Allosteric modulation of the rat adenosine A1 receptor: Differential effects on agonist and antagonist binding

The interaction of 2‐amino‐3‐benzoyl‐thiophene derivative, PD81,723, as well as other G protein‐coupled receptor (GPCR)‐modulating agents such as suramin (SUR), N‐ethylmaleimide (NEM), GTP, and NaCl with the rat adenosine A₁ receptor was investigated using kinetic, saturation, as well as displacement experiments of various agonists, partial agonists or antagonists. PD81,723 enhanced agonist (CPA, R‐PIA, NECA) binding ～2‐fold, while its effect on CPA binding was increased (4–11‐fold) with other modulators present. In contrast, binding of antagonists (DPCPX, CPT, N‐0840) was inhibited, while binding of partial agonists (8BCPA, MeSCPA) remained uninfluenced. The effect of PD81,723 is consistent with shifting A₁ receptors to an “active” (R*) state with high affinity for agonists and low for antagonists. Further, all described allosteric modulators influenced both agonist and antagonist binding. The IC₅₀ values observed for the agonist CPA, ranging from 4.7 nM in the presence of PD81,723 to a high value of 2.9 μM in the combined presence of NEM, NaCl, and GTP, represented a greater than 600‐fold affinity shift. We suggest that the latter micromolar IC₅₀ value may approximate CPA’s “true” affinity (K_A) for the rat adenosine A₁ receptor. Drug Dev. Res. 51:207–215, 2000. © 2001 Wiley‐Liss, Inc.     

Evaluation of 2‐benzylidene‐1‐tetralone derivatives as antagonists of A1 and A2A adenosine receptors

Antagonists of the adenosine receptors (A₁ and A_2A) are thought to be beneficial in neurological disorders, such as Alzheimer's and Parkinson's disease. The aim of this study was to explore 2‐benzylidene‐1‐tetralone derivatives as antagonists of A₁ and/or A_2A adenosine receptors. In general, the test compounds were found to be selective for the A₁ adenosine receptor, with only three test compounds possessing affinity for both the A₁ and A_2A adenosine receptor. The 2‐benzylidene‐1‐tetralones bearing a hydroxyl substituent at either position C5, C6 or C7 of ring A displayed favourable adenosine A₁ receptor binding, while C5 hydroxy substitution led to favourable A_2A adenosine receptor affinity. Interestingly, para‐hydroxy substitution on ring B in combination with ring A bearing a hydroxy at position C6 or C7 provided the 2‐benzylidene‐1‐tetralones with both A₁ and A_2A adenosine receptor affinity. Compounds 4 and 8 displayed the highest A₁ and A_2A adenosine receptor affinity with values below 7 μm . Both these compounds behaved as A₁ adenosine receptor antagonists in the performed GTP shift assays. In conclusion, the 2‐benzylidene‐1‐tetralone derivatives can be considered as lead compounds to design a new class of dual acting adenosine A₁/A_2A receptor antagonists that may have potential in treating both dementia and locomotor deficits in Parkinson's disease.     

Mapping the allosteric sites of the A2A adenosine receptor

The A_2A adenosine receptor (A_2AAR) is a G protein‐coupled receptor that is pharmacologically targeted for the treatment of inflammation, sepsis, cancer, neurodegeneration, and Parkinson's disease. Recently, we applied long‐timescale molecular dynamics simulations on two ligand‐free receptor conformations, starting from the agonist‐bound (PDB ID: 3QAK) and antagonist‐bound (PDB ID: 3EML) X‐ray structures. This analysis revealed four distinct conformers of the A_2AAR: the active, intermediate 1, intermediate 2, and inactive. In this study, we apply the fragment‐based mapping algorithm, FTMap, on these receptor conformations to uncover five non‐orthosteric sites on the A_2AAR. Two sites that are identified in the active conformation are located in the intracellular region of the transmembrane helices (TM) 3/TM4 and the G protein‐binding site in the intracellular region between TM2/TM3/TM6/TM7. Three sites are identified in the intermediate 1 and intermediate 2 conformations, annexing a site in the lipid interface of TM5/TM6. Five sites are identified in the inactive conformation, comprising a site in the intracellular region of TM1/TM7 and in the extracellular region of TM3/TM4 of the A_2AAR. We postulate that these sites on the A_2AAR be screened for allosteric modulators for the treatment of inflammatory and neurological diseases.     

4‐amino‐6‐alkyloxy‐2‐alkylthiopyrimidine derivatives as novel non‐nucleoside agonists for the adenosine A1 receptor

Three 4‐amino‐6‐alkyloxy‐2‐alkylthiopyrimidine derivatives ( 4 – 6 ) were investigated as potential non‐nucleoside agonists at human adenosine receptors (ARs). When tested in competition binding experiments, these compounds exhibited low micromolar affinity (K_i values comprised between 1.2 and 1.9 μm ) for the A₁ AR and no appreciable affinity for the A_2A and A₃ ARs. Evaluation of their efficacy profiles by measurement of intracellular cAMP levels revealed that 4 and 5 behave as non‐nucleoside agonists of the A₁ AR with EC₅₀ values of 0.47 and 0.87 μm , respectively. No clear concentration‐response curves could be instead obtained for 6 , probably because this compound modulates one or more additional targets, thus masking the putative effects exerted by its activation of A₁ AR. The three compounds were not able to modulate A_2B AR‐mediated cAMP accumulation induced by the non‐selective AR agonist NECA, thus demonstrating no affinity toward this receptor.     

In vitro functional evaluation of isolaureline,dicentrine and glaucine enantiomers at 5‐HT2 and α1 receptors

Compounds with activity at serotonin (5‐hydroxytryptamine) 5‐HT₂ and α₁ adrenergic receptors have potential for the treatment of central nervous system disorders, drug addiction or overdose. Isolaureline, dicentrine and glaucine enantiomers were synthesized, and their in vitro functional activities at human 5‐HT₂ and adrenergic α₁ receptor subtypes were evaluated. The enantiomers of isolaureline and dicentrine acted as antagonists at 5‐HT₂ and α₁ receptors with (R)‐isolaureline showing the greatest potency (pK_b = 8.14 at the 5‐HT_2C receptor). Both (R)‐ and (S)‐glaucine also antagonized α₁ receptors, but they behaved very differently to the other compounds at 5‐HT₂ receptors: (S)‐glaucine acted as a partial agonist at all three 5‐HT₂ receptor subtypes, whereas (R)‐glaucine appeared to act as a positive allosteric modulator at the 5‐HT_2A receptor.     

A2b adenosine receptors can change their spots

Recently, a central role for the A_2b adenosine receptor in a variety of cardiovascular functions including inflammation, erectile function, coronary artery dilation, asthma and cardioprotection has been demonstrated. Despite this evidence, the low-affinity A_2b adenosine receptor is still poorly understood. This receptor appears to be very promiscuous in its coupling. In most tissues, it couples to G_s much like its cousin, the A_2a adenosine receptor, but in mast cells and now, most recently, in cardiac fibroblasts, the A_2b receptor also couples to G_q. Because of its low affinity, this receptor was originally thought unlikely to play any important physiological role. But the sensitivity of A_2b adenosine receptors can be greatly increased by interaction with protein kinase C (PKC) making this receptor, under various conditions, both an activator and a target of PKC. We have recently documented a third coupling involving G_i. This plasticity and versatility of A_2b adenosine receptors position them as potential triggers of signalling in multiple signalling cascades in many physiological responses, making this a most interesting receptor indeed.This article is a commentary on Feng et al., pp. 1598–1607 of this issue. To view this paper visit http://dx.doi.org/10.1111/j.1476-5381.2009.00558.x     

1,3‐dialkylxanthine derivatives having high potency as antagonists at human A2B adenosine receptors

The structure–activity relationships (SAR) of alkylxanthine derivatives as antagonists at the recombinant human adenosine receptors were explored in order to identify selective antagonists of A_2B receptors. The effects of lengthening alkyl substituents from methyl to butyl at 1‐ and 3‐positions and additional substitution at the 7‐ and 8‐positions were probed. K_i values, determined in competition binding in membranes of HEK‐293 cells expressing A_2B receptors using ¹²⁵I‐ABOPX (¹²⁵I‐3‐(4‐amino‐3‐iodobenzyl)‐8‐(phenyl‐4‐oxyacetate)‐1‐propylxanthine), were approximately 10 to 100 nM for 8‐phenylxanthine functionalized congeners. Xanthines containing 8‐aryl, 8‐alkyl, and 8‐cycloalkyl substituents, derivatives of XCC (8‐[4‐[[[carboxy]methyl]oxy]phenyl]‐1,3‐dipropylxanthine) and XAC (8‐[4‐[[[[(2‐aminoethyl)amino]carbonyl]methyl]‐oxy]phenyl]‐1,3‐dipropylxanthine), containing various ester and amide groups, including L‐ and D‐amino acid conjugates, were included. Enprofylline was 2‐fold more potent than theophylline in A_2B receptor binding, and the 2‐thio modification was not tolerated. Among the most potent derivatives examined were XCC, its hydrazide and aminoethyl and fluoroethyl amide derivatives, XAC, N‐hydroxyethyl‐XAC, and the L‐citrulline and D‐p‐aminophenylalanine conjugates of XAC. An N‐hydroxysuccinimide ester of XCC (XCC‐NHS, MRS 1204) bound to A_2B receptors with a K_i of 9.75 nM and was the most selective (at least 20‐fold) in this series. In a functional assay of recombinant human A_2B receptors, four of these potent xanthines were shown to fully antagonize the effects of NECA‐induced stimulation of cyclic AMP accumulation. Drug Dev. Res. 47:45–53, 1999. Published 1999 Wiley‐Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.     

Synthesis and structure-activity relationships of 4-cycloalkylamino-1, 2, 4-triazolo[4, 3-a]quinoxalin-1- one derivatives as A1 and A3 adenosine receptor antagonists

In a previous paper we reported the synthesis and binding activity of 4-cycloalkylamino-1, 2, 4-triazolo[4, 3-a]quinoxalin-1-one derivatives, differently substituted on the appended 2-phenyl ring, some of which were potent and selective A(1) adenosine receptor (AR) antagonists. In the present paper several 4-cycloalkylamino-2-phenyl-1, 2, 4-triazolo[4, 3-a]quinoxalin-1-one derivatives (1-11), bearing simple substituents on the benzofused moiety, are reported. The binding data of bovine A(1) and A(2A) and human A(3) AR show that we have obtained highly potent A(1) AR antagonists. In particular, the 4-cyclohexylamino derivatives 1-5 show higher A(1) vs A(2A) selectivity than the parent compound A, which lacks substituents on the benzofused moiety. Moreover, compounds 1-11 display, in general, good A(3) AR affinity. Finally, SAR studies provide some new insights about the steric requirements of the A(3) receptor pocket, which accommodates the benzofused moiety of our 4-amino-triazoloquinoxalin-1-one derivatives.     

Design of high affinity cyclic pentapeptide ligands for κ‐opioid receptors

Abstract: Using results from our previously reported cyclic opioid peptide series and reliable models for μ‐, δ‐, and κ‐opioid receptors (MOR, DOR, and KOR, respectively) and their complexes with peptide ligands, we have designed and synthesized a series of cyclic pentapeptides of structure Tyr‐c[d ‐Cys‐Phe‐Phe‐X]‐NH₂, cyclized via disulfide, methylene, or ethylene dithioethers, and where X = d ‐ or l ‐Cys; or d ‐ or l ‐penicillamine (Pen; β,β‐dimethylcysteine). Determination of binding affinities to MOR, DOR, and KOR revealed that members of this series with X = d ‐ or l ‐Cys display KOR affinities in the low nanomolar range, demonstrating that a ‘DPDPE‐like’ tetrapeptide scaffold is suitable not only for DOR and MOR ligands, but also for KOR ligands. The cyclic pentapeptides reported here are not, however, selective for KOR, rather they display significant selectivity and high affinity for MOR. Indeed, peptide 8 , Tyr‐c[d ‐Cys‐Phe‐Phe‐Cys]‐NH₂‐cyclized via a methylene dithioether, shows picomolar binding affinity for MOR ( = 16 pm ) with more than 100‐fold selectivity for MOR vs. DOR or KOR, and may be of interest as a high affinity, high selectivity MOR ligand. Nonetheless, the high affinity KOR peptides in this series represent excellent leads for the development of structurally related, selective KOR ligands designed to exploit structurally specific features of KOR, MOR, and DOR.     

Inhibition of muscarinic K+ current in guinea-pig atrial myocytes by PD 81,723, an allosteric enhancer of adenosine binding to A1 receptors

1. PD 81,723 has been shown to enhance binding of adenosine to A₁ receptors by stabilizing G protein-receptor coupling (‘allosteric enhancement''). Evidence has been provided that in the perfused hearts and isolated atria PD 81,723 causes a sensitization to adenosine via this mechanism.
2. We have studied the effect of PD 81,723 in guinea-pig isolated atrial myocytes by use of whole-cell measurement of the muscarinic K⁺ current (I_K(ACh)) activated by different G_i-coupled receptors (A₁, M₂, sphingolipid). PD 81,273 caused inhibition of I_K(ACh) (IC₅₀≃5 μM) activated by either of the three receptors. Receptor-independent I_K(ACh) in cells loaded with GTP-γ-S and background I_K(ACh), which contributes to the resting conductance of atrial myocytes, were equally sensitive to PD 81,723. At no combination of concentrations of adenosine and PD 81,723 could an enhancing effect be detected.
3. The compound was active from the outside only. Loading of the cells with PD 81,723 (50 μM) via the patch pipette did not affect either I_K(ACh) or its sensitivity to adenosine. We suggest that PD 81,723 acts as an inhibitor of inward rectifying K⁺ channels; this is supported by the finding that ventricular I_K1, which shares a large degree of homology with the proteins (GIRK1/GIRK4) forming I_K(ACh) but is not G protein-gated, was also blocked by this compound.
4. It is concluded that the functional effects of PD 81,723 described in the literature are not mediated by the A₁ adenosine receptor-G_i-I_K(ACh) pathway.

     

Phenyl‐substituted N6‐phenyladenosines and N6‐phenyl‐5′‐N‐ethylcarboxamidoadenosines with high activity at human adenosine A2B receptors

A series of phenyl‐substituted N⁶‐phenyladenosines and N⁶‐phenyl‐5′‐N‐ethylcarboxamidoadenosines were synthesized and tested at adenosine receptor subtypes. EC₅₀ values were determined for cyclic AMP production in CHO cells expressing human A_2B receptors. Binding affinities were determined for rat A₁ and A_2A receptors and human A₃ receptors. N⁶‐phenyladenosine displayed an EC₅₀ value at A_2B receptors of 6.3 μM. Several N⁶‐phenyladenosine derivatives were more active than N⁶‐phenyladenosine, while two analogs were also more potent than 5′‐N‐ethylcarboxamidoadenosine (NECA, 0.76 μM), i.e., the 4‐iodophenyl ( 10 , 0.37 μM) and the 4‐aminosulfonylphenyl ( 20 , 0.44 μM) derivatives. N⁶‐phenyl‐NECA derivatives were as active as their analogous adenosine derivatives. Drug Dev. Res. 49:85–93, 2000. © 2000 Wiley‐Liss, Inc.     

Nuclear expression of μ‐opioid receptors in a human mesothelial cell line

1 Possibly acting via μ‐opioid receptors (MORs), morphine inhibits the formation of experimentally induced postoperative abdominal adhesions in rats. Mesothelial cells may participate in adhesion formation by secreting mediators that interfere negatively with fibrinolysis. Morphine may prevent adhesions by inhibiting the release of pro‐adhesion mediators from mesothelial cells. This study aimed to investigate whether human mesothelial cells express ΜΟR‐1; if so, such could constitute a site of action for morphine in adhesion prevention. 2 Cells from Met‐5A, a human mesothelial cell line were seeded and prepared for immunocytochemistry and Western blotting. 3 Immunocytochemistry showed MOR‐1 expression in mesothelial cells, predominantly in the nuclei. Western blotting showed two bands (c. 35 and 50 kDa) which correspond to those obtained with a control lysate from cells known to express MORs. In addition, we found MOR‐1 expression with nuclear and cytoplasmatic localization in biopsies from human abdominal adhesions. 4 The current findings may suggest that morphine could interact directly with mesothelial cells via MOR‐1 receptors, and thereby modulate adhesion formation, possibly by interfering with the release of pro‐adhesion factors from these cells.     

Radioligand binding and functional responses of ligands for human recombinant adenosine A(3) receptors

The binding and functional properties of adenosine receptor ligands were compared in Chinese hamster ovary cells transfected with human adenosine A(3) receptors. Inhibition of [(125)I]-aminobenzyl-5'-N-methylcarboamidoadenosine ([(125)I]-AB-MECA) binding by adenosine receptor ligands was examined in membrane preparations. Inhibition of forskolin-induced cAMP accumulation by agonists was measured using a cAMP enzyme immunoassay. The rank order of agonist potency for both assays was N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) > 5'-N-ethylcarboxamidoadenosine (NECA) > (-)-N(6)-[(R)-phenylisopropyl] adenosine (R-PIA) > 4-aminobenzyl-5'-N-methylcarboxamidoadenosine (AB-MECA) > N(6)-cyclopentyl adenosine (CPA) > adenosine. The radioligand binding rank order of antagonist potency was N-[9-chloro-2-(2-furanyl)[1,2,4]-triazolo[1,5-c]quinazolin-5-benzeneacetamide (MRS1220) > 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) > 8-phenyltheophylline (8-PT) > 8-(p-sulfophenyl)-theophylline (8-SPT). MRS1220 competitively inhibited the effect of IB-MECA on cAMP production, with a K(B) value of 0.35 nm. These data are characteristic of adenosine A(3) receptors. The absence of Mg(2+) and presence of guanosine 5'-(gamma-thio)triphosphate (GTPgammaS) significantly reduced agonist binding inhibition potency, indicating binding to high- and low-affinity states. The IB-MECA, NECA and R-PIA IC(50) values were greater for the cAMP assay than for radioligand binding, suggesting an efficient stimulus-response transduction pathway.     

The endocannabinoid 2-arachidonylglycerol is a negative allosteric modulator of the human A3 adenosine receptor

Studies of endogenous cannabinoid agonists, such as 2-arachidonylglycerol (2-AG), have revealed their potential to exert modulatory actions on other receptor systems in addition to their ability to activate cannabinoid receptors. This study investigated the effect of cannabinoid ligands on the human adenosine A₃ (hA₃R) receptor. The endocannabinoid 2-AG was able to inhibit agonist ([¹²⁵I]N⁶-(4-amino-3-iodobenzyl) adenosine-5′-(N-methyluronamide) - [¹²⁵I] AB MECA) binding at the hA₃R. This inhibition occurred over a narrow range of ligand concentration and was characterized by high Hill coefficients suggesting a non-competitive interaction. Furthermore, in the presence of 2-AG, the rate of [¹²⁵I] AB MECA dissociation was increased, consistent with an action as a negative allosteric modulator of the hA₃R. Moreover, by measuring intracellular cAMP levels, we demonstrate that 2-AG decreases both the potency of an agonist at the hA₃R and the basal signalling of this receptor. Since the hA₃R has been shown to be expressed in astrocytes and microglia, these findings may be particularly relevant in certain pathological states such as cerebral ischemia where levels of 2-AG and anandamide are raised.     

Acute withdrawal induced by adenosine A1‐receptor activation in isolated guinea‐pig ileum: role of opioid receptors and effect of cholecystokinin

Objectives In isolated guinea‐pig ileum, the μ‐opioid acute withdrawal response is under control of several neuronal systems, including the κ‐opioid and the A₁‐adenosine systems, which are involved in the μ‐withdrawal response inhibitory control. After μ‐opioid system stimulation, indirect activation of both κ‐opioid and A₁‐adenosine systems is prevented by the peptide cholecystokinin‐8 (CCk‐8). Guinea‐pig ileum exposed to A₁‐adenosine agonist (CPA), shows a withdrawal contracture precipitated by the A₁‐adenosine antagonist (CPT). We investigated this response. Methods We investigated the involvement of the opioid system in the A₁‐adenosine acute withdrawal response in guinea‐pig ileum, the potential induced cross‐dependence between the A₁ and the opioid system and also the interaction between the CCk‐8 and A₁ systems. Key findings We found that in the guinea‐pig ileum preparation exposed to CPA, μ‐ and κ‐opioid antagonists increased the withdrawal response to CPT. Tissues exposed to CPA showed a contractile response to the opioid receptor antagonist naloxone only after complete removal of the A₁‐agonist. In the presence of CPA, the response to CCk‐8 was inhibited while a significant increase in CPT response intensity was observed. Conclusions In guinea‐pig ileum, stimulation of the A₁ system indirectly activates both μ‐ and κ‐opioid systems; this indirect activation is significantly, albeit not completely, antagonised by CCk‐8. Cross dependence between A₁ and opioid systems was also observed.     

Roles of residues 3 and 4 in cyclic tetrapeptide ligand recognition by the κ‐opioid receptor

Abstract: A series of cyclic, disulfide‐ or dithioether‐containing tetrapeptides based on previously reported potent μ‐ and δ‐selective analogs has been explored with the aim of improving their poor affinity to the κ‐opioid receptor. Specifically targeted were modifications of tetrapeptide residues 3 and 4, as they presumably interact with residues from transmembrane helices 6 and 7 and extracellular loop 3 that differ among the three receptors. Accordingly, tetrapeptides were synthesized with Phe³ replaced by aliphatic (Gly, Ala, Aib, Cha), basic (Lys, Arg, homo‐Arg), or aromatic sides chains (Trp, Tyr, p‐NH₂Phe), and with d ‐Pen⁴ replaced by d ‐Cys⁴, and binding affinities to stably expressed μ‐, δ‐, and κ‐receptors were determined. In general, the resulting analogs failed to exhibit appreciable affinity for the κ‐receptor, with the exception of the tetrapeptide Tyr‐c[d ‐Cys‐Phe‐d ‐Cys]‐NH₂, cyclized via a disulfide bond, which demonstrated high binding affinity toward all opioid receptors (Ki^μ = 1.26 nm , Ki^δ = 16.1 nm , Ki^κ = 38.7 nm ). Modeling of the κ‐receptor/ligand complex in the active state reveals that the receptor‐binding pocket for residues 3 and 4 of the tetrapeptide ligands is smaller than that in the μ‐receptor and requires, for optimal fit, that the tripeptide cycle of the ligand assume a higher energy conformation. The magnitude of this energy penalty depends on the nature of the fourth residue of the peptide (d ‐Pen or d ‐Cys) and correlates well with the observed κ‐receptor binding affinity.     

New insights into the structure of the trace amine‐associated receptor 2: Homology modelling studies exploring the binding mode of 3‐iodothyronamine

Recent studies have further investigated the trace amine‐associated receptor type 2 (TAAR2) pharmacology, revealing its role not only at the olfactory sensory neurons but also at the immune system, being expressed in human leucocytes. In particular, the ability of this receptor to bind the unselective TAAR ligand 3‐iodo‐thyronamine ( T ₁ AM ) was elucidated, making in the meanwhile the discovery of selective compounds a urgent need to derive much more suitable tools for studying TAARs. In this context, we developed our work on TAAR2 applying a structure‐based computational protocol, including TAAR2 homology modelling and T ₁ AM docking studies. The results were compared with those we previously obtained about TAAR1, in order to point out new insights guiding for selectivity between TAAR1 and TAAR2. The in silico strategy applied allowed us to provide for the first time thorough TAAR2 homology models, which are expected to be useful tools for a further design process of more selective TAAR ligands.