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Synthesis and pharmacological characterization of [(125)I]MRS1898, a high-affinity, selective radioligand for the rat A(3) adenosine receptor.

ABSTRACT: A known selective agonist of the A(3) adenosine receptors (AR), MRS1898 [(1'R,2'R,3'S,4'R,5'S)-4-{2-chloro-6-[(3-iodophenylmethyl)amino]purin-9-yl}-1-(methylaminocarbonyl)bicyclo[3.1.0]hexane-2,3-diol], was synthesized in radioactive form and characterized pharmacologically. This agonist ligand series, based on nucleoside analogues containing a rigid, bicyclic ring system in place of the ribose moiety, was selected for radiolabeling due to its high A(3)AR affinity across species, with nanomolar binding at both rat and human A(3)ARs. The radioiodination of MRS1898 on its N (6)-3-iodobenzyl substituent was accomplished in 76% radiochemical yield by iododestannylation of a 3-(trimethylstannyl)benzyl precursor. [(125)I]MRS1898 bound to the rat A(3)AR with a K(d) value of 0.17 +/- 0.04 nM and a B(max) value of 0.66 +/- 0.15 pmol/mg protein. The competition binding profiles for other agonists and antagonists obtained with this radioligand are similar to those previously obtained with other radioligands. The advantages of [(125)I]MRS1898 compared with previously used radioligands are primarily its high selectivity and affinity for the rat A(3)AR and also its facile synthesis and radiochemical stability; however, a relatively high level of nonspecific binding presents a limitation. Thus, we have introduced the first selective radioligand for the rat A(3)AR.

SUBMITTER: Gao ZG

PROVIDER: S-EPMC2721771 | biostudies-other | 2009 Mar

REPOSITORIES: biostudies-other

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Pharmacological characterization of FE 201874, the first selective high affinity rat V1A vasopressin receptor agonist.

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| S-EPMC3834753 | biostudies-literature

Synthesis and characterization of [76Br]-labeled high-affinity A3 adenosine receptor ligands for positron emission tomography.

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| S-EPMC8677861 | biostudies-literature

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| S-EPMC4407721 | biostudies-literature

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Project description:The selective, high affinity A(2B) adenosine receptor (AdoR) antagonists that were synthesized by several research groups should aid in determining the role of the A(2B) AdoR in inflammatory diseases like asthma or rheumatoid arthritis (RA) and angiogenic diseases like diabetic retinopathy or cancer. CV Therapeutics scientists discovered the selective, high affinity A(2B) AdoR antagonist 10, a 8-(4-pyrazolyl)-xanthine derivative [CVT-6883, K(i)(hA(2B)) = 22 nM; K(i)(hA(1)) = 1,940 nM; K(i)(hA(2A)) = 3,280; and K(i)(hA(3)) = 1,070 nM] that has favorable pharmacokinetic (PK) properties (t (1/2) = 4 h and F > 35% rat). Compound 10 demonstrated functional antagonism at the A(2B) AdoR (K(B) = 6 nM) and efficacy in a mouse model of asthma. In two phase 1 clinical trials, CVT-6883 was found to be safe, well tolerated, and suitable for once daily dosing. A second compound 20, 8-(5-pyrazolyl)-xanthine, has been nominated for development from Baraldi's group in conjunction with King Pharmaceuticals that has favorable A(2B) AdoR affinity and selectivity [K(i)(hA(2B)) = 5.5 nM; K(i)(hA(1)) > 1,000 nM; K(i)(hA(2A)) > 1,000; and K(i)(hA(3)) > 1,000 nM], and it has been demonstrated to be a functional antagonist. A third compound 32, a 2-aminopyrimidine, from the Almirall group has high A(2B) AdoR affinity and selectivity [K(i)(hA(2B)) = 17 nM; K(i)(hA(1)) > 1,000 nM; K(i)(hA(2A)) > 2,500; and K(i)(hA(3)) > 1,000 nM], and 32 has been moved into preclinical safety testing. Since three highly selective, high affinity A(2B) AdoR antagonists have been nominated for development with 10 (CVT-6883) being the furthest along in the development process, the role of the A(2B) AdoR in various disease states will soon be established.

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Synthesis, structure-affinity relationships, and radiolabeling of selective high-affinity 5-HT4 receptor ligands as prospective imaging probes for positron emission tomography.

Project description:In a search for high-affinity receptor ligands that might serve for development as radioligands for the imaging of brain 5-HT(4) receptors in vivo with positron emission tomography (PET), structural modifications were made to the high-affinity 5-HT(4) antagonist (1-butylpiperidin-4-yl)methyl 8-amino-7-iodo-2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate (1, SB 207710). These modifications were made mainly on the aryl side of the ester bond to permit possible rapid labeling of the carboxylic acid component with a positron emitter, either carbon-11 (t(1/2) = 20.4 min) or fluorine-18 (t(1/2) = 109.7 min), and included (i) replacement of the iodine atom with a small substituent such as nitrile, methyl, or fluoro, (ii) methylation of the 8-amino group, (iii) opening of the dioxan ring, and (iv) alteration of the length of the N-alkyl goup. High-affinity ligands were discovered for recombinant human 5-HT(4) receptors with amenability to labeling with a positron emitter and potential for development as imaging probes. The ring-opened radioligand, (([methoxy-(11)C]1-butylpiperidin-4-yl)methyl 4-amino-3-methoxybenzoate; [(11)C]13), showed an especially favorable array of properties for future evaluation as a PET radioligand for brain 5-HT(4) receptors.

| S-EPMC2951497 | biostudies-literature

Biochemical and behavioural characterization of EMPA, a novel high-affinity, selective antagonist for the OX(2) receptor.

Project description:Background and purposeThe OX(2) receptor is a G-protein-coupled receptor that is abundantly found in the tuberomammillary nucleus, an important site for the regulation of the sleep-wake state. Herein, we describe the in vitro and in vivo properties of a selective OX(2) receptor antagonist, N-ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulphonyl)-amino]-N-pyridin-3-ylmethyl-acetamide (EMPA).Experimental approachThe affinity of [(3)H]EMPA was assessed in membranes from HEK293-hOX(2)-cells using saturation and binding kinetics. The antagonist properties of EMPA were determined by Schild analysis using the orexin-A- or orexin-B-induced accumulation of [(3)H]inositol phosphates (IP). Quantitative autoradiography was used to determine the distribution and abundance of OX(2) receptors in rat brain. The in vivo activity of EMPA was assessed by reversal of [Ala(11),D-Leu(15)]orexin-B-induced hyperlocomotion during the resting phase in mice and the reduction of spontaneous locomotor activity (LMA) during the active phase in rats.Key results[(3)H]EMPA bound to human and rat OX(2)-HEK293 membranes with K(D) values of 1.1 and 1.4 nmol x L(-1) respectively. EMPA competitively antagonized orexin-A- and orexin-B-evoked accumulation of [(3)H]IP at hOX(2) receptors with pA(2) values of 8.6 and 8.8 respectively. Autoradiography of rat brain confirmed the selectivity of [(3)H]EMPA for OX(2) receptors. EMPA significantly reversed [Ala(11),D-Leu(15)]orexin-B-induced hyperlocomotion dose-dependently during the resting phase in mice. EMPA, injected i.p. in rats during the active phase, reduced LMA dose-dependently. EMPA did not impair performance of rats in the rotarod procedure.Conclusions and implicationsEMPA is a high-affinity, reversible and selective OX(2) receptor antagonist, active in vivo, which should prove useful for analysis of OX(2) receptor function.

| S-EPMC2697736 | biostudies-literature

Characterization of human striatal A2-adenosine receptors using radioligand binding and photoaffinity labeling.

Project description:The adenosine agonist [3H]CGS21680 (2-[4-[[2-carboxyethyl]phenyl]ethylamino]-5'- N-ethylcarboxamidoadenosine) bound to A2 receptors in human striatal membranes with a Kd of 17.8 +/- 1.1 nM and a Bmax of 313 +/- 10 fmol/mg protein. The addition of 100 microM GTP diminished both the affinity of agonist radioligand for A2 adenosine binding sites and the total binding, resulting in Kd and Bmax values of 28.6 +/- 1.0 nM and 185 +/- 22 fmol/mg of protein. Adenosine ligands competed for [3H]CGS21680 with the expected potency order. The adenosine antagonist [3H]XAC (8-[4-[[[[(2-aminoethyl)-amino]carbonyl]methyl] oxy]phenyl]-1,3-dipropylxanthine), although A1-selective in the rat, binds to human striatal A2 receptors with high affinity. 25 nM CPX (8-cyclopentyl-1,3-dipropylxanthine), an A1-selective antagonist, was added to the incubation medium and effectively eliminated 91% of [3H]XAC (1 nM) binding to human A1 receptors, yet preserved 90% of binding to A2 receptors. [3H]XAC exhibited saturable, specific binding (50% of total) to A2 sites with a Kd of 2.98 +/- 0.54 nM and a Bmax of 0.71 +/- 0.23 pmol/mg protein (25 degrees C, non-specific binding defined with 100 microM NECA). The potency order for antagonists against 1 nM [3H]XAC was CGS15943A greater than XAC approximately PD115,119 greater than PAPA-XAC greater than CPX greater than HTQZ approximately XCC approximately CP-66,713 greater than theophylline approximately caffeine, indicative of an A2-type binding site. A2a-receptors were found to be present in the human cortex, albeit at a much lower density than in the striatum. Photoaffinity labeling using 125I-PAPA-APEC revealed a molecular weight of 45K, but proteolytic cleavage was observed, resulting in fragments of MW 43K and 37K. In the absence of proteolytic inhibitors the 37K fragment, which still bound 125I-PAPA-APEC, was predominant.

| S-EPMC3429337 | biostudies-literature

Human adenosine A2A receptor binds calmodulin with high affinity in a calcium-dependent manner.

Project description:Understanding how ligands bind to G-protein-coupled receptors and how binding changes receptor structure to affect signaling is critical for developing a complete picture of the signal transduction process. The adenosine A2A receptor (A2AR) is a particularly interesting example, as it has an exceptionally long intracellular carboxyl terminus, which is predicted to be mainly disordered. Experimental data on the structure of the A2AR C-terminus is lacking, because published structures of A2AR do not include the C-terminus. Calmodulin has been reported to bind to the A2AR C-terminus, with a possible binding site on helix 8, next to the membrane. The biological meaning of the interaction as well as its calcium dependence, thermodynamic parameters, and organization of the proteins in the complex are unclear. Here, we characterized the structure of the A2AR C-terminus and the A2AR C-terminus-calmodulin complex using different biophysical methods, including native gel and analytical gel filtration, isothermal titration calorimetry, NMR spectroscopy, and small-angle X-ray scattering. We found that the C-terminus is disordered and flexible, and it binds with high affinity (Kd = 98 nM) to calmodulin without major conformational changes in the domain. Calmodulin binds to helix 8 of the A2AR in a calcium-dependent manner that can displace binding of A2AR to lipid vesicles. We also predicted and classified putative calmodulin-binding sites in a larger group of G-protein-coupled receptors.

| S-EPMC4336363 | biostudies-literature

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