Selectivity is species-dependent: Characterization of standard agonists and antagonists at human,rat, and mouse adenosine receptors

Adenosine receptors (ARs) have emerged as new drug targets. The majority of data on affinity/potency and selectivity of AR ligands described in the literature has been obtained for the human species. However, preclinical studies are mostly performed in mouse or rat, and standard AR agonists and antagonists are frequently used for studies in rodents without knowing their selectivity in the investigated species. In the present study, we selected a set of frequently used standard AR ligands, 8 agonists and 16 antagonists, and investigated them in radioligand binding studies at all four AR subtypes, A₁, A_2A, A_2B, and A₃, of three species, human, rat, and mouse. Recommended, selective agonists include CCPA (for A₁AR of rat and mouse), CGS-21680 (for A_2A AR of rat), and Cl-IB-MECA (for A₃AR of all three species). The functionally selective partial A_2B agonist BAY60-6583 was found to additionally bind to A₁ and A₃AR and act as an antagonist at both receptor subtypes. The antagonists PSB-36 (A₁), preladenant (A_2A), and PSB-603 (A_2B) displayed high selectivity in all three investigated species. MRS-1523 acts as a selective A₃AR antagonist in human and rat, but is only moderately selective in mouse. The comprehensive data presented herein provide a solid basis for selecting suitable AR ligands for biological studies.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-015-9460-9) contains supplementary material, which is available to authorized users.     

Tritium-labeled agonists as tools for studying adenosine A<Subscript>2B</Subscript> receptors

A selective agonist radioligand for A_2B adenosine receptors (A_2BARs) is currently not available. Such a tool would be useful for labeling the active conformation of the receptors. Therefore, we prepared BAY 60-6583, a potent and functionally selective A_2BAR (partial) agonist, in a tritium-labeled form. Despite extensive efforts, however, we have not been able to establish a radioligand binding assay using [³H]BAY 60-6583. This is probably due to its high non-specific binding and its moderate affinity, which had previously been overestimated based on functional data. As an alternative, we evaluated the non-selective A_2BAR agonist [³H]NECA for its potential to label A_2BARs. [³H]NECA showed specific, saturable, and reversible binding to membrane preparations of Chinese hamster ovary (CHO) or human embryonic kidney (HEK) cells stably expressing human, rat, or mouse A_2BARs. In competition binding experiments, the AR agonists 2-chloroadenosine (CADO) and NECA displayed significantly higher affinity when tested versus [³H]NECA than versus the A_2B-antagonist radioligand [³H]PSB-603 while structurally diverse AR antagonists showed the opposite effects. Although BAY 60-6583 is an A_2BAR agonist, it displayed higher affinity versus [³H]PSB-603 than versus [³H]NECA. These results indicate that nucleoside and non-nucleoside agonists are binding to very different conformations of the A_2BAR. In conclusion, [³H]NECA is currently the only useful radioligand for determining the affinity of ligands for an active A_2BAR conformation.     

Common actions of adenosine receptor agonists in modulating human trabecular meshwork cell transport

A₁ adenosine receptors (ARs) reduce, and A₂ARs increase intraocular pressure, partly by differentially altering resistance to aqueous humor outflow. It is unknown whether the opposing effects of A₁AR and A₂AR agonists are mediated at different outflow-pathway cell targets or by opposing actions on a single cell target. We tested whether a major outflow-pathway cell, the trabecular meshwork (TM) cell might constitute the primary AR-agonist target and respond differentially to A₁, A_2A and A₃AR agonists. Receptor activation in human TM cells was identified by applying subtype-selective AR agonists: CPA and ADAC for A₁ARs, CGS 21680 and DPMA for A_2AARs, and Cl-IB-MECA and IB-MECA for A₃ARs. Stimulation of A₁, A_2A and A₃ARs elevated Ca²⁺, measured with fura-2. Whole-cell patch clamping indicated that AR agonists activated ion channels non-uniformly, possibly reflecting variability in magnitude of agonist-triggered second-messenger responses. A₁, A_2A and A₃AR agonists all reduced volume, determined by calcein cell imaging. The endogenous source of adenosine delivery to the outflow pathway could be the TM cells since these cells were stimulated to release ATP by hypotonic perfusion. We conclude that: (1) TM cells express functional A₁, A_2A and A₃ARs; and (2) the reported differential effects of AR agonists on aqueous humor outflow are not mediated by differential actions on TM-cell Ca²⁺ and volume, but likely by actions on separate cell targets. Reprint requests should be addressed to: Dr. Mortimer M. Civan, Dept. of Physiology, University of Pennsylvania, Richards Building, Philadelphia, PA 19104-6085. [Tel.: (215)-898-8773; Fax: (215)-573-5851]     

The role of activated adenosine receptors in degranulation of human LAD2 mast cells

Mast cell degranulation triggers hypersensitivity reactions at the body–environment interface. Adenosine modulates degranulation, but enhancement and inhibition have both been reported. Which of four adenosine receptors (ARs) mediate modulation, and how, remains uncertain. Also uncertain is whether adenosine reaches mast cell ARs by autocrine ATP release and ecto-enzymatic conversion. Uncertainties partly reflect species and cell heterogeneity, circumvented here by focusing on homogeneous human LAD2 cells. Quantitative PCR detected expression of A_2A, A_2B, and A₃, but not A₁, ARs. Nonselective activation of ARs with increasing NECA monotonically enhanced immunologically or C3a-stimulated degranulation. NECA alone stimulated degranulation slightly. Selective AR antagonists did not affect C3a-stimulated degranulation. NECA''s enhancement of C3a-triggered degranulation was partially inhibited by separate application of each selective antagonist, and abolished by simultaneous addition of antagonists to the three ARs. Only the A_2A antagonist separately inhibited NECA''s enhancement of immunologically stimulated degranulation, which was abolished by simultaneous addition of the three selective antagonists. Immunological or C3a activation did not stimulate ATP release. NECA also enhanced immunologically triggered degranulation of mouse bone marrow derived mast cells (BMMCs), which was partially reduced only by simultaneous addition of the three antagonists or by the nonselective antagonist {"type":"entrez-protein","attrs":{"text":"CGS15943","term_id":"875345334"}}CGS15943. BMMCs also expressed A_2A, A_2B, and A₃ ARs. but not A₁AR detectably. We conclude that (a) A₁AR is unnecessary for LAD2 degranulation or AR enhancement; (b) A_2A, A_2B, and A₃ ARs all contribute to pharmacologic AR enhancement of LAD2 and BMMC degranulation; and (c) LAD2 cells depend on microenvironmental adenosine to trigger AR modulation.     

Modulation of G protein-coupled adenosine receptors by strategically functionalized agonists and antagonists immobilized on gold nanoparticles

Gold nanoparticles (AuNPs) allow the tuning of pharmacokinetic and pharmacodynamic properties by active or passive targeting of drugs for cancer and other diseases. We have functionalized gold nanoparticles by tethering specific ligands, agonists and antagonists, of adenosine receptors (ARs) to the gold surface as models for cell surface interactions with G protein-coupled receptors (GPCRs). The AuNP conjugates with chain-extended AR ligands alone (PEGylated nucleosides and nonnucleosides, anchored to the Au via thioctic acid) were found to be insoluble in water due to hydrophobic entities in the ligand. Therefore, we added a second, biologically inactive pendant moiety to increase the water solubility, consisting of a PEGylated chain terminating in a carboxylic or phosphate group. The purity and stability of the immobilized biologically active ligand were examined by ultrafiltration and HPLC. Pharmacological receptor binding studies on these GPCR ligand-derivatized AuNPs (2–5 nm in diameter), performed using membranes of mammalian cells stably expressing human A₁, A_2A, and A₃ARs, showed that the desired selectivity was retained with K _i values (nanomolar) of A₃AR agonist 21b and A_2AAR antagonists 24 and 26a of 14 (A₃), 34 (A_2A), and 69 (A_2A), respectively. The corresponding monomers displayed K _i values of 37, 61, and 1,420 nM, respectively. In conclusion, we have synthesized stable, water-soluble AuNP derivatives of tethered A₃ and A_2AAR ligands that retain the biological properties of their monomeric ligands and are intended for therapeutic and imaging applications. This is the first prototypical application to gold carriers of small molecule (nonpeptide) GPCR ligands, which are under investigation for treatment of cancer and inflammatory diseases.     

Pharmacological characterization of DPTN and other selective A3 adenosine receptor antagonists

The A₃ adenosine receptor (AR) is emerging as an attractive drug target. Antagonists are proposed for the potential treatment of glaucoma and asthma. However, currently available A₃AR antagonists are potent in human and some large animals, but weak or inactive in mouse and rat. In this study, we re-synthesized a previously reported A₃AR antagonist, DPTN, and evaluated its affinity and selectivity at human, mouse, and rat ARs. We showed that DPTN, indeed, is a potent A₃AR antagonist for all three species tested, albeit a little less selective for mouse and rat A₃AR in comparison to the human A₃AR. DPTN’s K_i values at respective A₁, A_2A, A_2B, and A₃ receptors were (nM) 162, 121, 230, and 1.65 (human); 411, 830, 189, and 9.61 (mouse); and 333, 1147, 163, and 8.53 (rat). Its antagonist activity at both human and mouse A₃ARs was confirmed in a cyclic AMP functional assay. Considering controversial use of currently commercially available A₃AR antagonists in rats and mice, we also re-examined other commonly used and selective A₃AR antagonists under the same experimental conditions. The K_i values of MRS1523 were shown to be 43.9, 349, and 216 nM at human, mouse, and rat A₃ARs, respectively. MRS1191 and MRS1334 showed incomplete inhibition of [¹²⁵I]I-AB-MECA binding to mouse and rat A₃ARs, while potent human A₃AR antagonists, MRS1220, MRE3008F20, PSB10, PSB-11, and VUF5574 were largely inactive. Thus, we demonstrated that DPTN and MRS1523 are among the only validated A₃AR antagonists that can be possibly used (at an appropriate concentration) in mouse or rat to confirm an A₃AR-related mechanism or function.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11302-021-09823-5.     

Novel fluorescent triazinobenzimidazole derivatives as probes for labelling human A1 and A2B adenosine receptor subtypes

The expression levels and the subcellular localization of adenosine receptors (ARs) are affected in several pathological conditions as a consequence of changes in adenosine release and metabolism. In this respect, labelled probes able to monitor the AR expression could be a useful tool to investigate different pathological conditions. Herein, novel ligands for ARs, bearing the fluorescent 7-nitrobenzofurazan (NBD) group linked to the N¹ (1,2) or N¹⁰ (3,4) nitrogen of a triazinobenzimidazole scaffold, were synthesized. The compounds were biologically evaluated as fluorescent probes for labelling A₁ and A_2B AR subtypes in bone marrow-derived mesenchymal stem cells (BM-MSCs) that express both receptor subtypes. The binding affinity of the synthetized compounds towards the different AR subtypes was determined. The probe 3 revealed a higher affinity to A₁ and A_2B ARs, showing interesting spectroscopic properties, and it was selected as the most suitable candidate to label both AR subtypes in undifferentiated MSCs.Fluorescence confocal microscopy showed that compound 3 significantly labelled ARs on cell membranes and the fluorescence signal was decreased by the cell pre-incubation with the A₁ AR and A_2B AR selective agonists, R-PIA and BAY 60-6583, respectively, thus confirming the specificity of the obtained signal. In conclusion, compound 3 could represent a useful tool to investigate the expression pattern of both A₁ and A_2B ARs in different pathological and physiological processes. Furthermore, these results provide an important basis for the design of new and more selective derivatives able to monitor the expression and localization of each different ARs in several tissues and living cells.     

Allosteric modulators of human A2B adenosine receptor

Background

Among adenosine receptors (ARs) the A_2B subtype exhibits low affinity for the endogenous agonist compared with the A₁, A_2A, and A₃ subtypes and is therefore activated when concentrations of adenosine increase to a large extent following tissue damages (e.g. ischemia, inflammation). For this reason, A_2B AR represents an important pharmacological target.

Methods

We evaluated seven 1-benzyl-3-ketoindole derivatives (7–9) for their ability to act as positive or negative allosteric modulators of human A_2B AR through binding and functional assays using CHO cells expressing human A₁, A_2A, A_2B, and A₃ ARs.

Results

The investigated compounds behaved as specific positive or negative allosteric modulators of human A_2B AR depending on small differences in their structures. The positive allosteric modulators 7a,b and 8a increased agonist efficacy without any effect on agonist potency. The negative allosteric modulators 8b,c and 9a,b reduced agonist potency and efficacy.

Conclusions

A number of 1-benzyl-3-ketoindole derivatives were pharmacologically characterized as selective positive (7a,b) or negative (8c, 9a,b) allosteric modulators of human A_2B AR.

General significance

The 1-benzyl-3-ketoindole derivatives 7–9 acting as positive or negative allosteric modulators of human A_2B AR represent new pharmacological tools useful for the development of therapeutic agents to treat pathological conditions related to an altered functionality of A_2B AR.     

Role of extracellular cysteine residues in the adenosine A<Subscript>2A</Subscript> receptor

The G protein-coupled A_2A adenosine receptor represents an important drug target. Crystal structures and modeling studies indicated that three disulfide bonds are formed between ECL1 and ECL2 (I, Cys71^2.69-Cys159^45.43; II, Cys74^3.22-Cys146^45.30, and III, Cys77^3.25-Cys166^45.50). However, the A_2BAR subtype appears to require only disulfide bond III for proper function. In this study, each of the three disulfide bonds in the A_2AAR was disrupted by mutation of one of the cysteine residues to serine. The mutant receptors were stably expressed in Chinese hamster ovary cells and analyzed in cyclic adenosine monophosphate (cAMP) accumulation and radioligand binding studies using structurally diverse agonists: adenosine, NECA, CGS21680, and PSB-15826. Results were rationalized by molecular modeling. The observed effects were dependent on the investigated agonist. Loss of disulfide bond I led to a widening of the orthosteric binding pocket resulting in a strong reduction in the potency of adenosine, but not of NECA or 2-substituted nucleosides. Disruption of disulfide bond II led to a significant reduction in the agonists’ efficacy indicating its importance for receptor activation. Disulfide bond III disruption reduced potency and affinity of the small adenosine agonists and NECA, but not of the larger 2-substituted agonists. While all the three disulfide bonds were essential for high potency or efficacy of adenosine, structural modification of the nucleoside could rescue affinity or efficacy at the mutant receptors. At present, it cannot be excluded that formation of the extracellular disulfide bonds in the A_2AAR is dynamic. This might add another level of G protein-coupled receptor (GPCR) modulation, in particular for the cysteine-rich A_2A and A_2BARs.     

Characterization of human and rodent native and recombinant adenosine A2B receptors by radioligand binding studies

Adenosine A_2B receptors of native human and rodent cell lines were investigated using [³H]PSB-298 [(8-{4-[2-(2-hydroxyethylamino)-2-oxoethoxy]phenyl}-1-propylxanthine] in radioligand binding studies. [³H]PSB-298 showed saturable and reversible binding. It exhibited a K_D value of 60 ± 1 nM and limited capacity (B_max = 3.511 fmol per milligram protein) at recombinant human adenosine A_2B receptors expressed in human embryonic kidney cells (HEK-293). The addition of sodium chloride (100 mM) led to a threefold increase in the number of binding sites recognized by the radioligand. The curve of the agonist 5′-N-ethylcarboxamidoadenosine (NECA) was shifted to the right in the presence of NaCl, while the curve of the antagonist PSB-298 was shifted to the left, indicating that PSB-298 may be an inverse agonist at A_2B receptors. Adenosine A_2B receptors were shown to be the major adenosine A₂ receptor subtype on the mouse neuroblastoma x rat glioma hybrid cell line NG108-15 cells. Binding studies at rat INS-1 cells (insulin secreting cell line) demonstrated that [³H]PSB-298 is a selective radioligand for adenosine A_2B binding sites in this cell line.     

Antagonist binding and induced conformational dynamics of GPCR A2A adenosine receptor

The A_2A adenosine receptor (A_2AAR) is a unique G‐protein coupled receptor (GPCR), because besides agonist, its antagonist could also lead to therapeutic relevance. Based on A_2AAR‐antagonist crystal structure, we have studied the binding mechanism of two distinct antagonists, ZM241385 and KW6002, and dynamic behaviors of A_2AAR induced by antagonist binding. Key residues interacting with both antagonists and residues specifically binding to one of them are identified. ZM241385 specifically bound to S67^2.65, M177^5.38, and N253^6.55, while KW6002 binds to F62^2.60, A81^3.29, and H264^7.29. Moreover, interactions with L167^5.28 are found for both antagonists, which were not reported in agonist binding. The dynamic behaviors of antagonist bound holo‐A_2AARs were found to be different from the apo‐A_2AAR in three typical functional switches, (i) the “ionic lock” was in equilibrium between formation and breakage in apo‐A_2AAR, but stayed broken in holo‐A_2AARs; (ii) the “rotamer toggle switch,” T88^3.36/F242^6.44/W246^6.48, adopted different rotameric conformations in apo‐A_2AAR and holo‐A_2AARs; (iii) apo‐A_2AAR preferred α‐helical intracellular loop (IC)2 and flexible IC3, while holo‐A_2AARs had a flexible IC2 and α‐helical IC3. Our results indicated that antagonist binding induced different conformational rearrangements of these characteristic functional switches in apo‐A_2AAR and holo‐A_2AARs. Proteins 2013; 81:1399–1410. © 2013 Wiley Periodicals, Inc.     

Regulation of adenosine receptors expression in rat B lymphocytes by insulin

Development of diabetes is associated with altered expression of adenosine receptors (ARs). Some of these alterations might be attributed to changes in insulin concentration. This study was undertaken to investigate the possible insulin effect on ARs level, and to determine the signaling pathway utilized by insulin to regulate the expression of ARs in rat B lymphocytes. Western blot analysis of B lymphocytes protein extracts indicated that all four ARs were present at detectable levels in the cells cultured for 24 h without insulin (≤10^?11 M), although the protein band of A_2A‐AR was barely visible. Inclusion of insulin (10^?8 M) in the culture medium resulted in an increase of A₁‐AR and A_2A‐AR protein levels and a significant decrease of A_2B‐AR protein, whereas the protein level of A₃‐AR remained unchanged. Alterations in the ARs protein content were accompanied by changes in the ARs mRNA levels. Increase of the insulin concentration from 10^?11 to 10^?8 M resulted in 50% decrease of A_2B‐AR mRNA level and two‐, and threefold increase of A₁‐AR and A_2A‐AR mRNA levels, respectively. Pretreatment of B cells with cycloheximide completely blocked the insulin action on A₁‐AR and A_2A‐AR mRNA, but not on A_2B‐AR expression. Detailed pharmacological analysis demonstrated that insulin‐induced A₁‐AR and A_2A‐AR mRNA expression through the Ras/Raf‐1/MEK/ERK pathway. The insulin effect on A_2B‐AR expression was blocked by p38 MAP kinase inhibitor (SB 203580). Concluding, elevated insulin concentration differentially affects the expression of ARs in B lymphocytes in a fashion that might enhance the various immunomodulatory effects of adenosine. J. Cell. Biochem. 109: 396–405, 2010. © 2009 Wiley‐Liss, Inc.     

Adenosine A2B receptor antagonist suppresses differentiation to regulatory T cells without suppressing activation of T cells

Extracellular adenosine activates P1 receptors (A₁, A_2A, A_2B, A₃) on cellular membranes. Here, we investigated the involvement of P1 receptor-mediated signaling in differentiation to regulatory T cells (Treg). Treg were induced in vitro by incubating isolated CD4⁺CD62L⁺ naïve murine T cells under Treg-skewing conditions. Antagonists of A₁ and A_2B receptors suppressed the expression of Foxp3, a specific marker of Treg, and the production of IL-10, suggesting the involvement of A₁ and A_2B receptors in differentiation to Treg. We also investigated the effect of these antagonists on T cell activation, which is essential for differentiation to Treg, and found that A₁ antagonist, but not A_2B antagonist, suppressed T cell activation. We conclude that A₁ and A_2B receptors are both involved in differentiation to Treg, but through different mechanisms. Since A_2B antagonist blocked differentiation to Treg without suppressing T cell activation, it is possible that blockade of A_2B receptor would facilitate tumor immunity.     

A<Subscript>2B</Subscript>adenosine receptor activity is reduced in neutrophils from patients with systemic sclerosis

We conducted the present study to investigate protein expression and functioning of A_2A and A_2B adenosine receptors (ARs) in neutrophils of patients affected by systemic sclerosis (SSc). The presence of A_2A and A_2B ARs was assessed by immunoblotting using specific antibodies. Equilibrium A_2A and A_2B ARs binding parameters were evaluated by radioligand binding assay. Functional studies were conducted to investigate coupling of the A_2B AR to the adenylyl cyclase pathway. This is the first report of the use of Western blot analysis to confirm the presence of A_2A and A_2B ARs in human neutrophils. No significant changes in A_2A AR binding parameters or expression levels were detected between SSc patients and healthy control individuals. A significant decrease (65%) in the maximum density of A_2B AR binding sites occurred in SSc neutrophils, whereas no changes in the affinity constant values were found. Moreover, a decrease in A_2B AR mediated adenylyl cyclase activity was observed in patients with SSc. Our findings demonstrate the occurrence of selective alterations in A_2B AR density and signalling in SSc.     

Species dependence of A3 adenosine receptor pharmacology and function

Efforts to fully understand pharmacological differences between G protein-coupled receptor (GPCR) species homologues are generally not pursued in detail during the drug development process. To date, many GPCRs that have been successfully targeted are relatively well-conserved across species in amino acid sequence and display minimal variability of biological effects. However, the A₃ adenosine receptor (AR), an exciting drug target for a multitude of diseases associated with tissue injury, ischemia, and inflammation, displays as little as 70% sequence identity among mammalian species (e.g., rodent vs. primate) commonly used in drug development. Consequently, the pharmacological properties of synthetic A₃AR ligands vary widely, not only in binding affinity, selectivity, and signaling efficacy, but to the extent that some function as agonists in some species and antagonists in others. Numerous heterocyclic antagonists that have nM affinity at the human A₃AR are inactive or weakly active at the rat and mouse A₃ARs. Positive allosteric modulators, including the imidazo [4,5-c]quinolin-4-amine derivative LUF6000, are only active at human and some larger animal species that have been evaluated (rabbit and dog), but not rodents. A₃AR agonists evoke systemic degranulation of rodent, but not human mast cells. The rat A₃AR undergoes desensitization faster than the human A₃AR, but the human homologue can be completely re-sensitized and recycled back to the cell surface. Thus, comprehensive pharmacological evaluation and awareness of potential A₃AR species differences are critical in studies to further understand the basic biological functions of this unique AR subtype. Recombinant A₃ARs from eight different species have been pharmacologically characterized thus far. In this review, we describe in detail current knowledge of species differences in genetic identity, G protein-coupling, receptor regulation, and both orthosteric and allosteric A₃AR pharmacology.     

A2B adenosine receptor blockade inhibits growth of prostate cancer cells

The role of the A_2B adenosine receptor (AR) in prostate cell death and growth was studied. The A_2B AR gene expression quantified by real-time quantitative RT-PCR and Western blot analysis was the highest among four AR subtypes (A₁, A_2A, A_2B, and A₃) in all three commonly used prostate cancer cell lines, PC-3, DU145, and LNCaP. We explored the function of the A_2B AR using PC-3 cells as a model. The A_2B AR was visualized in PC-3 cells by laser confocal microscopy. The nonselective A_2B AR agonist NECA and the selective A_2B AR agonist BAY60-6583, but not the A_2A AR agonist CGS21680, concentration-dependently induced adenosine 3′,5′-cyclic monophosphate (cyclic AMP) accumulation. NECA diminished lactate dehydrogenase (LDH) release, TNF-α-induced increase of caspase-3 activity, and cycloheximide (CHX)-induced morphological changes typical of apoptosis in PC-3 cells, which were blocked by a selective A_2B AR antagonist PSB603. NECA-induced proliferation of PC-3 cells was diminished by siRNA specific for the A_2B AR. The selective A_2B AR antagonist PSB603 was shown to inhibit cell growth in all three cell lines. Thus, A_2B AR blockade inhibits growth of prostate cancer cells, suggesting selective A_2B AR antagonists as potential novel therapeutics.     

Adenosine A<Subscript>2A</Subscript> receptor as a drug target for treatment of sepsis

Sepsis is a generalized infection accompanied by response of the body that manifests in a clinical and laboratory syndrome, namely, in the systemic inflammatory response syndrome (SIRS) from the organism to the infection. Although sepsis is a widespread and life-threatening disease, the assortment of drugs for its treatment is mostly limited by antibiotics. Therefore, the search for new cellular targets for drug therapy of sepsis is an urgent task of modern medicine and pharmacology. One of the most promising targets is the adenosine A_2A receptor (A_2AAR). The activation of this receptor, which is mediated by extracellular adenosine, manifests in almost all types of immune cells (lymphocytes, monocytes, macrophages, and dendritic cells) and results in reducing the severity of inflammation and reperfusion injury in various tissues. The activation of adenosine A_2A receptor inhibits the proliferation of T cells and production of proinflammatory cytokines, which contributes to the activation of the synthesis of anti-inflammatory cytokines, thereby suppressing the systemic response. For this reason, various selective A_2AAR agonists and antagonists may be considered to be drug candidates for sepsis pharmacotherapy. Nevertheless, they remain only efficient ligands and objects of pre-clinical and clinical trials. This review examines the molecular mechanisms of inflammatory response in sepsis and the structure and functions of A_2AAR and its role in the pathogenesis of sepsis, as well as examples of using agonists and antagonists of this receptor for the treatment of SIRS and sepsis.     

Synthesis and pharmacological characterization of a new series of 5,7-disubstituted-[1,2,4]triazolo[1,5-a][1,3,5]triazine derivatives as adenosine receptor antagonists: A preliminary inspection of ligand–receptor recognition process

A new series of triazolotriazines variously substituted at the C5 and N7 (5–25) positions was synthesized and fully characterized at the four adenosine receptor (AR) subtypes. In particular, arylacetyl or arylcarbamoyl moieties were introduced at the N7 position, which enhanced affinity at the hA_2B and hA₃ ARs, respectively, when utilized on the pyrazolo-triazolopyrimidine nucleus as we reported in the past. In general, compounds with a free amino group at the 7 position (5, 6), showed good affinity at the rat (r) A_2A AR (range 18.3–96.5 nM), while the introduction of a phenylcarbamoyl moiety at the N7 position (12, 19, 24) slightly increased the affinity at the hA₃ AR (range 311–633 nM) with respect to the unsubstituted derivatives. The binding profiles of the synthesized analogues seemed to correlate with the substitutions at the C5 and N7 positions. At the hA_2B AR, derivative 5, which contained a free amino group at the 7 position, was the most potent (EC₅₀ 3.42 μM) and could represent a starting point for searching new non-xanthine hA_2B AR antagonists. Molecular models of the rA_2A and hA₃ ARs were constructed by homology to the recently reported crystallographic structure of the hA_2A AR. A preliminary receptor-driven structure–activity relationship (SAR) based on the analysis of antagonist docking has been provided.     

Pharmacochemistry of the platelet purinergic receptors

Platelets contain at least five purinergic G protein-coupled receptors, e.g., the pro-aggregatory P2Y₁ and P2Y₁₂ receptors, a P2Y₁₄ receptor (GPR105) of unknown function, and anti-aggregatory A_2A and A_2B adenosine receptor (ARs), in addition to the ligand-gated P2X1 ion channel. Probing the structure–activity relationships (SARs) of the P2X and P2Y receptors for extracellular nucleotides has resulted in numerous new agonist and antagonist ligands. Selective agents derived from known ligands and novel chemotypes can be used to help define the subtypes pharmacologically. Some of these agents have entered into clinical trials in spite of the challenges of drug development for these classes of receptors. The functional architecture of P2 receptors was extensively explored using mutagenesis and molecular modeling, which are useful tools in drug discovery. In general, novel drug delivery methods, prodrug approaches, allosteric modulation, and biased agonism would be desirable to overcome side effects that tend to occur even with receptor subtype-selective ligands. Detailed SAR analyses have been constructed for nucleotide and non-nucleotide ligands at the P2Y₁, P2Y₁₂, and P2Y₁₄ receptors. The thienopyridine antithrombotic drugs Clopidogrel and Prasugrel require enzymatic pre-activation in vivo and react irreversibly with the P2Y₁₂ receptor. There is much pharmaceutical development activity aimed at identifying reversible P2Y₁₂ receptor antagonists. The screening of chemically diverse compound libraries has identified novel chemotypes that act as competitive, non-nucleotide antagonists of the P2Y₁ receptor or the P2Y₁₂ receptor, and antithrombotic properties of the structurally optimized analogues were demonstrated. In silico screening at the A_2A AR has identified antagonist molecules having novel chemotypes. Fluorescent and other reporter groups incorporated into ligands can enable new technology for receptor assays and imaging. The A_2A agonist CGS21680 and the P2Y₁ receptor antagonist MRS2500 were derivatized for covalent attachment to polyamidoamine dendrimeric carriers of MW 20,000, and the resulting multivalent conjugates inhibited ADP-promoted platelet aggregation. In conclusion, a wide range of new pharmacological tools is available to control platelet function by interacting with cell surface purine receptors.     

Protein kinase Cμ mediates adenosine-stimulated steroidogenesis in primary rat adrenal cells

Adenosine (Ado), an endogenous nucleoside, can stimulate corticosterone synthesis in adrenal cells via the A_2A/A_2B adenosine receptors (ARs). This study evaluated the contribution of protein kinase C (PKC) isoforms in Ado-induced steroidogenesis. The PKC inhibitor calphostin c blocked Ado-induced steroidogenesis, the mitogen-activated protein kinase (MEK)-extracellular signal-related regulated kinase (ERK)-cyclic AMP responsive element-binding protein cascade, and the mRNA expression of steroidogenic acute regulatory protein and CYP11B1. Further analyses revealed that PKCμ was indeed activated by Ado. Moreover, downregulation of PKCμ by small interfering RNA (siRNA) inhibited Ado-stimulated steroidogenesis and ERK phosphorylation. Finally, inhibition of either A_2AAR or A_2BAR led to the suppression of PKCμ phosphorylation. Together, these findings suggest that A₂AR-PKCμ-MEK signaling mediates Ado-stimulated adrenal steroidogenesis.