Fluorescence Characteristics of Hydrophobic Partial Agonist Probes of the Cholecystokinin Receptor

2006 ◽  
Vol 26 (2) ◽  
pp. 89-100 ◽  
Author(s):  
Kaleeckal G. Harikumar ◽  
Delia I. Pinon ◽  
Laurence J. Miller
Keyword(s):  
Partial Agonist ◽  
Receptor Activation ◽  
Spectroscopic Studies ◽  
Dynamic Changes ◽  
Full Agonist ◽  
Cholecystokinin Receptor ◽  
Partial Agonists ◽  
Receptor Structure ◽  
Fluorescence Characteristics ◽  
Agonist And Antagonist

Fluorescence spectroscopic studies are powerful tools for the evaluation of receptor structure and the dynamic changes associated with receptor activation. Here, we have developed two chemically distinct fluorescent probes of the cholecystokinin (CCK) receptor by attaching acrylodan or a nitrobenzoxadiazole moiety to the amino terminus of a partial agonist CCK analogue. These two probes were able to bind to the CCK receptor specifically and with high affinity, and were able to elicit only submaximal intracellular calcium responses typical of partial agonists. The fluorescence characteristics of these probes were compared with those previously reported for structurally-related full agonist and antagonist probes. Like the previous probes, the partial agonist probes exhibited longer fluorescence lifetimes and increased anisotropy when bound to the receptor than when free in solution. The receptor-bound probes were not easily quenched by potassium iodide, suggesting that the fluorophores were protected from the extracellular aqueous milieu. The fluorescence characteristics of the partial agonist probes were quite similar to those of the analogous full agonist probes and quite distinct from the analogous antagonist probes. These data suggest that the partially activated conformational state of this receptor is more closely related to its fully active state than to its inactive state.

scholarly journals Design, Synthesis and Pharmacological Profile of LUF7746, a Novel Covalent Partial Agonist for the Adenosine A1 Receptor

2020 ◽  
Author(s):  
Xue Yang ◽  
Majlen Dilweg ◽  
Dion Osemwengie ◽  
Lindsey Burggraaff ◽  
Daan van der Es ◽  
...  
Keyword(s):  
G Protein ◽  
Partial Agonist ◽  
Receptor Activation ◽  
Adenosine A1 Receptor ◽  
Activation Process ◽  
Full Agonist ◽  
Partial Agonists ◽  
Activation Assay ◽  
Adenosine A1 ◽  
A1 Receptor

Partial agonists for G protein-coupled receptors (GPCRs) provide opportunities for novel pharmacotherapies with enhanced on-target safety compared to full agonists. For the human adenosine A1 receptor (hA1AR) this has led to the discovery of capadenoson, which has been in phase IIa clinical trials for heart failure. Accordingly, the design and profiling of novel hA1AR partial agonists has become an important research focus. In this study, we report on LUF7746, a capadenoson derivative bearing an electrophilic fluorosulfonyl moiety, as an irreversibly binding hA1AR modulator. Meanwhile, a nonreactive ligand bearing a methylsulfonyl moiety, LUF7747, was designed as a control probe in our study. In a radioligand binding assay, LUF7746’s apparent affinity increased to nanomolar range with longer pre-incubation time, suggesting an increasing level of covalent binding over time. Moreover, compared to the reference full agonist CPA, LUF7746 was a partial agonist in a hA1AR-mediated G protein activation assay and resistant to blockade with an antagonist/inverse agonist. An in silico structure-based docking study combined with site-directed mutagenesis of the hA1AR demonstrated that amino acid Y2717.36 was the primary anchor point for the covalent interaction. Additionally, a label-free whole-cell assay was set up to identify LUF7746’s irreversible activation of an A1 receptor-mediated cell morphological response. These results led us to conclude that LUF7746 is a novel covalent hA1AR partial agonist and a valuable chemical probe for further mapping the receptor activation process. It may also serve as a prototype for a therapeutic approach in which a covalent partial agonist may cause less on-target side effects, conferring enhanced safety compared to a full agonist.<br>

scholarly journals Design, Synthesis and Pharmacological Profile of LUF7746, a Novel Covalent Partial Agonist for the Adenosine A1 Receptor

2020 ◽  
Author(s):  
Xue Yang ◽  
Majlen Dilweg ◽  
Dion Osemwengie ◽  
Lindsey Burggraaff ◽  
Daan van der Es ◽  
...  
Keyword(s):  
G Protein ◽  
Partial Agonist ◽  
Receptor Activation ◽  
Adenosine A1 Receptor ◽  
Activation Process ◽  
Full Agonist ◽  
Partial Agonists ◽  
Activation Assay ◽  
Adenosine A1 ◽  
A1 Receptor

Partial agonists for G protein-coupled receptors (GPCRs) provide opportunities for novel pharmacotherapies with enhanced on-target safety compared to full agonists. For the human adenosine A1 receptor (hA1AR) this has led to the discovery of capadenoson, which has been in phase IIa clinical trials for heart failure. Accordingly, the design and profiling of novel hA1AR partial agonists has become an important research focus. In this study, we report on LUF7746, a capadenoson derivative bearing an electrophilic fluorosulfonyl moiety, as an irreversibly binding hA1AR modulator. Meanwhile, a nonreactive ligand bearing a methylsulfonyl moiety, LUF7747, was designed as a control probe in our study. In a radioligand binding assay, LUF7746’s apparent affinity increased to nanomolar range with longer pre-incubation time, suggesting an increasing level of covalent binding over time. Moreover, compared to the reference full agonist CPA, LUF7746 was a partial agonist in a hA1AR-mediated G protein activation assay and resistant to blockade with an antagonist/inverse agonist. An in silico structure-based docking study combined with site-directed mutagenesis of the hA1AR demonstrated that amino acid Y2717.36 was the primary anchor point for the covalent interaction. Additionally, a label-free whole-cell assay was set up to identify LUF7746’s irreversible activation of an A1 receptor-mediated cell morphological response. These results led us to conclude that LUF7746 is a novel covalent hA1AR partial agonist and a valuable chemical probe for further mapping the receptor activation process. It may also serve as a prototype for a therapeutic approach in which a covalent partial agonist may cause less on-target side effects, conferring enhanced safety compared to a full agonist.<br>

scholarly journals Identification of new gonadotrophin-releasing hormone partial agonists

2006 ◽  
Vol 189 (3) ◽  
pp. 509-517 ◽  
Author(s):  
Alfredo Leaños-Miranda ◽  
Alfredo Ulloa-Aguirre ◽  
Laura A Cervini ◽  
Jo Ann Janovick ◽  
Jean Rivier ◽  
...  
Keyword(s):  
Inositol Phosphate ◽  
Partial Agonist ◽  
Prolonged Treatment ◽  
Complete Suppression ◽  
Antagonist Activity ◽  
Gnrh Analogues ◽  
Full Agonist ◽  
Partial Agonists ◽  
Stimulation Of

GnRH agonists or antagonists are currently utilized as therapeutic agents in a number of diseases. A side-effect of prolonged treatment with GnRH analogues is hypoestrogenism. In this study, we tested the in vitro potency of different GnRH analogues originally found to be partial agonists (i.e. analogues with decreased efficacy for activating or stimulating their cognate receptor) as well as novel analogues, to identify compounds that might potentially be useful for partial blockade of gonadotrophin release. Cultured COS-7 cells transiently expressing the rat or human GnRH receptor (GnRHR) were exposed to increasing concentrations (10−8 to 10−5 M) of GnRH analogues (c(4–10)[Asp4,DNal6,Dpr10]-GnRH; c(4–10) [Dpr4,DNal6,Asp10]-GnRH; c(4–10)[Cys4,10,DNal6]-GnRH; c[Eaca1,DNal6]-GnRH; c[Gly1,DNal6]-GnRH; c[βAla1,DTrp6]-GnRH; c[Dava1,DNal6]-GnRH; c[Gaba1, DNal6]-GnRH), and the ability of these analogues to provoke or antagonize GnRH-stimulated inositol phosphate production was assessed. With both human and rat GnRHRs, c[Eaca1,DNal6]-GnRH, c[Gly1,DNal6]-GnRH, c[βAla1,DTrp6]-GnRH and c[Dava1,DNal6]-GnRH exhibited partial agonist activity (35–87% of the maximal efficacy shown by 10−6 M GnRH), whereas c[Gaba1,DNal6]-GnRH behaved as a partial agonist with the human GnRHR and as full agonist with the rat GnRHR. c(4–10)[Asp4, DNal6,Dpr10]-GnRH and c(4–10)[Dpr4,DNal6,Asp10]-GnRH exhibited full antagonist activity with both GnRHRs, and c(4–10) [Cys4,10,DNal6]-GnRH was a weak, partial agonist with the human GnRHR and a full antagonist with the rat GnRHR. With the exception of c[Gaba1,DNal6]-GnRH stimulation of the human GnRHR, and c[Dava1,DNal6]-GnRH and c[Gaba1, DNal6]-GnRH stimulation of the rat GnRHR, all partial agonists also exhibited antagonist activity in the presence of the exogenous full agonist. The results demonstrate that structurally similar analogues display variable potencies and efficacies in vitro for a specific GnRHR as well as for the human versus the rat GnRHR. Their ultimate in vivo usefulness to treat clinical conditions in which complete suppression of gonadotroph activity is not required remains to be investigated.

scholarly journals Predicting the Activation of the Androgen Receptor by Mixtures of Ligands Using Generalized Concentration Addition

2020 ◽  
Vol 177 (2) ◽  
pp. 466-475 ◽  
Author(s):  
Jennifer J Schlezinger ◽  
Wendy Heiger-Bernays ◽  
Thomas F Webster
Keyword(s):  
Androgen Receptor ◽  
Partial Agonist ◽  
Pharmacodynamic Model ◽  
Concentration Addition ◽  
Receptor Ligands ◽  
Receptor System ◽  
Competitive Antagonist ◽  
Full Agonist ◽  
Response Data ◽  
Partial Agonists

Abstract Concentration/dose addition is widely used for compounds that act by similar mechanisms. But it cannot make predictions for mixtures of full and partial agonists for effect levels above that of the least efficacious component. As partial agonists are common, we developed generalized concentration addition, which has been successfully applied to systems in which ligands compete for a single binding site. Here, we applied a pharmacodynamic model for a homodimer receptor system with 2 binding sites, the androgen receptor, that acts according to the classic homodimer activation model: Each cytoplasmic monomer protein binds ligand, undergoes a conformational change that relieves inhibition of dimerization, and binds to DNA response elements as a dimer. We generated individual dose-response data for full (dihydroxytestosterone, BMS564929) and partial (TFM-4AS-1) agonists and a competitive antagonist (MDV3100) using reporter data generated in the MDA-kb2 cell line. We used the Schild method to estimate the binding affinity of MDV3100. Data for individual compounds fit the homodimer pharmacodynamic model well. In the presence of a full agonist, the partial agonist had agonistic effects at low effect levels and antagonistic effects at high levels, as predicted by pharmacological theory. The generalized concentration addition model fits the empirical mixtures data—full/full agonist, full/partial agonist, and full agonist/antagonist—as well or better than relative potency factors or effect summation. The ability of generalized concentration addition to predict the activity of mixtures of different types of androgen receptor ligands is important as a number of environmental compounds act as partial androgen receptor agonists or antagonists.

Adenosine receptor activation in human fibroblasts: nucleoside agonists and antagonists

1980 ◽  
Vol 58 (6) ◽  
pp. 673-691 ◽  
Author(s):  
Robert F. Bruns
Keyword(s):  
Adenosine Receptor ◽  
Partial Agonist ◽  
Human Fibroblasts ◽  
Fold Increase ◽  
Receptor Activation ◽  
Prostaglandin E ◽  
Conformationally Restricted ◽  
Partial Agonists ◽  
High Concentrations ◽  
Noncompetitive Inhibitors

Adenosine (ED50 15 μM) causes a 50-fold increase in intracellular cyclic AMP in the VA13 human fibroblast line. A total of 128 nucleosides was tested as agonists and antagonists. Eight classes of compounds were found: full agonists (14 compounds), weak agonists (20), high-efficacy partial agonists (16), low-efficacy partial agonists (7), competitive inhibitors (11), noncompetitive inhibitors (3), partial agonist - noncompetitive inhibitors (3), and inactive compounds (54). The noncompetitive inhibitors antagonized the responses to adenosine, isoproterenol, and prostaglandin E1 and thus may have been adenylate cyclase inhibitors. The most potent noncompetitive inhibitor, 2′,5′-dideoxyadenosine, was a partial inhibitor, reducing the response to isoproterenol by only 77% even at very high concentrations. The most potent agonists, partial agonists, and pure antagonists had apparent affinities of about 5 μM. Although all positions were important for affinity at the adenosine receptor, only the 3′- and 5′-positions and to a much lesser extent the 6- and 8-positions had an effect on efficacy. The receptor tolerated bulky groups at the 6-position of adenosine, had an ethyl-sized "pocket" near the 5′-position, and had little bulk tolerance towards modifications at other positions. Among the full agonists, only one 5′-derivative and one 2-position derivative had higher apparent affinity than adenosine. Studies with conformationally restricted agonists and antagonists showed that adenosine must be in the anti conformation in order to bind to the receptor.

Thermodynamics of full agonist, partial agonist, and antagonist binding to wild-type and mutant adenosine A1 receptors

1998 ◽  
Vol 56 (11) ◽  
pp. 1437-1445 ◽  
Author(s):  
Alessandro Dalpiaz ◽  
Andrea Townsend-Nicholson ◽  
MargotW Beukers ◽  
PeterR Schofield ◽  
AdriaanP IJzerman
Keyword(s):  
Partial Agonist ◽  
Wild Type ◽  
Adenosine A1 Receptors ◽  
Full Agonist ◽  
Adenosine A1 ◽  
Agonist And Antagonist ◽  
Antagonist Binding

scholarly journals Probing the β2 Adrenoceptor Binding Site with Catechol Reveals Differences in Binding and Activation by Agonists and Partial Agonists

2005 ◽  
Vol 280 (23) ◽  
pp. 22165-22171 ◽  
Author(s):  
Gayathri Swaminath ◽  
Xavier Deupi ◽  
Tae Weon Lee ◽  
Wen Zhu ◽  
Foon Sun Thian ◽  
...  
Keyword(s):  
Binding Site ◽  
Aromatic Ring ◽  
Partial Agonist ◽  
Transmembrane Helix ◽  
Receptor Activation ◽  
Molecular Probe ◽  
Toggle Switch ◽  
Partial Agonists ◽  
Gpcr Activation ◽  
Multistep Process

The β2 adrenergic receptor (β2AR) is a prototypical family A G protein-coupled receptor (GPCR) and an excellent model system for studying the mechanism of GPCR activation. The β2AR agonist binding site is well characterized, and there is a wealth of structurally related ligands with functionally diverse properties. In the present study, we use catechol (1,2-benzenediol, a structural component of catecholamine agonists) as a molecular probe to identify mechanistic differences between β2AR activation by catecholamine agonists, such as isoproterenol, and by the structurally related non-catechol partial agonist salbutamol. Using biophysical and pharmacologic approaches, we show that the aromatic ring of salbutamol binds to a different site on the β2AR than the aromatic ring of catecholamines. This difference is important in receptor activation as it has been hypothesized that the aromatic ring of catecholamines plays a role in triggering receptor activation through interactions with a conserved cluster of aromatic residues in the sixth transmembrane segment by a rotamer toggle switch mechanism. Our experiments indicate that the aromatic ring of salbutamol does not activate this mechanism either directly or indirectly. Moreover, the non-catechol ring of partial agonists does not interact optimally with serine residues in the fifth transmembrane helix that have been shown to play an important role in activation by catecholamines. These results demonstrate unexpected differences in binding and activation by structurally similar agonists and partial agonists. Moreover, they provide evidence that activation of a GPCR is a multistep process that can be dissected into its component parts using agonist fragments.

Atropine blockade of cholinergic drugs on rabbit stomach muscle

1978 ◽  
Vol 56 (5) ◽  
pp. 873-876 ◽  
Author(s):  
L. Spero
Keyword(s):  
Smooth Muscle ◽  
Partial Agonist ◽  
Affinity Constant ◽  
Stomach Muscle ◽  
Competitive Effect ◽  
Full Agonist ◽  
Cholinergic Drugs ◽  
Partial Agonists

The competition between atropine and a full agonist, carbachol, was tested on dibenamine-pretreated smooth muscle of rabbit stomach. Even after extensive irreversible blockade by dibenamine, atropine blockade of the response to carbachol was still competitive and the atropine affinity constant was unchanged. This differed from the noncompetitive action of atropine in blocking the contractile effects of partial agonists such as pilocarpine or heptyl trimethylammonium. The results thus indicate that it is not possible to convert a full agonist into a partial agonist merely by reducing the number of available cholinergic receptors.The present study compares the competitive effect of atropine on the actions of the full agonist carbachol, under conditions of irreversible blockade, with the competitive effect of atropine on partial agonists such as pilocarpine or heptyl trimethylammonium.

scholarly journals Structurally Related Peptide Agonist, Partial Agonist, and Antagonist Occupy a Similar Binding Pocket within the Cholecystokinin Receptor

1999 ◽  
Vol 274 (8) ◽  
pp. 4778-4785 ◽  
Author(s):  
Maoqing Dong ◽  
Xi-Qin Ding ◽  
Delia I. Pinon ◽  
Elizabeth M. Hadac ◽  
Robert P. Oda ◽  
...  
Keyword(s):  
Partial Agonist ◽  
Binding Pocket ◽  
Cholecystokinin Receptor ◽  
Related Peptide ◽  
Agonist And Antagonist ◽  
Peptide Agonist

scholarly journals Predicting the activation of the androgen receptor by mixtures of ligands using Generalized Concentration Addition

2020 ◽  
Author(s):  
Jennifer J Schlezinger ◽  
Wendy Heiger-Bernays ◽  
Thomas F Webster
Keyword(s):  
Androgen Receptor ◽  
Partial Agonist ◽  
Pharmacodynamic Model ◽  
Model Fit ◽  
Concentration Addition ◽  
Receptor Ligands ◽  
Competitive Antagonist ◽  
Full Agonist ◽  
Response Data ◽  
Partial Agonists

AbstractConcentration/dose addition (CA) is widely used for compounds that act by similar mechanisms. But, CA cannot make predictions for mixtures of full and partial agonists for effect levels above that of the least efficacious component. As partial agonists are common, we developed Generalized Concentration Addition (GCA), which has been successfully applied to systems in which ligands compete for a single binding site. Here, we applied a pharmacodynamic model for a system with two binding sites, the androgen receptor (AR). AR acts according to the classic homodimer activation model: each cytoplasmic AR protein binds ligand, undergoes a conformational change that relieves inhibition of dimerization, and binds to DNA response elements as a dimer. We generated individual dose-response data for full (dihydroxytestosterone, BMS564929) and partial (TFM-4AS-1) agonists and a competitive antagonist (MDV3100) using reporter data generated in the MDA-kb2 cell line. We used the Schild method to estimate the binding affinity of AR for MDV3100. Data for individual compounds fit the AR pharmacodynamic model well. The partial agonist had agonistic effects at low effect levels and antagonistic effects at high levels, as predicted by pharmacological theory. The GCA model fit the empirical mixtures data—full/full agonist, full/partial agonist and full agonist/antagonist—as well or better than relative potency factors (a special case of CA) or effect summation. The ability of generalized concentration addition to predict the activity of mixtures of different types of androgen receptor ligands is important as a number of environmental compounds act as partial AR agonists or antagonists.

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