Targeting Adenosine Receptors: A Potential Pharmacological Avenue for Acute and Chronic Pain

Adenosine is a purine nucleoside, responsible for the regulation of multiple physiological and pathological cellular and tissue functions by activation of four G protein-coupled receptors (GPCR), namely A₁, A_2A, A_2B, and A₃ adenosine receptors (ARs). In recent years, extensive progress has been made to elucidate the role of adenosine in pain regulation. Most of the antinociceptive effects of adenosine are dependent upon A₁AR activation located at peripheral, spinal, and supraspinal sites. The role of A_2AAR and A_2BAR is more controversial since their activation has both pro- and anti-nociceptive effects. A₃AR agonists are emerging as promising candidates for neuropathic pain. Although their therapeutic potential has been demonstrated in diverse preclinical studies, no AR ligands have so far reached the market. To date, novel pharmacological approaches such as adenosine regulating agents and allosteric modulators have been proposed to improve efficacy and limit side effects enhancing the effect of endogenous adenosine. This review aims to provide an overview of the therapeutic potential of ligands interacting with ARs and the adenosinergic system for the treatment of acute and chronic pain.     

Adenosine and Inflammation: Here,There and Everywhere

Adenosine is a ubiquitous endogenous modulator with the main function of maintaining cellular and tissue homeostasis in pathological and stress conditions. It exerts its effect through the interaction with four G protein-coupled receptor (GPCR) subtypes referred as A₁, A_2A, A_2B, and A₃ adenosine receptors (ARs), each of which has a unique pharmacological profile and tissue distribution. Adenosine is a potent modulator of inflammation, and for this reason the adenosinergic system represents an excellent pharmacological target for the myriad of diseases in which inflammation represents a cause, a pathogenetic mechanism, a consequence, a manifestation, or a protective factor. The omnipresence of ARs in every cell of the immune system as well as in almost all cells in the body represents both an opportunity and an obstacle to the clinical use of AR ligands. This review offers an overview of the cardinal role of adenosine in the modulation of inflammation, showing how the stimulation or blocking of its receptors or agents capable of regulating its extracellular concentration can represent promising therapeutic strategies for the treatment of chronic inflammatory pathologies, neurodegenerative diseases, and cancer.     

Dual Actions of A2A and A3 Adenosine Receptor Ligand Prevents Obstruction-Induced Kidney Fibrosis in Mice

Kidney fibrosis is the final outcome of chronic kidney disease (CKD). Adenosine plays a significant role in protection against cellular damage by activating four subtypes of adenosine receptors (ARs), A₁AR, A_2AAR, A_2BAR, and A₃AR. A_2AAR agonists protect against inflammation, and A₃AR antagonists effectively inhibit the formation of fibrosis. Here, we showed for the first time that LJ-4459, a newly synthesized dual-acting ligand that is an A_2AAR agonist and an A₃AR antagonist, prevents the progression of tubulointerstitial fibrosis. Unilateral ureteral obstruction (UUO) surgery was performed on 6-week-old male C57BL/6 mice. LJ-4459 (1 and 10 mg/kg) was orally administered for 7 days, started at 1 day before UUO surgery. Pretreatment with LJ-4459 improved kidney morphology and prevented the progression of tubular injury as shown by decreases in urinary kidney injury molecular-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) excretion. Obstruction-induced tubulointerstitial fibrosis was attenuated by LJ-4459, as shown by a decrease in fibrotic protein expression in the kidney. LJ-4459 also inhibited inflammation and oxidative stress in the obstructed kidney, with reduced macrophage infiltration, reduced levels of pro-inflammatory cytokines, as well as reduced levels of reactive oxygen species (ROS). These data demonstrate that LJ-4459 has potential as a therapeutic agent against the progression of tubulointerstitial fibrosis.     

Insight into the Interactions between Novel Coumarin Derivatives and Human A3 Adenosine Receptors

A study focused on the discovery of new chemical entities based on the 3‐arylcoumarin scaffold was performed with the aim of finding new adenosine receptor (AR) ligands. Thirteen synthesized compounds were evaluated by radioligand binding (A₁, A_2A, and A₃) and adenylyl cyclase activity (A_2B) assays in order to study their affinity for the four human AR (hAR) subtypes. Seven of the studied compounds proved to be selective A₃AR ligands, with 3‐(4′‐methylphenyl)‐8‐(2‐oxopropoxy)coumarin ( 12 ) being the most potent (K_i=634 nM ). None of the compounds showed affinity for the A_2B receptor, while four compounds were found to be nonselective AR ligands for the other three subtypes. Docking simulations were carried out to identify the hypothetical binding mode and to rationalize the interaction of these types of coumarin derivatives with the binding site of the three ARs to which binding was observed. The results allowed us to conclude that the 3‐arylcoumarin scaffold composes a novel and promising class of A₃AR ligands. ADME properties were also calculated, with the results suggesting that these compounds are promising leads for the identification of new drug candidates.     

Generation of a Retargeted Oncolytic Herpes Virus Encoding Adenosine Deaminase for Tumor Adenosine Clearance

Background: Oncolytic viruses are immunotherapeutic agents that can be engineered to encode payloads of interest within the tumor microenvironment to enhance therapeutic efficacy. Their therapeutic potential could be limited by many avenues for immune evasion exerted by the tumor. One such is mediated by adenosine, which induces pleiotropic immunosuppression by inhibiting antitumor immune populations as well as activating tolerogenic stimuli. Adenosine is produced starting from the highly immunostimulatory ATP, which is progressively hydrolyzed to ADP and adenosine by CD39 and CD73. Cancer cells express high levels of CD39 and CD73 ectoenzymes, thus converting immunostimulatory purinergic signal of ATP into an immunosuppressive signal. For this reason, CD39, CD73 and adenosine receptors are currently investigated in clinical trials as targets for metabolic cancer immunotherapy. This is of particular relevance in the context of oncovirotherapy, as immunogenic cell death induced by oncolytic viruses causes the secretion of a high amount of ATP which is available to be quickly converted into adenosine. Methods: Here, we took advantage of adenosine deaminase enzyme that naturally converts adenosine into the corresponding inosine derivative, devoid of immunoregulatory function. We encoded ADA into an oncolytic targeted herpes virus redirected to human HER2. An engineered ADA with an ectopic signal peptide was also generated to improve enzyme secretion (ADA-SP). Results: Insertion of the expression cassette was not detrimental for viral yield and cancer cell cytotoxicity. The THV_ADA and THV_ADA-SP successfully mediated the secretion of functional ADA enzyme. In in vitro model of human monocytes THP1, this ability of THV_ADA and THV_ADA-SP resulted in the retrieval of eADO-exposed monocytes replication rate, suggesting the proficiency of the viruses in rescuing the immune function. Conclusions: Encoding ADA into oncolytic viruses revealed promising properties for preclinical exploitation.     

Identification of Adenosine Deaminase Inhibitors by Metal-binding Pharmacophore Screening

Adenosine deaminase (ADA) is a human mononuclear Zn²⁺ metalloenzyme that converts adenosine to inosine. ADA is a validated drug target for cancer, but there has been little recent work on the development of new therapeutics against this enzyme. The lack of new advancements can be partially attributed to an absence of suitable assays for high-throughput screening (HTS) against ADA. To facilitate more rapid drug discovery efforts for this target, an in vitro assay was developed that utilizes the enzymatic conversion of a visibly emitting adenosine analogue to the corresponding fluorescent inosine analogue by ADA, which can be monitored via fluorescence intensity changes. Utilizing this assay, a library of ∼350 small molecules containing metal-binding pharmacophores (MBPs) was screened in an HTS format to identify new inhibitor scaffolds against ADA. This approach yielded a new metal-binding scaffold with a K_i value of 26±1 μM.     

Targeting G Protein-Coupled Receptors with Magnetic Carbon Nanotubes: The Case of the A3 Adenosine Receptor

The A₃ adenosine receptor (AR) is a G protein-coupled receptor (GPCR) overexpressed in the membrane of specific cancer cells. Thus, the development of nanosystems targeting this receptor could be a strategy to both treat and diagnose cancer. Iron-filled carbon nanotubes (CNTs) are an optimal platform for theranostic purposes, and the use of a magnetic field can be exploited for cancer magnetic cell sorting and thermal therapy. In this work, we have conjugated an A₃AR ligand on the surface of iron-filled CNTs with the aim of targeting cells overexpressing A₃ARs. In particular, two conjugates bearing PEG linkers of different length were designed. A docking analysis of A₃AR showed that neither CNT nor linker interferes with ligand binding to the receptor; this was confirmed by in vitro preliminary radioligand competition assays on A₃AR. Encouraged by this result, magnetic cell sorting was applied to a mixture of cells overexpressing or not the A₃AR in which our compound displayed indiscriminate binding to all cells. Despite this, it is the first time that a GPCR ligand has been anchored to a magnetic nanosystem, thus it opens the door to new applications for cancer treatment.     

2‐Amino[1,2,4]triazolo[1,5‐c]quinazolines and Derived Novel Heterocycles: Syntheses and Structure–Activity Relationships of Potent Adenosine Receptor Antagonists

2‐Amino[1,2,4]triazolo[1,5‐c]quinazolines were identified as potent adenosine receptor (AR) antagonists. Synthetic strategies were devised to gain access to a broad range of derivatives including novel polyheterocyclic compounds. Potent and selective A₃AR antagonists were discovered, including 3,5‐diphenyl[1,2,4]triazolo[4,3‐c]quinazoline ( 17 , K_i human A₃AR 1.16 nm ) and 5′‐phenyl‐1,2‐dihydro‐3′H‐spiro[indole‐3,2′‐[1,2,4]triazolo[1,5‐c]quinazolin]‐2‐one ( 20 , K_i human A₃AR 6.94 nm ). In addition, multitarget antagonists were obtained, such as the dual A₁/A₃ antagonist 2,5‐diphenyl[1,2,4]triazolo[1,5‐c]quinazoline ( 13 b , K_i human A₁AR 51.6 nm , human A₃AR 11.1 nm ), and the balanced pan‐AR antagonists 5‐(2‐thienyl)[1,2,4]triazolo[1,5‐c]quinazolin‐2‐amine ( 11 c , K_i human A₁AR 131 nm , A_2AAR 32.7 nm , A_2BAR 150 nm , A₃AR 47.5 nm ) and 9‐bromo‐5‐phenyl[1,2,4]triazolo[1,5‐c]quinazolin‐2‐amine ( 11 q , K_i human A₁AR 67.7 nm , A_2AAR 13.6 nm , A_2BAR 75.0 nm , A₃AR 703 nm ). In many cases, significantly different affinities for human and rat receptors were observed, which emphasizes the need for caution in extrapolating conclusions between different species.     

Guanosine-Mediated Anxiolytic-Like Effect: Interplay with Adenosine A1 and A2A Receptors

Acute or chronic administration of guanosine (GUO) induces anxiolytic-like effects, for which the adenosine (ADO) system involvement has been postulated yet without a direct experimental evidence. Thus, we aimed to investigate whether adenosine receptors (ARs) are involved in the GUO-mediated anxiolytic-like effect, evaluated by three anxiety-related paradigms in rats. First, we confirmed that acute treatment with GUO exerts an anxiolytic-like effect. Subsequently, we investigated the effects of pretreatment with ADO or A₁R (CPA, CCPA) or A_2AR (CGS21680) agonists 10 min prior to GUO on a GUO-induced anxiolytic-like effect. All the combined treatments blocked the GUO anxiolytic-like effect, whereas when administered alone, each compound was ineffective as compared to the control group. Interestingly, the pretreatment with nonselective antagonist caffeine or selective A₁R (DPCPX) or A_2AR (ZM241385) antagonists did not modify the GUO-induced anxiolytic-like effect. Finally, binding assay performed in hippocampal membranes showed that [³H]GUO binding became saturable at 100–300 nM, suggesting the existence of a putative GUO binding site. In competition experiments, ADO showed a potency order similar to GUO in displacing [³H]GUO binding, whereas AR selective agonists, CPA and CGS21680, partially displaced [³H]GUO binding, but the sum of the two effects was able to displace [³H]GUO binding to the same extent of ADO alone. Overall, our results strengthen previous data supporting GUO-mediated anxiolytic-like effects, add new evidence that these effects are blocked by A₁R and A_2AR agonists and pave, although they do not elucidate the mechanism of GUO and ADO receptor interaction, for a better characterization of GUO binding sites in ARs.     

Molecular and Pharmacological Characterization of the Interaction between Human Geranylgeranyltransferase Type I and Ras-Related Protein Rap1B

Geranylgeranyltransferase type-I (GGTase-I) represents an important drug target since it contributes to the function of many proteins that are involved in tumor development and metastasis. This led to the development of GGTase-I inhibitors as anti-cancer drugs blocking the protein function and membrane association of e.g., Rap subfamilies that are involved in cell differentiation and cell growth. In the present study, we developed a new NanoBiT assay to monitor the interaction of human GGTase-I and its substrate Rap1B. Different Rap1B prenylation-deficient mutants (C181G, C181S, and ΔCQLL) were designed and investigated for their interaction with GGTase-I. While the Rap1B mutants C181G and C181S still exhibited interaction with human GGTase-I, mutant ΔCQLL, lacking the entire CAAX motif (defined by a cysteine residue, two aliphatic residues, and the C-terminal residue), showed reduced interaction. Moreover, a specific, peptidomimetic and competitive CAAX inhibitor was able to block the interaction of Rap1B with GGTase-I. Furthermore, activation of both Gα_s-coupled human adenosine receptors, A_2A (A_2AAR) and A_2B (A_2BAR), increased the interaction between GGTase-I and Rap1B, probably representing a way to modulate prenylation and function of Rap1B. Thus, A_2AAR and A_2BAR antagonists might be promising candidates for therapeutic intervention for different types of cancer that overexpress Rap1B. Finally, the NanoBiT assay provides a tool to investigate the pharmacology of GGTase-I inhibitors.     

Purinergic Signaling: Impact of GPCR Structures on Rational Drug Design

The purinergic signaling system includes membrane-bound receptors for extracellular purines and pyrimidines, and enzymes/transporters that regulate receptor activation by endogenous agonists. Receptors include: adenosine (A₁, A_2A, A_2B, and A₃) and P2Y (P2Y₁, P2Y₂, P2Y₄, P2Y₆, P2Y₁₁, P2Y₁₂, P2Y₁₃, and P2Y₁₄) receptors (all GPCRs), as well as P2X receptors (ion channels). Receptor activation, especially accompanying physiological stress or damage, creates a temporal sequence of signaling to counteract this stress and either mobilize (P2Rs) or suppress (ARs) immune responses. Thus, modulation of this large signaling family has broad potential for treating chronic diseases. Experimentally determined structures represent each of the three receptor families. We focus on selective purinergic agonists (A₁, A₃), antagonists (A₃, P2Y₁₄), and allosteric modulators (P2Y₁, A₃). Examples of applying structure-based design, including the rational modification of known ligands, are presented for antithrombotic P2Y₁R antagonists and anti-inflammatory P2Y₁₄R antagonists and A₃AR agonists. A₃AR agonists are a potential, nonaddictive treatment for chronic neuropathic pain.     

A Body of Circumstantial Evidence for the Irreversible Ectonucleotidase Inhibitory Action of FSCPX,an Agent Known as a Selective Irreversible A1 Adenosine Receptor Antagonist So Far

In previous studies using isolated, paced guinea pig left atria, we observed that FSCPX, known as a selective A₁ adenosine receptor antagonist, paradoxically increased the direct negative inotropic response to A₁ adenosine receptor agonists (determined using concentration/effect (E/c) curves) if NBTI, a nucleoside transport inhibitor, was present. Based on mathematical modeling, we hypothesized that FSCPX blunted the cardiac interstitial adenosine accumulation in response to nucleoside transport blockade, probably by inhibiting CD39 and/or CD73, which are the two main enzymes of the interstitial adenosine production in the heart. The goal of the present study was to test this hypothesis. In vitro CD39 and CD73 inhibitor assays were carried out; furthermore, E/c curves were constructed in isolated, paced rat and guinea pig left atria using adenosine, CHA and CPA (two A₁ adenosine receptor agonists), FSCPX, NBTI and NBMPR (two nucleoside transport inhibitors), and PSB-12379 (a CD73 inhibitor), measuring the contractile force. We found that FSCPX did not show any inhibitory effect during the in vitro enzyme assays. However, we successfully reproduced the paradox effect of FSCPX in the rat model, mimicked the “paradox” effect of FSCPX with PSB-12379, and demonstrated the lipophilia of FSCPX, which could explain the negative outcome of inhibitor assays with CD39 and CD73 dissolved in a water-based solution. Taken together, these three pieces of indirect evidence are strong enough to indicate that FSCPX possesses an additional action besides the A₁ adenosine receptor antagonism, which action may be the inhibition of an ectonucleotidase. Incidentally, we found that POM-1 inhibited CD73, in addition to CD39.     

Effect of Linker Length and Composition on Heterobivalent Ligand‐Mediated Receptor Cross‐Talk between the A1 Adenosine and β2 Adrenergic Receptors

Heterobivalent ligands that possess pharmacophores designed to interact with both the A₁ adenosine receptor (A₁AR) and the β₂ adrenergic receptor (β₂AR) were prepared. More specifically, these ligands contain an adenosine moiety that is linked via its N⁶‐position to the amino group of the saligenin‐substituted ethanolamine moiety present in the well‐known β₂AR agonist, salbutamol. The affinities of these ligands were determined at both receptors and found to vary with linker length and composition. With all compounds, affinity and functional potencies were found to have selectivity for the A₁AR over the β₂AR. In all cases, cAMP accumulation (a β₂AR‐mediated response) was mainly observed when the A₁AR was blocked or its function decreased by pertussis toxin or chronic agonist treatment. This suggests that heterobivalent compounds for receptors that mediate opposite responses might be useful for elucidating the mechanisms of receptor cross‐talk and how this interaction, in terms of responsiveness, may change under pathophysiological conditions.     

Novel,Dual Target-Directed Annelated Xanthine Derivatives Acting on Adenosine Receptors and Monoamine Oxidase B

Annelated purinedione derivatives have been shown to act as possible multiple-target ligands, addressing adenosine receptors and monoaminooxidases. In this study, based on our previous results, novel annelated pyrimido- and diazepino[2,1-f]purinedione derivatives were designed as dual-target-directed ligands combining A_2A adenosine receptor (AR) antagonistic activity with blocking monoamine oxidase B. A library of 19 novel compounds was synthesized and biologically evaluated in radioligand binding studies at AR subtypes and for their ability to inhibit MAO-B. This allowed 9-(2-chloro-6-fluorobenzyl)-3-ethyl-1-methyl-6,7,8,9-tetrahydropyrimido[2,1-f]purine-2,4(1H,3H)-dione ( 13 e ; K_i human A_2AAR: 264 nM and IC₅₀ human MAO-B: 243 nM) to be identified as the most potent dual-acting ligand from this series. ADMET parameters were estimated in vitro, and analysis of the structure-activity relationships was complemented by molecular-docking studies based on previously published X-ray structures of the protein targets. Such dual-acting ligands, by selectively blocking A_2A AR, accompanied by the inhibition of dopamine metabolizing enzyme MAO-B, might provide symptomatic and neuroprotective effects in, among others, the treatment of Parkinson disease     

High-Molecular-Weight Hyaluronic Acid Inhibits IL-1β-Induced Synovial Inflammation and Macrophage Polarization through the GRP78-NF-κB Signaling Pathway

Recent evidence has suggested that synovial inflammation and macrophage polarization were involved in the pathogenesis of osteoarthritis (OA). Additionally, high-molecular-weight hyaluronic acid (HMW-HA) was often used clinically to treat OA. GRP78, an endoplasmic reticulum (ER) stress chaperone, was suggested to contribute to the hyperplasia of synovial cells in OA. However, it was still unclear whether HMW-HA affected macrophage polarization through GRP78. Therefore, we aimed to identify the effect of HMW-HA in primary synovial cells and macrophage polarization and to investigate the role of GRP78 signaling. We used IL-1β to treat primary synoviocytes to mimic OA, and then treated them with HMW-HA. We also collected conditioned medium (CM) to culture THP-1 macrophages and examine the changes in the phenotype. IL-1β increased the expression of GRP78, NF-κB (p65 phosphorylation), IL-6, and PGE2 in primary synoviocytes, accompanied by an increased macrophage M1/M2 polarization. GRP78 knockdown significantly reversed the expression of IL-1β-induced GRP78-related downstream molecules and macrophage polarization. HMW-HA with GRP78 knockdown had additive effects in an IL-1β culture. Finally, the synovial fluid from OA patients revealed significantly decreased IL-6 and PGE2 levels after the HMW-HA treatment. Our study elucidated a new form of signal transduction for HMW-HA-mediated protection against synovial inflammation and macrophage polarization and highlighted the involvement of the GRP78-NF-κB signaling pathway.     

Expression and Regulation of CD73 during the Estrous Cycle in Mouse Uterus

Cluster of differentiation 73 (CD73, also known as ecto-5′-nucleotidase) is an enzyme that converts AMP into adenosine. CD73 is a surface enzyme bound to the outside of the plasma membrane expressed in several cells and regulates immunity and inflammation. In particular, it is known to inhibit T cell-mediated immune responses. However, the regulation of CD73 expression by hormones in the uterus is not yet clearly known. In this study, we investigated the expression of CD73 in ovariectomized mice treated with estrogen or progesterone and its regulation in the mouse uterus during the estrous cycle. The level of CD73 expression was dynamically regulated in the uterus during the estrous cycle. CD73 protein expression was high in proestrus, estrus, and diestrus, whereas it was relatively low in the metestrus stage. Immunofluorescence revealed that CD73 was predominantly expressed in the cytoplasm of the luminal and glandular epithelium and the stroma of the endometrium. The expression of CD73 in ovariectomized mice was gradually increased by progesterone treatment. However, estrogen injection did not affect its expression. Moreover, CD73 expression was increased when estrogen and progesterone were co-administered and was inhibited by the pretreatment of the progesterone receptor antagonist RU486. These findings suggest that the expression of CD73 is dynamically regulated by estrogen and progesterone in the uterine environment, and that there may be a synergistic effect of estrogen and progesterone.     

Purinergic Signaling in Pancreas—From Physiology to Therapeutic Strategies in Pancreatic Cancer

The purinergic signaling has an important role in regulating pancreatic exocrine secretion. The exocrine pancreas is also a site of one of the most serious cancer forms, the pancreatic ductal adenocarcinoma (PDAC). Here, we explore how the network of purinergic and adenosine receptors, as well as ecto-nucleotidases regulate normal pancreatic cells and various cells within the pancreatic tumor microenvironment. In particular, we focus on the P2X7 receptor, P2Y₂ and P2Y₁₂ receptors, as well as A₂ receptors and ecto-nucleotidases CD39 and CD73. Recent studies indicate that targeting one or more of these candidates could present new therapeutic approaches to treat pancreatic cancer. In pancreatic cancer, as much as possible of normal pancreatic function should be preserved, and therefore physiology of purinergic signaling in pancreas needs to be considered.     

Adenosine and Cordycepin Accelerate Tissue Remodeling Process through Adenosine Receptor Mediated Wnt/β-Catenin Pathway Stimulation by Regulating GSK3b Activity

Adenosine is a cellular metabolite with diverse derivatives that possesses a wide range of physiological roles. We investigated the molecular mechanisms of adenosine and cordycepin for their promoting effects in wound-healing process. The mitochondrial energy metabolism and cell proliferation markers, cAMP responsive element binding protein 1 (CREB1) and Ki67, were enhanced by adenosine and cordycepin in cultured dermal fibroblasts. Adenosine and cordycepin stimulated adenosine receptor signaling via elevated cAMP. The phosphorylation of mitogen-activated protein kinase kinase (MEK) 1/2, mammalian target of rapamycin (mTOR) and glycogen synthase kinase 3 beta (Gsk3b) and Wnt target genes such as bone morphogenetic protein (BMP) 2/4 and lymphoid enhancer binding factor (Lef) 1 were activated. The enhanced gene expression by adenosine and cordycepin was abrogated by adenosine A_2A and A_2B receptor inhibitors, ZM241385 and PSH603, and protein kinase A (PKA) inhibitor H89, indicating the involvement of adenosine receptor A_2A, A_2B and PKA. As a result of Wnt/β-catenin pathway activation, the secretion of growth factors such as insulin-like growth factor (IGF)-1 and transforming growth factor beta (TGFβ) 3 was increased, previously reported to facilitate the wound healing process. In addition, in vitro fibroblast migration was also increased, demonstrating their possible roles in facilitating the wound healing process. In conclusion, our data strongly demonstrate that adenosine and cordycepin stimulate the Wnt/β-catenin signaling through the activation of adenosine receptor, possibly promoting the tissue remodeling process and suggest their therapeutic potential for treating skin wounds.     

Uncovering the Mechanisms of Adenosine Receptor-Mediated Pain Control: Focus on the A3 Receptor Subtype

Agonists of the G_i protein-coupled A₃ adenosine receptor (A₃AR) have shown important pain-relieving properties in preclinical settings of several pain models. Active as a monotherapy against chronic pain, A₃AR agonists can also be used in combination with classic opioid analgesics. Their safe pharmacological profile, as shown by clinical trials for other pathologies, i.e., rheumatoid arthritis, psoriasis and fatty liver diseases, confers a realistic translational potential, thus encouraging research studies on the molecular mechanisms underpinning their antinociceptive actions. A number of pathways, involving central and peripheral mechanisms, have been proposed. Recent evidence showed that the prototypical A₃AR agonist Cl-IB-MECA and the new, highly selective, A₃AR agonist MRS5980 inhibit neuronal (N-type) voltage-dependent Ca²⁺ currents in dorsal root ganglia, a known pain-related mechanism. Other proposed pathways involve reduced cytokine production, immune cell-mediated responses, as well as reduced microglia and astrocyte activation in the spinal cord. The aim of this review is to summarize up-to-date information on A₃AR in the context of pain, including cellular and molecular mechanisms underlying this effect. Based on their safety profile shown in clinical trials for other pathologies, A₃AR agonists are proposed as novel, promising non-narcotic agents for pain control.     

Derivatives of benzimidazol-2-ylquinoline and benzimidazol-2-ylisoquinoline as selective A1 adenosine receptor antagonists with stimulant activity on human colon motility

A number of quinolines and isoquinolines connected in various ways to a substituted benzimidazol-2-yl system were synthesized and evaluated as novel antagonists of adenosine receptors (ARs) by competition experiments using human A(1), A(2A), and A(3) ARs. The new compounds were designed based on derivatives of 2-(benzimidazol-2-yl)quinoxaline, previously reported as potent and selective antagonists of A(1) and A(3) ARs. Among these, 3-[4-(ethylthio)-1H-benzimidazol-2-yl]isoquinoline 4b exhibited the best combination of potency toward the A(1) AR (K(i) =1.4 nM) and selectivity against the A(2A) (K(i) >10 μM), A(2B) (K(i)>10 μM), and A(3) ARs (K(i)>1 μM). Functional experiments in circular smooth muscle preparations of isolated human colon showed that 4b behaves as a potent and selective antagonist of the A(1) AR in the neuromuscular compartment of this intestinal region. Biological and pharmacological data suggest that 4b is a suitable starting point for the development of novel agents endowed with stimulant properties on colonic activity.