Insight into the binding model of new antagonists of kappa receptor using docking and molecular dynamics simulation

PF-4455242 and its analogues represent a new series of kappa opioid selective antagonists that demonstrate high selectivity and potency. We investigated their binding mode to the κ-receptor via docking and molecular dynamics simulations. The ranking of the predicted binding free energies is consistent with experimental results. Detailed binding free energies between antagonists and individual protein residues were calculated, and key residues involved in binding were identified. Deviation of the active site residues was investigated, and the results show that Gln115, Leu135, Tyr139, Trp287 and Tyr313 deviate greatly from the reference structure. Information obtained from molecular modeling studies will aid in the design of potent kappa receptor antagonists.     

Rate constants of agonist binding to muscarinic receptors in rat brain medulla. Evaluation by competition kinetics

The method of competition kinetics, which measures the binding kinetics of an unlabeled ligand through its effect on the binding kinetics of a labeled ligand, was employed to investigate the kinetics of muscarinic agonist binding to rat brain medulla pons homogenates. The agonists studied were acetylcholine, carbamylcholine, and oxotremorine, with N-methyl-4-[3H]piperidyl benzilate employed as the radiolabeled ligand. Our results suggested that the binding of muscarinic agonists to the high affinity sites is characterized by dissociation rate constants higher by 2 orders of magnitude than those of antagonists, with rather similar association rate constants. In contrast, the major differences between the kinetic binding parameters of agonists and antagonists to the low affinity agonist binding sites are in the association rate constants, which were 2-5 orders of magnitude lower for agonists. This demonstrates that there are basic differences in the interactions of agonists with the low and high affinity sites. Our findings also suggest that isomerization of the muscarinic receptors following ligand binding is significant in the case of antagonists, but not of agonists. Moreover, it is demonstrated that in the medulla pons preparation, agonist-induced interconversion between high and low affinity bindings sites does not occur to an appreciable extent.     

Anterior pituitary dopamine receptors. Demonstration of interconvertible high and low affinity states of the D-2 dopamine receptor

The interactions of dopaminergic agonists and antagonists with binding sites in bovine anterior pituitary membranes have been investigated with radioligand-binding techniques and computer-modeling procedures. 3H-labeled agonist binding is stereospecific, reversible, saturable, and of high affinity. The rank order of catecholamines, phenothiazines, and related drugs in competing for 3H-agonist binding is indicative of interactions with a D-2 dopamine receptor. Both agonist/3H-agonist and antagonist/3H-agonist competition curves are monophasic and noncooperative (nH = 1) with computer analysis indicating a single class of binding sites. Specific 3H-agonist binding can be completely inhibited by guanine nucleotides. GppNHp us the most potent nucleotide followed by GTP and GDP which are equipotent. The equilibrium binding capacity for 3H-labeled antagonists is twice that for 3H-agonists. Unlabeled antagonists inhibit 3H-antagonist binding competitively and exhibit antagonist/3H-antagonist competition curves which model best to a state of homogeneous affinity. In contrast, unlabeled agonists inhibit 3H-antagonist binding in a heterogeneous fashion displaying multiphasic (nH less than 1) competition curves which can be resolved into high and low affinity binding sites. In the presence of saturating concentrations of guanine nucleotides, however, the agonist/3H-antagonist curves model best to a single affinity state which is identical with the low affinity state seen in control curves. The binding data can be explained by postulating two states of the D-2 dopamine receptor, inducible by agonists but not antagonists and modulated by guanine nucleotides.     

Adenosine uptake sites in dog heart and brain; interaction with calcium antagonists

[3H] Nitrobenzylthioinosine (NBI) binding is characterized in dog heart and brain. Evidence is presented suggesting that [3H]NBI is binding to the adenosine uptake site in both tissues. Physiologic studies in open-chested dogs clearly demonstrate that NBI acts as a coronary vasodilator, consistent with an action at the adenosine uptake site. The binding is reversible, saturable and of high affinity (KD = 0.78 +/- .06 nM for heart and 0.52 +/- .05 nM for brain). Both dipyridamole and hexobendine are high potency inhibitors of [3H]NBI binding in heart and brain while other antihypertensives and vasodilators such as propranolol and nitroglycerin have no effect. The inhibition of [3H]NBI binding observed with dipyridamole was competitive indicating that both agents are acting at the same site. The dihydropyridine calcium antagonists also inhibited binding with a lower potency than the adenosine uptake blockers. Non-dihydropyridine calcium antagonists were much less potent in this regard. The inhibition of [3H]NBI binding observed with the dihydropyridine calcium antagonists was non-competitive suggesting that the calcium channel and adenosine uptake site may be coupled to each other.     

Parallel strategies for the preparation and selection of liver-targeted glucocorticoid receptor antagonists

Libraries of mifepristone analogs, MP-Acids, were designed and synthesized to increase the chances of identifying GR antagonists that possess liver-selective pharmacological profiles. MP-Acids were uniformly potent GR antagonists in binding and in cell-based functional assays. A high throughput pharmacokinetic selection strategy that employs the cassette dosing of MP-Acids was developed to identify liver-targeting compounds. Thus, resource-intensive in vivo assays to measure liver-selective pharmacology were enriched with GR antagonists that achieve high concentrations in the liver.     

Glucocorticoid-receptor interactions. Discrimination between glucocorticoid agonists and antagonists by means of receptor-binding kinetics.

The kinetics of binding to the molybdate-stabilized glucocorticoid receptor of rat thymus cytosol were determined at 0 degrees C for a number of glucocorticoid agonists and antagonists. Equilibrium constants derived from the rate constants for association and dissociation were in good agreement with those determined directly or by competition under equilibrium conditions. Kinetics parameters for the slowly dissociating form of binding detected by a non-equilibrium dextran/charcoal competitive binding assay reflected the nature and extent of functional-group substitution on the steroid nucleus, but bore no relation to the classification of steroids as glucocorticoid agonists or antagonists. It is concluded that the binding of antagonists that is detected by such methods is agonist-like binding, which is not relevant to their antiglucocorticoid actions. Both agonists and antagonists displayed Michaelis--Menten association kinetics, but this behaviour was much more pronounced for antagonists. This is attributed to the existence of a second form of steroid-receptor complex, which escapes detection by the usual assay methods as a result of a high rate of dissociation and which is quantitatively antagonist-specific under equilibrium conditions. Direct evidence for the existence of two forms of antagonist-receptor complex was provided by results showing that the dissociation of the glucocorticoid antagonist progesterone from the receptor was biphasic.     

Specific binding of a calcium channel activator, [3H]BAY k 8644, to membranes from cardiac muscle and brain

BAY k 8644 is a member of a new class of drugs that directly activates Ca2+ channels. This 1,4-dihydropyridine was found to bind to both high and low affinity sites on rabbit ventricular microsomes and guinea pig brain synaptosomes. The dissociation constant obtained from Scatchard analysis with [3H]BAY k 8644 was 2 to 3 nM for the high affinity binding site, and the estimated maximal number of binding sites was 0.8 and 0.4 pmol/mg protein for heart and brain membranes, respectively, at 15 degrees C. Competition between nitrendipine and [3H]BAY k 8644 indicated a common high affinity binding site for Ca2+ channel activators and antagonists. The results suggest that the 1,4-dihydropyridine Ca2+ channel antagonists do not act as simple channel plugs.     

The (--)[3H]dihydroalprenolol binding to rat adipocyte membranes: an explanation of curvilinear Scatchard plots and implications for quantitation of beta-adrenergic sites

In rat adipocyte membranes, both beta-adrenergic agonists and beta-adrenergic antagonists competed with (--)[3H]dihydroalprenolol for high affinity (KD 2-4 nM) and low capacity binding sites. The antagonists but not the agonists competed with (--)[3H]dihydroalprenolol for lower affinity and higher capacity sites. The present studies were performed in order to characterize the adipocyte beta-adrenergic receptor and distinguish it from low affinity, higher capacity sites which were heat-labile and not stereoselective. When isoproterenol was used to define the nonspecific binding, saturation studies showed a single binding site with a capacity of approximately 100 fmol/mg membrane protein (corresponding to approximately 50,000 sites/adipocyte). Binding was saturated by 10 nM (--)[3H]dihydroalprenolol. Approximate KD's of 204 nM were observed. Kinetic analysis of (--)[3H]dihydroalprenolol binding provided an independent measurement of KD between 0.75 and 1.1 nM. This binding site had the characteristics of a beta 1-adrenergic receptor with the potency of isoproterenol greater than norepinephrine greater than or equal to epinephrine as competitors of binding. Furthermore, the KD of inhibition of (--)[3H]dihydroalprenolol binding correlated with the Ki of inhibition by antagonists or Ka of activation by agonists of glycerol release in isolated adipocytes (r = 0.968, P less than 0.001). These results suggest that beta-adrenergic agonists compete with (--)[3H]dihydroalprenolol for the high affinity binding site which represents the physiological site. Furthermore, the use of antagonists (propranolol, alprenolol) to define specific beta-binding includes nonspecific site(s) as well as the beta-adrenergic site. Previous characterization and quantitation of beta receptors in rat fat cell membranes may have been in error by incorporating both types of binding in their measurement.     

6,7-Dichloroquinoxaline-2,3-Dione is a Competitive Antagonist Specific to Strychnine-Insensitive [3H]Glycine Binding Sites on the N-Methyl-D-Aspartate Receptor Complex

Multiple binding sites on the N-methyl-D-aspartate (NMDA) receptor complex were examined using rat brain synaptic membranes treated with Triton X-100. Binding of [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne ([3H]MK-801), a noncompetitive NMDA antagonist, in the presence of 10 microM L-glutamate not only was inhibited by different types of antagonists, such as 6,7-dichloro-3-hydroxy-2-quinoxaline-carboxylate, 7-chlorokynurenate, and 6,7-dichloroquinoxaline-2,3-dione (DCQX), but also was abolished by non-NMDA antagonists, including 6-cyano-7-nitroquinoxaline-2,3-dione and 6,7-dinitroquinoxaline-2,3-dione. The inhibition of [3H]MK-801 binding by these compounds was invariably reversed or attenuated by addition of 10 microM glycine. Among these novel antagonists with an inhibitory potency on [3H]MK-801 binding, only DCQX abolished [3H]glycine binding without inhibiting [3H]glutamate and [3H](+-)-3-(2-carboxypiperazine-4-yl)propyl-1-phosphonate bindings. Other antagonists examined were all effective as displacers of the latter two bindings. These results suggest that DCQX is an antagonist highly selective to the strychnine-insensitive glycine binding sites with a relatively high affinity.     

Guanine nucleotides modulate the affinity of antagonists at beta-adrenergic receptors

Investigation of the properties of the binding of the radiolabelled antagonists (125I)-iodohydroxybenzylpindolol, (125I)-iodopindolol, and (125I)-iodocyanopindolol to beta-adrenergic receptors of L6 myoblast membranes revealed that guanine nucleotides caused a 2 to 4.5 fold increase in the apparent affinity of these antagonists. No significant effects of GTP were observed on the density of binding sites determined with each radioligand. GTP, GDP, and GMPPNP were of similar high affinity in producing this effect, while GMP was much less potent, and ATP was without effect. Under similar assay conditions GTP reduced the apparent binding affinity of the agonist isoproterenol for the beta-adrenergic receptors of L6 cells. The results indicate that, contrary to previous observations, guanine nucleotides affect not only the interactions of agonists with beta-adrenergic receptors, but also the interaction of antagonists with these adenylate cyclase-linked receptors.     

Synthesis and characterization of 5,6,7,8-tetrahydroquinoline C5a receptor antagonists

A novel series of substituted 2-aryl-5-amino-5,6,7,8-tetrahydroquinoline C5a receptor antagonists is reported. Synthetic routes were developed that allow the substituents on the tetrahydroquinoline core to be efficiently varied, facilitating determination of structure-activity relationships. Members of the series display high binding affinity for the C5a receptor and are potent functional antagonists.     

Muscarinic Autoreceptors of Torpedo Electric Organ Are of the M1 Subtype: Evidence by Radioligand Binding Using Selective Antagonists

The presynaptic muscarinic autoreceptor of Torpedo marmorata electric organ has been characterised by radioligand binding studies using the subtype-selective antagonists pirenzepine, (+)-telenzepine, methoctramine, and AF-DX 116. The presynaptic receptor had relatively high affinity for the M1 antagonists pirenzepine and (+)-telenzepine (Ki = 35 and 7 nM, respectively) and lower affinities for the M2 antagonists AF-DX 116 and methoctramine (Ki = 311 and 277 nM, respectively). Comparison of these binding data with those from an M2 receptor (rat heart membranes) assayed under identical conditions and with data in the recent literature suggests that the Torpedo muscarinic autoreceptor has a pharmacology most similar to the M1 pharmacological subtype of muscarinic acetylcholine receptor.     

Identification of Phe313 of the gonadotropin-releasing hormone (GnRH) receptor as a site critical for the binding of nonpeptide GnRH antagonists

The dog GnRH receptor was cloned to facilitate the identification and characterization of selective nonpeptide GnRH antagonists. The dog receptor is 92% identical to the human GnRH receptor. Despite such high conservation, the quinolone-based nonpeptide GnRH antagonists were clearly differentiated by each receptor species. By contrast, peptide antagonist binding and functional activity were not differentiated by the two receptors. The basis of the differences was investigated by preparing chimeric receptors followed by site-directed mutagenesis. Remarkably, a single substitution of Phe313 to Leu313 in the dog receptor explained the major differences in binding affinities and functional activities. The single amino acid replacement of Phe313 of the human receptor with Leu313 resulted in a 160-fold decrease of binding affinity of the nonpeptide antagonist compound 1. Conversely, the replacement of Leu313 of the dog receptor with Phe313 resulted in a 360-fold increase of affinity for this compound. These results show that Phe313 of the GnRH receptor is critical for the binding of this structural class of GnRH antagonists and that the dog receptor can be "humanized" by substituting Leu for Phe. This study provides the first identification of a critical residue in the binding pocket occupied by nonpeptide GnRH antagonists and reinforces cautious extrapolation of ligand activity across highly conserved receptors.     

Pharmacologic methods for identification of receptors

Determinations of apparent equilibrium dissociation constants of drug-receptor interactions are made from both functional and radioligand binding studies. In each type of study, reversible reactions are assumed and the mass action law is applied. Functional studies are frequently used to determine the dissociation constant of a competitive antagonist but are less frequently used to obtain this constant for agonist compounds since the latter determination requires an experimental procedure that irreversibly inactivates a fraction of the receptors. In the present report, values of dissociation constant for prototype agonists and antagonists, determined from binding and from functional studies, are examined in two classical isolated preparations, rabbit aorta and guinea-pig ileum. In each preparation the dissociation constants from binding and functional experiments agree well for the antagonists but differ markedly for the agonists. Further, the dissociation constant values from binding are seen to be greater for the agonists than for the antagonists. When a chronic treatment regimen in the rabbit resulted in a pronounced change in the functional dissociation constant of subsequently administered norepinephrine, there was no significant change in either the binding constant of this agonist or in the pA2 value of the alpha antagonist, phentolamine. These, and the previously described results, are shown to be compatible with a simple two-state receptor model in which agonists bind with high and low affinity to each state while antagonists do not distinguish between the states. In this model, the ratio of low to high affinity states accounts for the failure of the binding procedure to detect changes in the agonists dissociation constant that are highly significant in the functional study. Whereas the model is based on data for these two classical preparations only, and may not be more generally applicable, the findings demonstrate the necessity for employing both functional and radioligand binding experiments when characterizing drug receptors.     

Specific receptor sites for platelet activating factor on rat liver plasma membranes

The binding of 3H-labeled 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine (PAF) to isolated rat liver plasma membranes and its inhibition by PAF agonists and receptor antagonists was demonstrated. The specific binding was readily saturable with a high affinity. The equilibrium dissociation constant (KD) value was 0.51 (+/- 0.14) nM and the maximal number of binding sites (Bmax) was estimated to be 141 (+/- 18) fmol/mg protein. The binding site was PAF specific-biologically inactive enantiomer was practically inactive. Two PAF-like receptor antagonists, Ono-6240 and CV-3988, and two PAF-unlike receptor antagonists, L-652,731 and kadsurenone, also displaced the binding of [3H]PAF to rat liver plasma membranes but their relative potencies in this system differed from those found in other receptor systems. Mg2+ potentiated [3H]PAF binding but inhibited it at concentrations higher than 10 mM. Both Na+ and K+ inhibited the Mg2+-potentiated binding, an ionic effect which was different from that found in rabbit platelets. These results suggest that rat livers contain PAF-specific receptors, and the receptors in rat livers are different from those found in other receptor systems.     

Identification and optimization of novel 1,3,4-oxadiazole EP1 receptor antagonists

A novel series of oxadiazole EP1 receptor antagonists was identified by replacing the amide of a known glycine sulfonamide derivative with a 1,3,4-oxadiazole. Optimization of the substitution patterns on the three aromatic rings led to the identification of high affinity EP1 receptor antagonists. The derivative with highest affinity displayed a binding IC50 of 2.5 nM (pIC50 8.6).     

Heterogeneity of calcium channel agonist binding sites in the coronary artery

The binding properties (3H) BAY k 8644 a 1,4-dihydropyridine calcium channel agonist were studied in the subcellular membrane fraction isolated from the coronary artery by differential centrifugation. The specific binding of (3H) BAY k 8644 to microsomal membranes of the coronary smooth muscle was rapid, saturable, reversible and of both high and low affinity. The dissociation constants obtained from Scatchard analysis with (3H) BAY k 8644 and nitrendipine were 0.60 +/- 0.02 nmol.l-1 and 9.1 +/- 0.1 nmol.l-1 for the high and low affinity binding site respectively and the estimated maximal numbers of binding sites in the plasma membrane fraction were 0.76 +/- 0.02 and 3.15 +/- 0.18 pmol.mg-1 of protein respectively. The substituted dihydropyridine calcium channel antagonists nitrendipine and nifedipine competitively inhibited specific (3H)BAY k 8644 binding suggesting a common high affinity 1,4-dihydropyridine binding site in the coronary microsomal fraction for calcium channel activator and antagonists. The low affinity agonist binding sites were significantly inhibited by adding nucleoside carrier inhibitors, 2-deoxyadenosine and dipyridamole, and by -SH alkylating agent N-ethylmaleimide. The results suggests that the coronary artery contains both high and low affinity calcium channel binding sites (in a 1:5 ratio) with the low affinity calcium channel agonist binding sites being associated with nucleoside carrier and/or with-SH groups.     

Comparison of glucocorticoid receptors liganded with dexamethasone or progesterone

The initial goal of this work was to examine directly the properties of glucocorticoid receptors bound with antagonists. Cortexolone, progesterone, and R-5020 were the antagonists studied. The tritiated agonists, dexamethasone and triamcinolone acetonide, served as controls. Although the three antiglucocorticoids interfered with agonist binding to the glucocorticoid receptor, direct binding of the tritiated antagonists could not be reproducibility demonstrated using either a charcoal assay or rapid techniques like high performance liquid chromatography or vertical tube rotor ultracentrifugation. Ultraviolet radiation was used to attach covalently tritiated steroid to the receptor. This technique allowed the identification of species that bound agonist or antagonist. That the two classes of steroids bound to the same receptor was shown using a monoclonal antibody directed against the glucocorticoid receptor. These labeled species had the same physical properties upon ultracentrifugation, DEAE cellulose chromatography, and high performance liquid chromatography. It is concluded that although the interaction of antiglucocorticoids like progesterone with the glucocorticoid receptor may be fleeting, antagonists do interact with the glucocorticoid receptor and form complexes with grossly similar properties as those derived from an interaction with agonists.     

Mass Ligand Binding Screening for Receptor Antagonists: Prototype New Drugs and Blind Alleys

Abstract

The ligand binding assay is a powerful tool in the search for antagonists for novel receptors, and for identification of novel classes of antagonists for well-known receptors. Ligand binding mass screening can be adapted for very high throughput. In order for mass screening to be useful, it is necessary to strictly define the binding characteristics for a compound to be considered a putative receptor antagonist. In practice, we have found that synthetic pursuit of a compound with a K_i of ± 1 uM is likely to lead down a blind alley unless very good evidence for specificity is available. Even potent competitors for binding should be thoroughly evaluated in assays of biological activity before a synthetic program is initiated in earnest.     

Pyrrolidinones as potent functional antagonists of the human melanocortin-4 receptor

A series of pyrrolidinones derived from phenylalaninepiperazines were synthesized and characterized as potent and selective antagonists of the melanocortin-4 receptor. In addition to their high binding affinities, these compounds displayed high functional potencies. 12a had a K(i) of 0.94 nM in binding and IC(50) of 21 nM in functional activity. 12a also demonstrated efficacy in a mouse cachexia model.