Pharmacological Character Research Articles

Ligand-induced changes in estrogen receptor conformation as measured by site-directed spin labeling.

Site-directed spin labeling (SDSL), the site-specific incorporation of nitroxide spin-labels into a protein, has allowed us to investigate ligand-induced conformational changes in the ligand-binding domain of human estrogen receptor alpha (hERalpha-LBD). EPR (electron paramagnetic resonance) spectroscopy of the nitroxide probe attached to ER produces different spectra depending upon the identity of the bound ligand; these differences are indicative of changes in the type and degree of motional character of the spin-label induced by different ligand-induced conformations of labeled ER. Visual inspection of EPR spectra, construction of B versus C cross-correlation plots, and cross-comparison of spectral pairs using a relative squared difference (RSD) calculation allowed receptor-ligand complexes to be profiled according to their conformational character. Plotting B and C parameters allowed us to evaluate the liganded receptor according to the motional characteristics of the attached spin-label, and they were particularly illustrative for the receptor labeled at position 530, which had motion between the fast and intermediate regimes. RSD analysis allowed us to directly compare the similarity or difference between two different spectra, and these comparisons produced groupings that paralleled those seen in B versus C cross-correlation plots, again relating meaningfully with the pharmacological nature of the bound ligand. RSD analysis was also particularly useful for qualifying differences seen with the receptor labeled at position 417, which had motion between the intermediate and slow motional regimes. This work demonstrates that B and C formulas from EPR line shape theory are useful for qualitative analysis of spectra with differences subtler than those that are often analyzed by EPR spectroscopists. This work also provides evidence that the ER can exist in a range of conformations, with specific conformations resulting from preferential stabilization of ER by the bound ligand. Furthermore, it documents the complexity and uniqueness of the ligand-receptor structure, and highlights the fact that structural differences exist between the receptor bound with ligands of different pharmacological character that, nevertheless, produce similar crystal structures.

Molecular sensors of estrogen receptor conformations and dynamics.

The ligand-induced conformation of a nuclear receptor ligand-binding domain (LBD) is a principal factor leading to transcriptional activity and determining the pharmacological response. Using the estrogen receptor (ER) LBD-labeled site specifically with a fluorophore, we demonstrate that effects of ligand binding on the conformation and dynamics of this domain can be studied directly, in a quantitative and convenient fashion, by various fluorescence methods. Estrogen ligands of different pharmacological character-agonists, selective ER modulators (SERMs), and pure antagonists-each produce distinctive spectroscopic signatures, characteristic of the conformational or dynamic features of their ER-LBD complexes. We can directly follow the equilibrium of helix 12 positions through the degree of local fluorophore rotational freedom and receptor helicity near the C terminus of helix 11. We observe differences even between ligands within a specific pharmacological class, such as the SERMs raloxifene and trans-4-hydroxytamoxifen, highlighting the ability of these fluorescent receptor sensors to detect unique ER conformations induced even by closely related ligands, yet ones that produce distinctive biological activities in estrogen target cells. Fluorophore-labeled LBDs can serve as versatile molecular sensors predictive of ligand pharmacological character and should be broadly applicable to other members of the nuclear receptor superfamily.

Physicochemical prediction of a brain–blood distribution profile in polycyclic amines

Recent investigation into the pharmacological character of the pentacyclo[5.4.0.0 2,6.0 3,10.0 5,9]undecyl and related polycyclic amines has revealed interesting facts regarding their possible use as neuroprotective agents. At this stage however, a clear shortcoming in the quest for further development of this novel class of compounds is the lack of concrete data on their ability to cross the blood-brain barrier (BBB). Working towards the aim of predicting BBB permeability, a series of related N-substituted 8-amino-8,11-oxapentacyclo[5.4.0.0 2,6.0 3,10.0 5,9]undecanes were synthesised. Compounds were characterised by both experimental and calculative methods, followed by biological assessment and statistical manipulation of the results obtained. In doing so, a simple biological model was established for the comparative evaluation of brain–blood distribution properties within the class. A highly sensitive ESI-MS.MS analytical procedure was developed for the detection of these compounds in biological tissues, indicating significant drug concentrations in the brain after intraperitoneal administration to C57Bl/6 mice. Stepwise multiple linear regression analysis of all data yielded two meaningful models ( R 2=0.9996 and R 2=0.7749) depicting lipophilicity (log P oct), solvent accessible molecular volume (SV), molar refractivity (MR) and system energy as the prime determinants of the brain–blood profile for these amines. The inherently high lipophilicity potential within the series is attributed to strong hydrophobic influences dominating hydrogen bonding effects. A possible conformational and energy dependent preference at the site of permeation is also suggested. The proposed estimations allow for the expedient and reliable prediction of brain partitioning behaviour for related polycyclic amines, facilitating the early rejection of unsuitable candidates and enabling research to focus on neuroprotective activity.

Site-Specific Fluorescent Labeling of Estrogen Receptors and Structure–Activity Relationships of Ligands in Terms of Receptor Dimer Stability

Publisher Summary The estrogen receptor (ER) is a ligand-regulated transcription factor that belongs to the nuclear receptor (NR) superfamily and acts as a dimeric species. There are two subtypes of ER—ERα and ERβ—which are both mainly regulated by the endogenous estrogen—estradiol (E2). ER modulation is involved in the development and regulation of reproductive, cardiovascular, and bone health, in addition to controlling the various aspects of cognitive function. Ligands that bind to the ER can be categorized into three pharmacological classes: agonists, mixed agonist—antagonists, and pure antagonists. The mixed agonist–antagonists are also referred to as “selective estrogen receptor modulators (SERMs)” because of their tissue-selective agonist or antagonist activities. Nuclear receptors (NRs) form strong dimers that are essential for their functioning as transcription factors. NRs that bind steroid ligands (i.e., ER) typically function as dimers, either homodimers between identical receptor monomers or between closely related subtypes. NRs that bind nonsteroidal ligands, however, typically function as heterodimers with the retinoid X receptor (RXR). Using fluorescence resonance energy transfer (FRET) and fluorescent-labeled ERα-LBDs, the regulation of homodimer stability by ligands and coactivator peptides is monitored. This technique provides structure–activity relationships (SARs) of ligands in terms of ER dimer stability and can be used as a functional assay to identify the pharmacological character of novel synthetic or environmental ER ligands. Fluorescent-labeled ERβ-LBDs can be used to study homo- and heterodimers of both ER subtypes and the manner in which SERMs and ER subtype-selective ligands (both agonists and antagonists) modulate differential homo- and heterodimer stability. Site-specific fluorescent ERs can also be used for the development of other types of assays to characterize receptor conformation, conformational dynamics, and ligand or coregulator interactions.

The Frizzled-1/(beta(2))-adrenergic receptor chimera: pharmacological properties of a unique G protein-linked receptor.

The frizzled gene family of Wnt receptors encodes proteins that have a seven-transmembrane-spanning motif characteristic of G-protein-coupled receptors. Using a chimeric receptor composed of the exofacial and the transmembrane, ligand-binding domain of the beta(2)-adrenergic receptor (beta2AR) fused with the corresponding cytoplasmic domains of the rat Frizzled-1 receptor (Rfz1), we created a unique chimera between distant members of the superfamily of G protein-coupled receptors. Herein, we describe the pharmacological properties of the chimera, which represents a receptor in which the exofacial and cytoplasmic domains are minimized in length. This unique chimera retains much of the pharmacological character of the native beta2AR, whereas the coupling can be ascribed to Rfz1 domains which operate via G alpha q and not G alpha s. The Rfz1 chimera demonstrates a robust agonist (isoproterenol)-induced sequestration. Since the Rfz1 cytoplasmic domains possess canonical sites for several protein kinases, we were able to investigate the effects of kinase inhibitors on Rfz1 chimera sequestration. Only the protein kinase A inhibitor KT5720, but not inhibitors of protein kinase C, calcium/calmodulin-sensitive kinase-2, casein kinase-2, and Src, inhibited agonist-induced sequestration. Expression of a dominant-negative form of beta-arrestin blocked sequestration of the beta2AR, but only reduced modestly that of the Rfz1 chimera. These data demonstrate that the Frizzled-1 chimera displays cardinal features of a G protein-coupled receptor, including agonist-induced sequestration, but appears to do so largely even in the presence of dominant-negative beta-arrestins.

Vascular α1-Adrenoceptor Subtype Selectivity and α1-Blocker-Induced Orthostatic Hypotension

Newly developed α1-adrenoceptor antagonists including naftopidil are free from the “prazosin-like” side effect of orthostatic hypotension and associated symptoms. We investigated the mechanism for the differential effects of naftopidil and prazosin on the development of postural hypotension, with special attention on their selectivity for the α1-adrenoceptor subtype. We observed that head-up tilt caused a similar extent of drop in mean arterial pressure in control, naftopidil (1 mg/kg)- or prazosin (10 μg/kg)-treated rats; however, the tilt-induced postural hypotension was recovered within 2 min in the naftopidil-treated group, but not in the prazosin-treated group. Comparing an inhibitory effect on noradrenaline-induced contraction in the rat aorta and portal vein, we found that naftopidil was sixfold less potent in the portal vein, while prazosin showed similar potency in both tissues. Reverse transcription-polymerase chain reaction analysis showed that the expression of α1d-adrenoceptor mRNA predominated in the aorta, while that of α1b-adrenoceptor mRNA predominated in the portal vein. Using cloned rat α1- adrenoceptor subtypes, we found that naftopidil was selective for the α1d-subtype with approximately ninefold higher affinity than at the other subtypes. These results show that the pharmacological character of naftopidil, combined with the differential expression of the α1-adrenoceptor subtype in the artery and the vein, may partly explain the differential effect of naftopidil and prazosin on head-up tilt-induced hemodynamic responses.

Structure-activity relationship of the agonist-antagonist transition on the type 1 angiotensin II receptor; the search for inverse agonists.

Peptidic angiotensin II (Ang) antagonists have been mostly reported to behave in a more or less competitive fashion. Thus, reinforcing the view of competitive analogues being compounds which can reversibly bind without producing any biological response to the receptor as well as competing with an agonist (e.g. Ang or other peptidic agonists) for the same site. Recently, a new concept was introduced that changes the classical view of agonist-antagonist action. This concept presents the receptor as a dynamic structure capable of undergoing a conformational change between a biologically active and an inactive form. A bound ligand may shift the equilibrium to either side, according to its pharmacological character as an agonist or an antagonist: An antagonist favoring the inactive form of the receptor represents what is called an “inverse agonist”. All peptidic Ang analogues bind to the same locus on the AT1 receptor but non-peptidic AT1 binding compounds (e.g. L-158,809 and DuP 753) seem to bind to different loci. Furthermore, it has also been shown that non-peptidic Ang antagonists do not possess the ability to recognize Ang receptors from amphibian or avian origins. In the present contribution, we attempt to fathom the molecular parameters that bring the transition from an agonistic to an antagonistic behaviour in order to select the compounds that display most profoundly these antagonistic features. We believe to possess the necessary tools (enlarged but planar aromatic side-chains in position 8) in order to explore the concept of inverse agonism on the mammalian AT1 receptor.KeywordsIntrinsic ActivityInverse AgonistFull AgonistRabbit AortaRecombinant Cell LineThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Brain nicotinic acetylcholine receptors and their functions in special reference to central cardiovascular regulation

The nicotinic acetylcholine receptors present in the mammalian brain have received less attention than those in other areas of the nervous system. However, recent studies have reported the pharmacological character and regional distribution of a high-affinity binding of [3H]acetylcholine or [3H]nicotine. In this review, the pharmacological characters and physiological functions of the brain nicotinic receptors were discussed with special reference to central cardiovascular regulation.

Dynorphin (1–17): Lack of analgesia but evidence for non-opiate electrophysiological and motor effects

Dynorphin and an opiate-inactive fragment des-Tyr-dynorphin produced similar effects on EEG, motor function and hippocampal unit firing. Naloxone had no effect on the actions of dynorphin in these systems and dynorphin failed to produce analgesia upon central administration. These results suggest that dynorphin has a pharmacological character that differs significantly from the classic narcotics.

Intraseptally injected opiate agents: Effects on morphine-induced behaviour of cats

Behavioural effects of intraseptally administered opiate agents were analyzed in cats pretreated with an intraperitoneal injection of morphine. In this way, it became possible to investigate (1) the involvement of septal opiate receptors in the behavioural response of cats to systemic administration of morphine, and (2) the pharmacological character of septal opiate receptors. The following results were obtained with intraseptal injections 15–16 min after intraperitoneal morphine: (1) naloxone decreased frequencies of head and limb movements, and (2) morphine was ineffective. The following results were obtained with intraseptal injections 40–41 min after intraperitoneal morphine: (1) β-endorphin and, to a lesser extent, fentanyl increased frequencies of locomotor patterns, (2) morphine and Met-enkephalin were ineffective, (3) naloxone and naltrexone decreased frequencies of locomotor patterns in a dose-dependent way, (4) naloxone and naltrexone antagonized the effects of β-endorphin and fentanyl, and (5) morphine did not attenuate the effect of naloxone. The intraseptal injections affected only the frequencies of the systemically evoked behaviour patterns; the nature of the behaviour patterns remained unchanged. It is concluded that (1) systemically administered morphine does not affect behaviour via a direct action on septal opiate receptors, and (2) the receptors mediating the septally evoked effects are most probably ϵ-type opiate receptors. The hypothesis is put forward that systemic administration of morphine results in an increased release of β-endorphin from hypothalamo-septal neurons and, as a consequence, changes the β-endorphin activity at the ϵ-type opiate receptors in the septum.

Double pharmacological character of therapy with benzylpenicillin diethylaminoethyl ester

Der zur Depotbehandlung angewandte Benzylpenicillin-diathylaminoathanolester ist, pharmakodynamisch betrachtet, einLokalanaestheticum, das in seiner Esterform antibiotisch unwirksam ist. Erst durch Hydrolyse entsteht im Organismus daraus das antibiotische, aktive Penicillin G, also ein Pharmakon anderen Wirkungstyps und ohne lokalanaesthetische Qualitaten. Als Lokalanaestheticum besitzt der Benzylpenicillin-diathylaminoathanolester intravenos blutdrucksenkende, chinidinartige, teilweise auch ganglioplege Wirkungen. Hinzu kommt auf Grund eigener elektrophysiologischer Untersuchungen an Lungenvagusnerven von Meerschweinchen und Kaninchen, sowie am Bezold-Jarisch-Reflex der Katze eineendoanaesthetische Wirkung auf normal tatige und auf chemisch sensibilisierte Lungendehnungsreceptoren. Die Bedeutung des „lokalanaesthetischen“ Wirkungscharakters des ungespaltenen Esters wird hinsichtlich Verteilung und Wirkung diskutiert.