[Does budesonide affect growth?].
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Administration, Topical↗
Biomedical subjects
K Mohr
Publications and source records attributed to K Mohr.
Synthesis, pharmacological and biophysical characterization, and membrane-interaction QSAR analysis of cationic amphiphilic model compounds.
Cationic amphiphilic drugs have a propensity to interact with biological interphases. This study was designed to gain more insight into the molecular properties of catamphiphilic drugs which govern this type of interaction. A series of phenylpropylamine model compounds were synthesized in which modifications were incorporated at the aromatic part of the molecule. The replacement of (45)Ca(2+) from phosphatidylserine monolayers served to monitor drug binding to the phospholipid. The influence on the phase-transition temperature of liposomes of dipalmitoylphosphatidic acid was measured to assess the perturbing action of the drugs on the structural organization of phospholipid assemblies. The antiarrhythmic activity of the compounds was determined in Langendorff preparations of guinea pig hearts to assess the membrane-stabilizing action. Quantitative structure-activity relationship (QSAR) models for these endpoints were developed using both intra- and intermolecular QSAR descriptors. Intermolecular membrane-interaction descriptors were derived from molecular dynamics simulations of the compounds in a model phospholipid monolayer. QSAR models were derived for all endpoints using partial least-squares regression (PLS) and a genetic algorithm tool, the genetic function approximation (GFA). Membrane-interaction descriptors appear to be of a particular importance in explaining the influence of the compounds on the phase-transition temperature of DPPA liposomes, while the other endpoints can be adequately modeled by intramolecular descriptors. The calcium-displacing activity at phosphatidylserine monolayers is governed by the electrostatic properties of the compounds. Measures of lipophilicity and molecular size are of particular importance for antiarrhythmic activity. Possible improvements to both the molecular modeling and the applied computational protocol of membrane-solute systems are identified and discussed.
Animals↗
Bisquaternary ligands of the common allosteric site of M2 acetylcholine receptors: search for the minimum essential distances between the pharmacophoric elements.
Structurally diverse molecules, such as alcuronium, gallamine, and tubocurarine as well as W84 and WDUO, are known to interact allosterically with ligand binding to muscarinic M2 acetylcholine receptors. Preliminary molecular modeling studies revealed two positive charges in the middle and two lateral aromatic areas to be essential elements of a high allosteric potency. To find out the optimum distances between these pharmacophoric elements, a systematic variation of the spacer in the series of W84, WDUO, and IWDUO compounds was performed. The allosteric reduction of the rate of dissociation of the antagonist [3H]-N-methylscopolamine from porcine heart M2 receptors served as a test system. The minimal essential distance between the positive charges was found to be 10 A. The length of the peripheral spacers connecting the positive charge and the lateral aromatic moiety appears to depend on the chemical functionality; the peripheral spacers have to be long and flexible enough to position the aromatic skeletons in the spatial neighborhood of the alkane middle chain: in the case of an oxime ether containing peripheral spacer, six atoms are required, and in the case of an alkane chain, four carbon atoms are necessary to adopt the pharmacophoric S-shape conformation.
Allosteric Site↗
Using a radioalloster to test predictions of the cooperativity model for gallamine binding to the allosteric site of muscarinic acetylcholine M(2) receptors.
The muscarinic M(2) receptor contains an orthosteric and an allosteric site. Binding of an allosteric agent may induce a shift alpha of the equilibrium dissociation constant K(D) of a radioligand for the orthosteric site. According to the cooperativity model, the K(A) of alloster binding is expected to be shifted to an identical extent depending on whether the orthosteric site is occupied by the orthoster or not. Here, the novel radioalloster [(3)H]dimethyl-W84 (N,N'-bis[3-(1,3-dihydro-1, 3-dioxo-4-methyl-2H-isoindol-2-yl)propyl]-N,N,N',N'-tetramethyl-1, 6-hexanediaminium diiodide) was applied to directly measure the K(A) shift induced for the prototype allosteric modulator gallamine by binding of N-methylscopolamine (NMS) to the orthosteric site of porcine heart M(2) receptors (4 mM Na(2)HPO(4), 1 mM KH(2)PO(4), pH 7.4; 23 degrees C; data are means +/- S.E.). First, in the common way, the concentration-dependent inhibition by gallamine of [(3)H]NMS equilibrium binding was measured and analyzed using the cooperativity model, which yielded for the affinity of gallamine binding at free receptors a pK(A)= 8.35 +/- 0.09 and a cooperativity factor alpha = 46 (n = 5). The dissociation constant for gallamine binding at NMS-occupied receptors was predicted as p(alpha. K(A)) = 6.69. Labeling of the allosteric site by [(3)H]dimethyl-W84 allowed the measure of competitive displacement curves for gallamine. The K(i) for gallamine at free receptors amounted to pK(i,-NMS) = 8.27 +/- 0.39 (n = 5), which is in line with the prediction of the cooperativtiy model. In the presence of 1 microM NMS, to occupy the orthosteric site, gallamine displaced [(3)H]dimethyl-W84 with pK(i, +NMS) = 6.60 +/- 0.19 (n = 3). Thus, the NMS-induced pK(i) shift amounted to 47, which matches the predicted value of alpha = 46. These results validate the cooperativity model.
Allosteric Site↗
Ex-chiral pool synthesis and pharmacological aspects of 3-pyrrolidinylisoxazoles.
Employing the dopamine autoreceptor agonist (-)-3-PPP (3) as well as the cholinergic receptor ligands 4 and 5 as lead compounds the 3-pyrrolidinylisoxazoles 2a,b as well as its optical antipodes ent 2a,b were synthesized from (R)-aspartic acid (6) and (S)-aspartic acid (ent-6), respectively. Pharmacological properties of the target compounds were evaluated employing dopamine D2 receptor binding studies and functional experiments on muscarinic M2 receptors.
Animals↗
[3H]Pramipexole: a selective radioligand for the high affinity dopamine D2 receptor in bovine striatal membranes.
The characterization of [3H]pramipexole binding to bovine striatal membranes is reported in full experimental detail. According to kinetic experiments, saturation and competition studies a single binding site can be selectively labeled which turned out to be the high affinity D2 receptor. Addition of GPP(NH)P resulted in almost complete loss of specific binding. The bovine D2 subtype shows high sequence identity with the human D2 receptor indicating that the heterologous competition assays are of interest for the evaluation of neurotropic drug candidates. Using the representative D2 agonists (+)-7-OH-DPAT, (-)-3-PPP and (S)-7-dipropylaminotetrahydroindolizine the same rank order of affinities was determined as described for rat striata labeled with [3H]pramipexole, however, the Ki values turned out to be significantly higher. Furthermore, the system facilitates structure activity relationship studied on D2 affinity modulating peptides. Using L-prolyl-L-leucyl-glycinamide as an example a significant increase of specific radioligand binding could be measured.
Animals↗
Interaction of Mg2+ with the allosteric site of muscarinic M2 receptors.
Mg2+-ions have been suspected to attenuate the inhibitory effect of allosteric modulators on the dissociation of orthosteric ligands from muscarinic M2 receptors. It was aimed to gain more insight into the molecular events underlying the effect of Mg2+. The interaction of Mg2+ with the allosteric model compounds W84 (hexane-1,6-bis [dimethyl-3'-phthalimidopropylammonium bromide]) and Chin3/6 (hexane-1 ,6-bis[dimethyl-3'-¿4-oxo-2-phenyl-3,4-dihydro-2H-quinazolin-1-yl propylammonium bromide]) was studied in porcine heart muscarinic receptors, the primary binding site of which was occupied by the ligand [3H]N-methylscopolamine ([3H]NMS). The incubation buffer was composed of 4 mM Na2HPO4 and 1 mM KH2PO4 (pH 7.4, 23 degrees C). The retardation of [3H]NMS dissociation (control t1/2=5.6 min) induced by the allosteric test compounds was diminished by 3 mM Mg2+ to a greater extent than to be expected with regard to its contribution to the ionic strength of the buffer solution. Concentration-effect curves for the allosteric retardation of [3H]NMS dissociation by W84 (half maximal effective concentration EC0.5=24 nM in the absence of Mg2+) and by Chin3/6 (EC0.5=28 nM) were shifted by Mg2+ to the right in a parallel fashion. The curve-shift was compatible with a competitive interplay between Mg2+ and the modulators. The pKb-values as a measure of the antagonistic potency of Mg2+, however, differed depending on the modulator, i.e. pKb=3.4 with W84 and pKb=2.8 with Chin3/6. Mg2+ itself was capable of slowing the dissociation of [3H]NMS; the maximal retardation of [3H]NMS dissociation was about 3 fold, the concentration-effect relationship was compatible with a two-site model using the above-mentioned pKb-values as affinity constants. Since the equilibrium-binding of [3H]NMS remained unchanged up to a Mg2+-concentration of 3 mM, the cation appears to inhibit the association and dissociation of [3H]NMS to the same extent in this concentration range. Taken together, the findings indicate that Mg2+ may bind to the allosteric region of muscarinic M2 receptors and that more than one site is involved in this interaction. The sites of action may represent divalent cation binding sites.
Allosteric Site↗
Contribution of lateral substituents in heptane-bisammonium derivatives to the allosteric stabilization of antagonist binding to M2-receptors.
Phthalimide-containing heptane-bisammonium-type compounds retard the dissociation of the antagonist [3H]-N-methylscopolamine ([3H]NMS) from muscarinic M2-receptor allosterically with high potency. To study the contribution of the lateral substituents to this effect, a series of derivatives was synthesized in which the phthalimide moiety was truncated. The potency of the compounds to delay [3H]NMS dissociation was measured in porcine heart homogenates (50 mM Tris-HCl, 3 mM MgHPO4, pH 7.3, 37 degrees C). Potency declined with diminuition of the lateral substituents, e.g. loss of the aromatic ring of the phthalimide resulted in a 400fold reduction in potency. In the hexahydrophthalimide derivatives, the cis-stereoisomer was about fivefold more potent than the trans-isomer. In conclusion, almost flat hydrophobic lateral moieties appear to be pivotal for high allosteric potency, suggesting a hydrophobic interaction of these parts of the molecule with the [3H]NMS occupied receptor protein.
Allosteric Regulation↗
M2 receptor binding of the selective antagonist AF-DX 384: possible involvement of the common allosteric site.
The hypothesis was tested that M2-selective antagonists partially utilize the allosteric site of muscarinic M2 receptors. The interactions of the allosteric agent W84 (hexane-1, 6-bis[dimethyl-3'-phthalimidopropyl-ammonium bromide]) were studied with the M2/M4-selective AF-DX 384 [(+/-)-5, 11-dihydro-11-([(2-(2-[(dipropylamino)methyl]-1-piperidinyl)ethyl)amino]carbonyl)-6H-pyrido(2,3-b)(1,4)-benzodiazepine-6-one], the nonselective N-methylscopolamine (NMS), and a number of other muscarinic antagonists. In isolated paced guinea pig atria, the antagonistic effect of W84 against oxotremorine- and arecaidine propargyl ester-induced negative inotropic actions reached a limiting value at higher W84 concentrations, revealing negative cooperativity (factors of cooperativity alpha = 311 and alpha = 495, respectively). The antagonistic potency of W84 in this M2 receptor model (W84 binding constant KA approximately 160 nM) was higher than at M1/M4-like receptors of rabbit vas deferens (KB approximately 800 nM) and at M3 receptors of guinea pig ileum (KB approximately 4,000 nM). In paced atria, combinations of W84 with muscarinic antagonists yielded more-than-additive antagonistic effects against oxotremorine in case of conventional antagonists such as NMS (alpha = 18) but less-than-additive effects with the M2-preferring AF-DX 384 (alpha = 444). In guinea pig heart homogenates, the equilibrium binding of [3H]NMS was only partially inhibited by W84 (alpha = 2.4), whereas [3H]AF-DX 384 binding could be suppressed completely (alpha = 194). The difference in cooperativity reflects that W84 inhibits [3H]NMS dissociation with a approximately 40-fold higher potency (ECdiss = 900 nM) than [3H]AF-DX 384 dissociation (ECdiss = 33,300 nM). [3H]NMS dissociation also could be retarded by AF-DX 384 (ECdiss = 22,000 nM), probably via an interaction with the site used by W84. The results suggest that the binding domain of AF-DX 384 partially overlaps with the common allosteric site of the M2 receptor protein.
Allosteric Regulation↗
Identification of a [3H]Ligand for the common allosteric site of muscarinic acetylcholine M2 receptors.
Muscarinic acetylcholine receptors bind allosteric modulators at a site apart from the orthosteric site used by conventional ligands. We tested in cardiac tissue whether modulator binding to ligand-occupied muscarinic M2 receptors is a preferential event that can be detected using a radioactive allosteric agent. The newly synthesized dimethyl-W84 (N,N'-bis[3-(1,3-dihydro-1, 3-dioxo-4-methyl-2H-isoindol-2-yl)propyl]-N,N,N',N'-tetramethyl-1, 6-hexanediaminium diiodide) has a particular high potency at M2 receptors occupied by the conventional antagonist N-methylscopolamine (NMS); dissociation of [3H]NMS is half-maximally retarded at an EC50,diss value of 3 nM. Using obidoxime as an "allosteric antagonist," evidence was found that dimethyl-W84 interacts with the postulated common allosteric site. Binding of [3H]dimethyl-W84 (0.3 nM; specific activity, 168 Ci/mmol) was measured in porcine heart homogenates (4 mM Na2HPO4, 1 mM KH2PO4, pH 7.4, 23 degrees) in the presence of 1 microM NMS. Homologous competition experiments revealed two components of saturable radioligand binding: one with a high affinity (KD = 2 nM) and small capacity ( approximately 30% of total saturable binding) and the other with a 20,000-fold lower affinity. The Bmax value of the high affinity sites (68 fmol/mg protein) matched muscarinic receptor density as determined by [3H]NMS (79 fmol/mg). Prototype allosteric agents, alcuronium, W84 (the parent compound of the radioligand), and gallamine, displaced high affinity [3H]dimethyl-W84 binding concentration-dependently (pKi values = 8.62, 7.83, and 6.72, respectively). The binding affinities of the modulators were in excellent correlation with their potencies to allosterically stabilize NMS/receptor complexes (EC50,diss = 8.40, 7.72, and 6.74, respectively). We conclude that high affinity binding of [3H]dimethyl-W84 reflects occupation of the common allosteric site of M2 receptors.
Allosteric Site↗
Molecular rigidity and potency of bispyridinium type allosteric modulators at muscarinic M2-receptors.
Several bispyridinium compounds have been shown to be potent allosteric modulators of ligand binding to muscarinic M2-receptors. ,,Uno compounds" are benzyl derivatives of the bispyridinium "TMB4" (trimethylene-bis-[4-hydroxy-iminomethyl-pyridinium]). To gain more insight into structure activity relationships, eleven derivatives with varying structure of the oxime-linked aromatic substituent were tested for their ability to inhibit the equilibrium-binding of [3H]N-methylscopolamine ([3H]NMS) in guinea pig cardiac membranes and to retard [3H]NMS-dissociation allosterically. At a concentration of 3 microM, all compounds reduced [3H]NMS-binding to about 40 % of the control level, indicating a similar potency to inhibit the association of [3H]NMS. Allosteric retardation of [3H]NMS-dissociation required higher concentrations. Comparing the effects of the compounds at 30 and 300 microM, respectively, revealed considerable differences in potency. Therefore, the concentration-dependency of the delay of [3H]NMS-dissociation was determined for selected compounds. The results indicate that introduction of a benzyl-moiety into TMB4 leads to a 20-fold increase in allosteric potency. A further increment by a factor of 10 is obtained with the 2,6-dichlorobenzyl-substitution and with the naphthyl-derivative. The other compounds were less potent. An inverse correlation was found between the rotational freedom of the aromatic substituent and the allosteric potency. In conclusion, the aromatic moiety of non-symmetric bispyridinium-type modulators does not seem to be part of the pharmacophore involved in the inhibitory effect on the association of [3H]NMS. In contrast, a rigid aromatic lateral moiety appears to be essential for the interaction with the recognition site mediating the allosteric delay of [3H]NMS dissociation from muscarinic M2-receptors.
Allosteric Regulation↗
Detection of immunoreactive parathyroid hormone-related protein in human cerebrospinal fluid.
Immunoreactive parathyroid hormone-related protein (PTH-rP) was measured in simultaneously obtained cerebrospinal fluid (CSF) and plasma from 51 patients suspected of suffering from a prolapsed intervertebral disc. Endocrine or psychiatric diseases were excluded. In addition, immunoreactive parathyroid hormone (PTH) in the CSF samples was measured. Both, PTH-rP and PTH were assayed by immunoradiometric assay. The results indicate the presence of both, PTH-rP and PTH in CSF. The following concentrations (mean values +/- SD) were found: PTH-rP (pmol/l) in CSF without pleocytosis (n = 17) 0.432 +/- 0.157, with pleocytosis (n = 34) 0.654 +/- 0.675; in plasma (pmol/l) 54.1 +/- 14.632; PTH (nmol/l) in CSF without pleocytosis (n = 17) 0.454 +/- 0.099, with pleocytosis (n = 34) 0.437 +/- 0.140, and in plasma 4.272 +/- 0.794. The concentrations of both, PTH-rP and PTH, in CSF with and without pleocytosis were not significantly different. No correlation was found between PTH-rP and PTH values. The present study demonstrated PTH-rP as a normal constituent in human CSF.
Adolescent↗
Divergent modes of action among cationic allosteric modulators of muscarinic M2 receptors.
We tested the hypothesis that structurally related modulators of ligand binding to muscarinic M2 receptors may not use a common recognition site. The applied test compounds are potent allosteric modulators [i.e., two bispyridinium model compounds substituted symmetrically either with phthalimidomethyl (WDuo3) or dichlorobenzyl (Duo3), a phthalimidoethyl-substituted hexamethonium compound (W84), alcuronium, and, for sake of comparison, gallamine]. As introduced by Ellis and Seidenberg as a tool to check for a common allosteric site [Mol. Pharmacol. 42:638-641 (1992)], obidoxime was used to antagonize the actions of the test compounds. The allosteric delay of the dissociation of [3H]N-methylscopolamine ([3H]NMS) from porcine heart muscarinic receptors was measured in 5 mM sodium/potassium phosphate buffer (4 mM Na2HPO4 and 1 mM KH2PO4, pH 7.4) at 23 degrees (control t1/2 = 4 min). The concentration-effect curve of obidoxime, which has a weak potency and submaximal efficacy to allosterically retard [3H]NMS dissociation, was better described with a two-site model than with a one-site model. The concentration-effect curves of the test compounds for the allosteric delay of [3H]NMS dissociation were shifted to the right in the presence of obidoxime, yet to a different extent. For WDuo3, W84, alcuronium, and gallamine, the shift induced by increasing concentrations of obidoxime was compatible with a competitive interplay. The pKb values of obidoxime against these modulators lay in a narrow range from pKb = 4.70 with gallamine to pKb = 4.16 with WDuo3. In contrast, the ability of obidoxime to shift the concentration-effect curve of Duo3 was weak (pA2 = 3.00) and not compatible with a competitive interplay. In conclusion, cationic allosteric modulators may stabilize [3H]NMS binding to M2 receptors by divergent modes of allosteric action. The findings suggest that the M2 receptor protein contains more than one allosteric recognition site on its extracellular face.
Animals↗
Evidence for a multiple binding mode of bispyridinium-type allosteric modulators of muscarinic receptors.
The ligand binding properties of muscarinic receptors can be modulated by allosterically acting compounds. Here, a set of novel bispyridinium-type compounds was investigated which were designed to study structure-activity relationships and to provide more insight into the molecular events underlying the allosteric delay of the dissociation of [3H]N-methylscopolamine from muscarinic M2 receptors in porcine cardiac membranes. The parent compound, a non-substituted bispyridinium oxime, displayed a weak allosteric potency and was unable to prevent radioligand dissociation at maximum concentrations. Introduction of either a phthalimidomethyl-moiety or a dichlorobenzyl-moiety at one end of the parent compound led to a considerable increase of the allosteric activity with regard to both the potency and the maximum effect. In these unilaterally ring-substituted bispyridiniums, homologous contralateral non-aromatic modifications were accompanied by divergent potency shifts depending on whether the unilateral ring was phthalimidomethyl or dichlorobenzyl. The findings point to a multiple binding mode of bispyridinium compounds at M2 receptors in the [3H]N-methylscopolamine-occupied state, i.e., different orientations of the compounds at the allosteric binding area or even an interaction with distinct allosteric recognition sites.
Allosteric Regulation↗
[Simvastatin].
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Anticholesteremic Agents↗
Opposite effects of alcuronium on agonist and on antagonist binding to muscarinic receptors.
Alcuronium is known to retard allosterically the dissociation of [3H]N-methylscopolamine from muscarinic M2 receptors, thereby augmenting the binding of this antagonist. Functionally, alcuronium behaves as a weak antimuscarinic agent and induces in combination with N-methylscopolamine an overadditive antimuscarinic action with oxotremorine-M as the agonist. The effect of alcuronium on the binding of [3H]oxotremorine-M was studied in porcine heart homogenates. Agonist binding was concentration dependently inhibited with a Ki = 0.48 +/- 0.03 microM (means +/- S.D., n = 3). Under identical conditions [3H]N-methylscopolamine binding was elevated. Alcuronium, 100 microM, which nearly prevented the dissociation of [3H]N-methylscopolamine, retarded the rate of dissociation of [3H]oxotremorine-M only by a factor of two. These findings support the notion that the overadditive antimuscarinic action of alcuronium in conjunction with N-methylscopolamine is based on a shift by alcuronium of the interplay between agonist and antagonist in favour of the antagonist.
Alcuronium↗
Two-point kinetic experiments to quantify allosteric effects on radioligand dissociation.
Allosteric modulation of a receptor may be proved experimentally by demonstrating an altered radioligand dissociation in the presence of the allosteric modulator. Two-point kinetic experiments provide a screening-type approach to determine the delay of radioligand dissociation caused by allosteric modulation. In this article, Evi Kostenis and Klaus Mohr describe a pitfall in the data analysis that may lead to a suboptimum determination of the allosteric potency in the case of monophasic dissociations, and suggest how this problem may be resolved.
Allosteric Regulation↗
[Calcium and magnesium salts in leg cramps?].
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Calcium↗