectivity. These data cast further doubt on the identification of SK1 as encoding for the K(+) channel responsible for the apamin-insensitive sAHP.     

New arylpiperazine 5-HT(1A) receptor ligands containing the pyrimido[2,1-f]purine fragment: synthesis, in vitro, and in vivo pharmacological evaluation

New 1H,3H-pyrimido[2,1-f]purine-2,4-dione derivatives of arylpiperazine (11-22) were prepared and evaluated in vitro for their affinity for 5-HT(1A), 5-HT(2A), alpha(1), and D(2) receptors. The tested compounds showed high affinity for 5-HT(1A) and alpha(1) receptors (K(i) = 1.1-87 and 10-62 nM, respectively) and moderate to low affinity for 5-HT(2A) (K(i) = 56-881 nM) and D(2) receptors (K(i) = 94-1245 nM). Compounds 14, 15, 18, 19, and 21, mostly 3'-chlorophenylpiperazine derivatives, can be classified as mixed 5-HT(1A)/5-HT(2A)/alpha(1) ligands. Compound 13, which showed the highest 5-HT(1A) receptor affinity (K(i) = 1.1 nM), was 50-fold selective in relation to alpha(1) adrenoceptors and at least 250-fold over 5-HT(2A) and D(2) sites. On the basis of in vivo functional tests, 8-phenylpiperazinoethylamino (11), 8-(2'-methoxyphenylpiperazino)ethylamino (13), and 8-phenylpiperazinopropylamino (14) derivatives of 1,3-dimethyl-1H,3H-pyrimido[2,1-f]purine-2,4-dione were identified as potent pre- and postsynaptic 5-HT(1A) receptor antagonists. 1,3-Dimethyl-7-bromo-8-(phenylpiperazinopropylamino)-1H,3H-pyrimido[2,1-f]purine-2,4-dione (20) behaved like an agonist of presynaptic and as a partial agonist of postsynaptic 5-HT(1A) receptors and resembled ipsapirone in terms of functional intrinsic activity. It revealed marked anxiolytic-like activity in the Vogel test in rats, comparable to that of the reference drug diazepam, and exhibited antidepressant-like activity in the Porsolt test in rats. The sedative effect of 20, evaluated in the open field test in rats, appeared at doses twice as high as those inducing a minimal anxiolytic-like effect and was similar to the effects of diazepam.     

Structure-activity relationship studies of CNS agents. Part VII. The effect of the imidazole fragment in 2-substituted 1-[3-(4-aryl-1-piperazinyl)propyl]imidazoles on their interaction modes with 5-HT1A and 5-HT2 receptors.

The synthesis and the 5-HT1A and 5-HT2 receptor affinity of 2-substituted 1-[3-(4-aryl-1-piperazinyl)propyl]-imidazoles (1-8) has been described. It has been shown that both the N-3 imidazole atom and the N-1 piperazine one should be considered as possible protonation centers under physiological conditions. It has been found that the folded conformations of 1-8 exist predominantly in solution. Moreover, three different modes of interaction of the analyzed compounds with 5-HT1A and 5-HT2 receptor sites have been proposed.