A2B adenosine receptor signaling and regulation.

The A2B adenosine receptor (A2BR) is one of the four adenosine-activated G protein-coupled receptors. In addition to adenosine, protein kinase C (PKC) was recently found to activate the A2BR. The A2BR is coupled to both Gs and Gi, as well as Gq proteins in some cell types. Many primary cells and cell lines, such as bladder and breast cancer, bronchial smooth muscle, skeletal muscle, and fat cells, express the A2BR endogenously at high levels, suggesting its potentially important role in asthma, cancer, diabetes, and other conditions. The A2BR has been characterized as both pro- and anti-inflammatory, inducing cell type-dependent secretion of IL-6, IL-8, and IL-10. Theophylline and enprofylline have long been used for asthma treatment, although it is still not entirely clear if their A2BR antagonism contributes to their therapeutic effects or side effects. The A2BR is required in ischemic cardiac preconditioning by adenosine. Both A2BR and protein kinase C (PKC) contribute to cardioprotection, and both modes of A2BR signaling can be blocked by A2BR antagonists. Inhibitors of PKC and A2BR are in clinical cancer trials. Sulforaphane and other isothiocyanates from cruciferous vegetables such as broccoli and cauliflower have been reported to inhibit A2BR signaling via reaction with an intracellular A2BR cysteine residue (C210). A full, A2BR-selective agonist, critical to elucidate many controversial roles of the A2BR, is still not available, although agonist-bound A2BR structures have recently been reported.

Adenosine receptors in breast cancer.

Adenosine receptors are important in the normal physiological function of cells and the pathogenesis of various cancer cells, including breast cancer cells. The activity of adenosine receptors in cancer cells is related to cell proliferation, angiogenesis, metastasis, immune system evasion, and interference with apoptosis. Considering the different roles of adenosine receptors in cancer cells, we intend to investigate the function of adenosine receptors and their biological pathways in breast cancer to improve understanding of therapeutically relevant signaling pathways.

Exploring Biginelli-based scaffolds as A2B adenosine receptor antagonists: Unveiling novel structure-activity relationship trends, lead compounds, and potent colorectal anticancer agents.

Antagonists of the A2B adenosine receptor have recently emerged as targeted anticancer agents and immune checkpoint inhibitors within the realm of cancer immunotherapy. This study presents a comprehensive evaluation of novel Biginelli-assembled pyrimidine chemotypes, including mono-, bi-, and tricyclic derivatives, as A2BAR antagonists. We conducted a comprehensive examination of the adenosinergic profile (both binding and functional) of a large compound library consisting of 168 compounds. This approach unveiled original lead compounds and enabled the identification of novel structure-activity relationship (SAR) trends, which were supported by extensive computational studies, including quantum mechanical calculations and free energy perturbation (FEP) analysis. In total, 25 molecules showed attractive affinity (Ki < 100 nM) and outstanding selectivity for A2BAR. From these, five molecules corresponding to the new benzothiazole scaffold were below the Ki < 10 nM threshold, in addition to a novel dual A2A/A2B antagonist. The most potent compounds, and the dual antagonist, showed enantiospecific recognition in the A2BAR. Two A2BAR selective antagonists and the dual A2AAR/A2BAR antagonist reported in this study were assessed for their impact on colorectal cancer cell lines. The results revealed a significant and dose-dependent reduction in cell proliferation. Notably, the A2BAR antagonists exhibited remarkable specificity, as they did not impede the proliferation of non-tumoral cell lines. These findings support the efficacy and potential that A2BAR antagonists as valuable candidates for cancer therapy, but also that they can effectively complement strategies involving A2AAR antagonism in the context of immune checkpoint inhibition.

Clinically relevant therapeutic approaches against acetaminophen hepatotoxicity and acute liver failure.

Liver injury and acute liver failure caused by an acetaminophen (APAP) overdose is a significant clinical problem in western countries. With the introduction of the mouse model of APAP hepatotoxicity in the 1970 s, fundamental mechanisms of cell death were discovered. This included the recognition that part of the APAP dose is metabolized by cytochrome P450 generating a reactive metabolite that is detoxified by glutathione. After the partial depletion of glutathione, the reactive metabolite will covalently bind to sulfhydryl groups of proteins, which is the initiating event of the toxicity. This insight led to the introduction of N-acetyl-L-cysteine, a glutathione precursor, as antidote against APAP overdose in the clinic. Despite substantial progress in our understanding of the pathomechanisms over the last decades viable new antidotes only emerged recently. This review will discuss the background, mechanisms of action, and the clinical prospects of the existing FDA-approved antidote N-acetylcysteine, of several new drug candidates under clinical development [4-methylpyrazole (fomepizole), calmangafodipir] and examples of additional therapeutic targets (Nrf2 activators) and regeneration promoting agents (thrombopoietin mimetics, adenosine A2B receptor agonists, Wharton's Jelly mesenchymal stem cells). Although there are clear limitations of certain therapeutic approaches, there is reason to be optimistic. The substantial progress in the understanding of the pathophysiology of APAP hepatotoxicity led to the consideration of several drugs for development as clinical antidotes against APAP overdose in recent years. Based on the currently available information, it is likely that this will result in additional drugs that could be used as adjunct treatment for N-acetylcysteine.

Ecto-5'-nucleotidase (Nt5e/CD73)-mediated adenosine signaling attenuates TGFß-2 induced elastin and cellular contraction.

Arterial calcification due to deficiency of CD73 (ACDC) is a rare genetic disease caused by a loss-of-function mutation in the NT5E gene encoding the ecto-5'-nucleotidase (cluster of differentiation 73, CD73) enzyme. Patients with ACDC develop vessel arteriomegaly, tortuosity, and vascular calcification in their lower extremity arteries. Histological analysis shows that patients with ACDC vessels exhibit fragmented elastin fibers similar to that seen in aneurysmal-like pathologies. It is known that alterations in transforming growth factor ß (TGFß) pathway signaling contribute to this elastin phenotype in several connective tissue diseases, as TGFß regulates extracellular matrix (ECM) remodeling. Our study investigates whether CD73-derived adenosine modifies TGFß signaling in vascular smooth muscle cells (SMCs). We show that Nt5e-/- SMCs have elevated contractile markers and elastin gene expression compared with Nt5e+/+ SMCs. Ecto-5'-nucleotidase (Nt5e)-deficient SMCs exhibit increased TGFß-2 and activation of small mothers against decapentaplegic (SMAD) signaling, elevated elastin transcript and protein, and potentiate SMC contraction. These effects were diminished when the A2b adenosine receptor was activated. Our results identify a novel link between adenosine and TGFß signaling, where adenosine signaling via the A2b adenosine receptor attenuates TGFß signaling to regulate SMC homeostasis. We discuss how disruption in adenosine signaling is implicated in ACDC vessel tortuosity and could potentially contribute to other aneurysmal pathogenesis.

Blocking the A2B adenosine receptor alleviates myocardial damage by inhibiting spleen-derived MDSC mobilisation after acute myocardial infarction.

BACKGROUND: Myeloid-derived suppressor cell (MDSC) mobilisation is an important immune event in acute myocardial infarction (AMI). The A2B adenosine receptor (A2BAR) plays key role in regulating MDSC function, but its specific involvement in MDSC mobilisation in AMI remains unclear. METHODS: In AMI patients, the circulating MDSC ratio and A2BAR mRNA expression were measured. A mouse AMI model was established by left anterior descending coronary artery (LADCA) ligation. MDSCs were analysed by FACS and immunofluorescence staining (of heart tissue). A2BAR mRNA expression was assessed by qRT-PCR. Myocardial injury was detected by HE staining. Myocardial cell apoptosis was analysed by immunohistochemistry. Cardiac systolic function was evaluated by transthoracic echocardiography. RESULTS: In AMI patients, the circulating MDSC ratio was increased and positively correlated with A2BAR mRNA expression (r = 0.86, p < 0.01). In AMI model mice, the percentage of MDSCs was increased in the circulation and infarcted heart and decreased in the spleen. MRS-1754-mediated A2BAR inhibition decreased the MDSC ratio in the circulation and infarcted heart and prevented the decrease in MDSC number in the spleens of mice with AMI. A2BAR blockade inhibited myocardial cell apoptosis, alleviated myocardial inflammatory injury, and improved myocardial systolic function in the AMI mouse model. Similar results were found in mice after splenectomy. Additionally, spleen-derived MDSC injection increased the MDSC ratio in the infarcted heart, increased myocardial cell apoptosis, aggravated myocardial injury, and decreased cardiac systolic function in mice with AMI. CONCLUSION: Blocking A2BAR alleviates myocardial damage and improves myocardial systolic function through inhibition of spleen-derived MDSC mobilisation after AMI. Key MessagesSpleen-derived MDSC mobilisation aggravates myocardial inflammatory injury within 24 h of AMI.A2BAR promotes spleen-derived MDSC mobilisation within 24 h of AMI.Blocking A2BAR improves myocardial systolic function through inhibition of spleen-derived MDSC mobilisation.

Design, synthesis, and biological evaluation of triazole-pyrimidine-methylbenzonitrile derivatives as dual A2A/A2B adenosine receptor antagonists.

A series of novel dual A2A/A2B AR antagonists based on the triazole-pyrimidine-methylbenzonitrile core were designed and synthesised. The A2A AR antagonist cAMP functional assay results were encouraging for most target compounds containing quinoline or its open-ring bioisosteres. In addition, compound 7i displayed better inhibitory activity on A2B AR (IC50 14.12 nM) and higher potency in IL-2 production than AB928. Moreover, molecular docking studies were carried out to explain the rationality of molecular design and the activity of compound 7i. Further studies on 7f and 7i revealed good liver microsomes stabilities and acceptable in vivo PK profiles. This study provides insight into the future development of dual A2A/A2B AR antagonists for cancer immunotherapy.

Activation of A2B adenosine receptor protects against demyelination in a mouse model of schizophrenia.

The purpose of the present study was to explore the effects of A2B adenosine receptor (A2BAR) on learning, memory and demyelination in a dizocilpine maleate (MK-801)-induced mouse model of schizophrenia (SCZ). BAY 60-6583, an agonist of A2BAR, or PSB 603, an antagonist of A2BAR, was used to treat SCZ in this model. The Morris Water Maze (MWM) was utilized to determine changes in cognitive function. Moreover, western blotting, immunohistochemistry and immunofluorescence were conducted to investigate the myelination and oligodendrocyte (OL) alterations at differentiation and maturation stages. The MWM results showed that learning and memory were impaired in SCZ mice, while subsequent treatment with BAY 60-6583 alleviated these impairments. In addition, western blot analysis revealed that myelin basic protein (MBP) and chondroitin sulphate proteoglycan 4 (NG2) expression levels were significantly decreased in MK-801-induced mice, while the expression of G protein-coupled receptor 17 (GPR17) was increased. Additionally, the number of anti-adenomatous polyposis coli clone CC-1/OL transcription factor 2 (CC-1+/Olig2+) cells was also decreased. Notably, BAY 60-6583 administration could reverse these changes, resulting in a significant increase in MBP and NG2 protein expression, and in the number of CC-1+/Olig2+ cells, while GPR17 protein expression levels were decreased. The present study indicated that the selective activation of A2BAR using BAY 60-6583 could improve the impaired learning and memory of SCZ mice, as well as protect the myelin sheath from degeneration by regulating the survival and maturation of OLs.

Kinetic profiling and functional characterization of 8-phenylxanthine derivatives as A2B adenosine receptor antagonists.

A2B adenosine receptor (A2BAR) antagonists have therapeutic potential in inflammation-related diseases such as asthma, chronic obstructive pulmonary disease and cancer. However, no drug is currently clinically approved, creating a demand for research on novel antagonists. Over the last decade, the study of target binding kinetics, along with affinity and potency, has been proven valuable in early drug discovery stages, as it is associated with improved in vivo drug efficacy and safety. In this study, we report the synthesis and biological evaluation of a series of xanthine derivatives as A2BAR antagonists, including an isothiocyanate derivative designed to bind covalently to the receptor. All 28 final compounds were assessed in radioligand binding experiments, to evaluate their affinity and for those qualifying, kinetic binding parameters. Both structure-affinity and structure-kinetic relationships were derived, providing a clear relationship between affinity and dissociation rate constants. Two structurally similar compounds, 17 and 18, were further evaluated in a label-free assay due to their divergent kinetic profiles. An extended cellular response was associated with long A2BAR residence times. This link between a ligand's A2BAR residence time and its functional effect highlights the importance of binding kinetics as a selection parameter in the early stages of drug discovery.

A2B adenosine receptor inhibition ameliorates hypoxic-ischemic injury in neonatal mice via PKC/Erk/Creb/HIF-1α signaling pathway.

Periventricular leukomalacia (PVL), the dominant cerebral white matter injury disease, is induced by hypoxia-ischemia and inflammation in premature infants. The activation of A2B adenosine receptor (A2BAR) is shown to involve into inflammation, ischemia, and other typical stress reactions, but its exact function in PVL has not been clarified. We gained initial insight from PVL mouse model (P9) by the induction of hypoxia-ischemia with right carotid ligation followed by exposure to hypoxia and intraperitoneal (i.p.) injection of Lipopolysaccharide (LPS). The results showed that treatment of PSB-603, an A2BAR selective antagonist, greatly ameliorated cerebral ischemic injury by increasing bodyweights, reducing infarct volume, brain injury,inflammation andcontributing to long-term learning memory functionalrecoveryof the PVL mice. Meanwhile, PSB-603 treatment suppressed neurons apoptosis as characterized byreducing of Caspase-3 level, inhibited microglia activation and attenuated hypomyelination through promoting MBP expression and oligodendrocytes differentiation. A2BAR inhibition also augmented PKC expression, the activity of PKC downstream signaling molecules were then explored. Erk expression and Creb phosphorylation exhibited upregulation in PSB-603 treatment group compared with the control group. Hypoxia Inducible Factor-1α (HIF-1α), a direct target of hypoxia, which is a key regulator of adenosine signaling by binding to the A2BAR promoter to induce expression of A2BAR, was shown to be decreased by PSB-603. Taken together, A2BAR inhibition can ameliorate hypoxic-ischemic injury in PVL mice maybe through PKC/Erk/Creb/HIF-1α signaling pathway.

Adenosine Receptor Antagonists to Combat Cancer and to Boost Anti-Cancer Chemotherapy and Immunotherapy.

Extracellular adenosine accumulates in the environment of numerous tumors. For years, this fact has fueled preclinical research to determine whether adenosine receptors (ARs) could be the target to fight cancer. The four ARs discovered so far, A1, A2A, A2B and A3, belong to the class A family of G protein-coupled receptors (GPCRs) and all four have been involved in one way or another in regulating tumor progression. Prompted by the successful anti-cancer immunotherapy, the focus was placed on the ARs more involved in regulation of immune cell differentiation and activation and that are able to establish molecular and functional interactions. This review focuses on the potential of A2A and A2B receptor antagonists in cancer control and in boosting anti-cancer chemotherapy and immunotherapy. The article also overviews the ongoing clinical trials in which A2AR and A2BR ligands are being tested in anti-cancer therapy.

Hypoxia-inducible factor-1alpha protects the liver against ischemia-reperfusion injury by regulating the A2B adenosine receptor.

Hepatic ischemia-reperfusion injury (IRI) is an inevitable complication associated with liver surgical procedures, and its pathological process remains elusive. Therefore, the present study investigated the role and mechanism of hypoxia-inducible factor-1alpha (HIF-1α) in hepatic IRI. Here, we constructed rat models with hepatic IRI and BRL-3A cell models with hypoxia/reoxygenation (H/R) insult. The extent of liver injury was assayed by measuring serum ALT/AST levels and performing H&E staining; the levels of SOD, MDA, MPO, IL-6 and TNF-α were determined using commercial kits; apoptosis was detected using the TUNEL assay and flow cytometry; and the expression of HIF-1α/A2BAR signaling-related molecules and apoptosis-associated indicators was detected using Western blotting or qRT-PCR. The expression level of HIF-1α was significantly upregulated in the liver of rats subjected to IRI, as well as in BRL-3A cells treated with H/R. HIF-1α overexpression exerted a protective effect on hepatic IRI or H/R insult by reducing serum aminotransferase levels and hepatic necrosis, inhibiting inflammation and apoptosis of hepatocytes, and alleviating oxidative stress. In contrast, inhibition of HIF-1α expression exacerbated hepatic injury induced by IR or H/R. Mechanistically, the expression level of A2BAR was markedly increased during hepatic IRI or H/R insult. Moreover, A2BAR expression increased with HIF-1α upregulation and decreased with HIF-1α downregulation. Importantly, inhibition of A2BAR signaling abolished HIF-1α overexpression-mediated hepatoprotection. Taken together, HIF-1α exerts protective effects on hepatic IRI by attenuating liver necrosis, the inflammatory response, oxidative stress and apoptosis, and its mechanism may be related to the upregulation of A2BAR signaling.

Simvastatin attenuates acute lung injury by activation of A2B adenosine receptor.

To explore the protective mechanism of simvastatin in acute lung injury (ALI), the lipopolysaccharide (LPS) induced (5 mg/kg) ALI rat model was used to examine the effects of simvastatin. Following simvastatin treatment, the histopathological evaluation of lung tissues was made using hematoxylin and eosin (H&E) staining. Also, myeloperoxidase (MPO) activity and the levels of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß), and IL-10 were determined by ELISA. Blood gas analyses of arterial blood samples were performed to assess the pulmonary gas exchange. Moreover, the neutrophil count and total protein content were determined in the bronchoalveolar lavage (BAL) fluid. The ratio of wet lung to dry lung (W/D) and the alveolar fluid clearance (AFC) were calculated to estimate the severity of edema. Lastly, the levels of A2BAR, CFTR, claudin4, and claudin18 were also measured by qRT-PCR and Western blotting. Simvastatin treatment, in a dose-related manner, markedly improved the lung histological injury and decreased the levels of TNF-α, IL-1ß, and increased IL-10 in LPS induced ALI. Also, pulmonary neutrophil count was alleviated. Besides, a decreased ratio of W/D lung also confirmed the simvastatin intervention. Notably, simvastatin reduced the levels of A2BAR, CFTR, and claudin18 but upregulated claudin4 in lung tissues. Additionally, treatment with PSB1115, an antagonist of A2BAR, countered the protective effect of simvastatin in ALI. Our study demonstrates that simvastatin has a protective effect against LPS-induced ALI by activating A2BAR and should be exploited as a novel therapeutic target for the treatment of ALI.

PSB 603 - a known selective adenosine A2B receptor antagonist - has anti-inflammatory activity in mice.

A2B adenosine receptors are present in a wide spectrum of tissues, especially on cells of the immune system. Since these particular receptors have the lowest, of all adenosine receptor subtypes, affinity for adenosine they are believed to play a special role in immunological processes associated with elevated adenosine levels such as inflammation. The aim of this preliminary study was to determine the potential anti-inflammatory properties of compound PSB-603, a potent and selective adenosine A2B receptor antagonist, in two different experimental models of local and systemic inflammation. In a model of inflammation induced by local carrageenan administration paw edema was measured using a pletysmometer. Additionally, levels of C-reactive protein (CRP), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α) and reactive oxygen species (ROS) were determined in the inflamed paw. Using the mouse model of peripheral inflammation induced by intraperitoneal (ip) administration of zymosan A, the influence of the A2B antagonist on the infiltration of neutrophils into the peritoneum and its effect on the plasma levels of CRP, TNF-α, and IL-6 were investigated. The results showed that PSB-603 administered at a dose of 5 mg/kg b.w. ip significantly reduced inflammation in both tested models. Particularly, it significantly decreased levels of the inflammatory cytokines IL-6, TNF-α and of ROS in the inflamed paw and reduced inflammation of the peritoneum by significantly decreasing the infiltration of leukocytes. Additionally, in the latter model, no statistically significant difference was observed in the CRP level between the control group without inflammation and the group which has been treated with the PSB-603 compound. Thus, the results may indicate the anti-inflammatory activity of adenosine A2B receptor antagonists in two different models of inflammation.

Novel positive allosteric modulators of A2B adenosine receptor acting as bone mineralisation promoters.

Small-molecules acting as positive allosteric modulators (PAMs) of the A2B adenosine receptor (A2B AR) could potentially represent a novel therapeutic strategy for pathological conditions characterised by altered bone homeostasis, including osteoporosis. We investigated a library of compounds (4-13) exhibiting different degrees of chemical similarity with three indole derivatives (1-3), which have been recently identified by us as PAMs of the A2B AR able to promote mesenchymal stem cell differentiation and bone formation. Evaluation of mineralisation activity of 4-13 in the presence and in the absence of the agonist BAY60-6583 allowed the identification of lead compounds with therapeutic potential as anti-osteoporosis agents. Further biological characterisation of one of the most performing compounds, the benzofurane derivative 9, confirmed that such a molecule behaves as PAM of the A2B AR.

Purinergic signalling in liver diseases: Pathological functions and therapeutic opportunities.

Extracellular nucleotides, including ATP, are essential regulators of liver function and serve as danger signals that trigger inflammation upon injury. Ectonucleotidases, which are expressed by liver-resident cells and recruited immune cells sequentially hydrolyse nucleotides to adenosine. The nucleotide/nucleoside balance orchestrates liver homeostasis, tissue repair, and functional restoration by regulating the crosstalk between liver-resident cells and recruited immune cells. In this review, we discuss our current knowledge on the role of purinergic signals in liver homeostasis, restriction of inflammation, stimulation of liver regeneration, modulation of fibrogenesis, and regulation of carcinogenesis. Moreover, we discuss potential targeted therapeutic strategies for liver diseases based on purinergic signals involving blockade of nucleotide receptors, enhancement of ectonucleoside triphosphate diphosphohydrolase activity, and activation of adenosine receptors.

Electroacupuncture improves myocardial ischemia injury via activation of adenosine receptors.

Electroacupuncture (EA) can improve myocardial ischemia (MI) injury; nevertheless, the mechanism is not entirely clear. And there were disagreements about whether the effect of EA at acupoint in disease-affected meridian is better than EA at acupoint in non-affected meridian and sham acupoint. Here, we showed that the effect of EA at Neiguan (PC6) is better than EA at Hegu (LI4) and sham acupoint in affecting RPP and ECG, increasing ATP and ADO production, decreasing AMP production, and upregulating the mRNA expression levels of A1AR, A2aAR, and A2bAR; knockdown of A1AR or A2bAR reversed the effect of EA at PC6 in alleviating MI injury; knockdown of A2aAR had no influence on the cardiac protection of EA at PC6; thus, the cardioprotective effect of EA at PC6 needs A1AR and A2bAR, instead of A2aAR; considering that the cardio protection of adenosine receptor needs activation of other adenosine receptors, one of the reasons may be that after silence of A1AR or A2bAR, EA at PC6 could not impact the expression levels of the other two adenosine receptors, and after silence of A2aAR, EA at PC6 could impact the expression levels of A1AR and A2bAR. These results suggested that EA at PC6 may be a potential and effective treatment for MI by activation of A1AR and A2bAR.

Development of a Radiofluorinated Adenosine A2B Receptor Antagonist as Potential Ligand for PET Imaging.

The adenosine A2B receptor has been proposed as a novel therapeutic target in cancer, as its expression is drastically elevated in several tumors and cancer cells. Noninvasive molecular imaging via positron emission tomography (PET) would allow the in vivo quantification of this receptor in pathological processes and most likely enable the identification and clinical monitoring of respective cancer therapies. On the basis of a bicyclic pyridopyrimidine-2,4-dione core structure, the new adenosine A2B receptor ligand 9 was synthesized, containing a 2-fluoropyridine moiety suitable for labeling with the short-lived PET radionuclide fluorine-18. Compound 9 showed a high binding affinity for the human A2B receptor (Ki(A2B) = 2.51 nM), along with high selectivities versus the A1, A2A, and A3 receptor subtypes. Therefore, it was radiofluorinated via nucleophilic aromatic substitution of the corresponding nitro precursor using [18F]F-/K2.2.2./K2CO3 in DMSO at 120 °C. Metabolic studies of [18F]9 in mice revealed about 60% of radiotracer intact in plasma at 30 minutes p.i. A preliminary PET study in healthy mice showed an overall biodistribution of [18F]9, corresponding to the known ubiquitous but low expression of the A2B receptor. Consequently, [18F]9 represents a novel PET radiotracer with high affinity and selectivity toward the adenosine A2B receptor and a suitable in vivo profile. Subsequent studies are envisaged to investigate the applicability of [18F]9 to detect alterations in the receptor density in certain cancer-related disease models.

Exploiting the Indole Scaffold to Design Compounds Binding to Different Pharmacological Targets.

Several indole derivatives have been disclosed by our research groups that have been collaborating for nearly 25 years. The results of our investigations led to a variety of molecules binding selectively to different pharmacological targets, specifically the type A γ-aminobutyric acid (GABAA) chloride channel, the translocator protein (TSPO), the murine double minute 2 (MDM2) protein, the A2B adenosine receptor (A2B AR) and the Kelch-like ECH-associated protein 1 (Keap1). Herein, we describe how these works were conceived and carried out thanks to the versatility of indole nucleus to be exploited in the design and synthesis of drug-like molecules.

Intraglomerular Monocyte/Macrophage Infiltration and Macrophage-Myofibroblast Transition during Diabetic Nephropathy Is Regulated by the A2B Adenosine Receptor.

Diabetic nephropathy (DN) is considered the main cause of kidney disease in which myofibroblasts lead to renal fibrosis. Macrophages were recently identified as the major source of myofibroblasts in a process known as macrophage-myofibroblast transition (MMT). Adenosine levels increase during DN and in vivo administration of MRS1754, an antagonist of the A2B adenosine receptor (A2BAR), attenuated glomerular fibrosis (glomerulosclerosis). We aimed to investigate the association between A2BAR and MMT in glomerulosclerosis during DN. Kidneys/glomeruli of non-diabetic, diabetic, and MRS1754-treated diabetic (DM+MRS1754) rats were processed for histopathologic, transcriptomic, flow cytometry, and cellular in vitro analyses. Macrophages were used for in vitro cell migration/transmigration assays and MMT studies. In vivo MRS1754 treatment attenuated the clinical and histopathological signs of glomerulosclerosis in DN rats. Transcriptomic analysis demonstrated a decrease in chemokine-chemoattractants/cell-adhesion genes of monocytes/macrophages in DM+MRS1754 glomeruli. The number of intraglomerular infiltrated macrophages and MMT cells increased in diabetic rats. This was reverted by MRS1754 treatment. In vitro cell migration/transmigration decreased in macrophages treated with MRS1754. Human macrophages cultured with adenosine and/or TGF-ß induced MMT, a process which was reduced by MRS1754. We concluded that pharmacologic blockade of A2BAR attenuated some clinical signs of renal dysfunction and glomerulosclerosis, and decreased intraglomerular macrophage infiltration and MMT in DN rats.