Allosteric modulators of human A2B adenosine receptor

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

A series of novel and known 5-substituted 7-amino-2-(2-furyl)[1,2,4]triazolo[1,5-a][1,3,5]triazine derivatives were synthesized and tested for adenosine receptor antagonism in radioligand binding assays at all four adenosine receptor subtypes and for inhibition of the agonist-induced cyclic AMP response at human A2B receptors. The known potent adenosine A2A receptor antagonist, 7-amino-2-(2-furyl)-5-[2-(4-hydroxyphenyl)ethyl]amino[1,2,4]triazolo[1,5-a][1,3,5]triazine (ZM241385, KiA2A = 1.78 nM) had a Ki value of 16.5 nM at A2B receptors in radioligand binding studies on Chinese hamster ovary cells expressing A2B receptors. A pA2 value of 7.9 was measured for the inhibition of the cyclic AMP response by A2B receptors induced by 5′-N-ethylcarboxamidoadenosine (NECA). In a series of 5-phenyl(alkyl)amino analogs the 5-(2-phenylethyl)amino analog LUF5452 and the 5-benzylamino analog LUF5451 were both more potent than ZM241385 in the cyclic AMP assay at A2B receptors. Moreover, Ki values of 9.9 and 7.6 nM were found in binding studies at this receptor subtype, indicating that LUF5451 and LUF5452 are more potent A2B receptor antagonists than ZM241385. The affinity of LUF5451 for the A2A receptor (Ki value = 13 nM) showed that the selectivity for this receptor subtype was lost and that a modest A2B receptor selectivity was achieved. The 5-(2-phenylhydrazino) derivative LUF5475 showed a high A2B receptor affinity (Ki = 7.6 nM), while it was equally active at A2A receptors, being A2B receptor-selective with respect to A1 and A3 receptors. Drug Dev. Res. 48:95–103, 1999. © 1999 Wiley-Liss, Inc.

Macrophages play an important role in the ankylosing spondylitis (AS) auto-inflammatory responses and fibrocartilage destruction. Adenosine is a key modulator of inflammatory conditions. The various effects of adenosine are mediated by its interaction with adenosine receptors (AR). In this study, we investigated the mRNA expression of A1, A2A, A2B, and A3 adenosine receptors, ectonucleoside triphosphate diphosphohydrolase-1 (CD39), and ecto-5'-nucleotidase (CD73) in the monocyte-derived macrophages from AS patients in comparison to healthy controls. We also explored the correlation between analyzed gene expression and patients' clinical manifestations. Whole blood-separated monocytes from 23 healthy controls and 23 active AS patients were stimulated by macrophage colony-stimulating factor (M-CSF) for 7days and differentiated to macrophages. Monocyte and macrophage markers were analyzed by flow cytometry. Analysis of adenosine receptors (ADORA1، ADORA2A، ADORA2B، ADORA3), CD39 and CD73 gene expression was performed by SYBR green real-time PCR. Our results demonstrated monocyte-derived macrophages from AS patients expressed increased level of A2AAR and reduced level of A1, A2BAR, and CD39 mRNA compared to healthy controls. We found an inverse correlation between A2AAR mRNA expression and Bath Ankylosing Spondylitis Functional Index (BASFI) score in AS patients. According to our results, altered expression level of adenosine-relying system would be involved in AS macrophage dysfunction and inflammation and correlated with functional status in AS patients.

The A1 adenosine receptor (A1AR) exerts important biological effects in the mammalian biology. To provide insights into the role A1AR action in human physiology, we characterized the pharmacologic properties of the human A1AR, examined somatic sites of A1AR gene expression, and identified the chromosomal location of the human A1AR gene. Using stably transfected CHO cells, the ligand binding properties of human and rat A1ARs were directly compared. Saturation studies showed that the human and rat A1ARs had similar high affinity for the A1 agonist [(3)H]CCPA (human, K(d)=517±64 pM; B(max) 438±29 fmol/mg of protein; rat, K(d)=429±69 pM; B(max) 358±76 fmol/mg of protein). Competition studies performed using seven adenosine agonists and four adenosine antagonists also did not detect differences in the ligand binding properties among the rat and human A1ARs. Northern analysis of 16 human tissues revealed the presence of a single hybridizing transcript of 2.5 kb. Human A1AR receptor mRNA expression was greatest in brain and testis; lower levels of A1AR mRNA were present in heart, pancreas, kidney and spleen. Southern blotting and PCR analysis of human-rodent somatic cell hybrids showed that the A1AR gene is on human chromosome 1. Using fluorescence in situ hybridization, the human A1AR gene was further localized to the 1q32.1 region. These observations show that the human A1AR is a high affinity receptor that has ligand binding properties similar to the rat A1AR, human A1AR mRNA is heavily expressed in brain and testis, and the gene encoding the human A1AR is present on the long arm of chromosome 1.

The purpose of this study was to determine whether there is a cross-talk between opioid receptors (OPRs) and adenosine receptors (ADRs) in remifentanil preconditioning (R-Pre) and, if so, to investigate the types of ADRs involved in the cross-talk. Isolated rat hearts received 30 min of regional ischemia followed by 2 hr of reperfusion. OPR and ADR antagonists were perfused from 10 min before R-Pre until the end of R-Pre. The heart rate, left ventricular developed pressure (LVDP),velocity of contraction (+dP/dtmax), and coronary flow (CF) were recorded. The area at risk and area of necrosis were measured. After reperfusion, the LVDP, +dP/dtmax,and CF showed a significant increase in the R-Pre group compared with the control group (no intervention before or after regional ischemia). These increases in the R-Pre group were blocked by naloxone, a nonspecific ADR antagonist, an A1 ADR antagonist, and an A2B ADR antagonist. The infarct size was reduced significantly in the R-Pre group compared with the control group. The infarct-reducing effect in the R-Pre group was blocked by naloxone, the nonspecific ADR antagonist, the A1 ADR antagonist, and the A2B ADR antagonist. The results of this study demonstrate that there is cross-talk between ADRs and OPRs in R-Pre and that A1 ADR and A2B ADR appear to be involved in the cross-talk.

Of 12 naturally occurring, activating mutations in the seven-transmembrane (7TM) domain of the human Ca2+ receptor (CaR) identified previously in subjects with autosomal dominant hypocalcemia (ADH), five appear at the junction of TM helices 6 and 7 between residue Ile819 and Glu837. After identifying a sixth activating mutation in this region, V836L, in an ADH patient, we studied the remaining residues in this region to determine whether they are potential sites for activating mutations. Alanine-scanning mutagenesis revealed five additional residues in this region that when substituted by alanine led to CaR activation. We also found that, whereas E837A did not activate the receptor, E837D and E837K mutations did. Thus, region Ile819-Glu837 of the 7TM domain represents a "hot spot" for naturally occurring, activating mutations of the receptor, and most of the residues in this region apparently maintain the 7TM domain in its inactive configuration. Unique among the residues in this region, Pro823, which is highly conserved in family 3 of the G protein-coupled receptors, when mutated to either alanine or glycine, despite good expression severely impaired CaR activation by Ca2+. Both the P823A mutation and NPS 2143, a negative allosteric modulator that acts on the 7TM through a critical interaction with Glu837, blocked activation of the CaR by various ADH mutations. These results suggest that the 7TM domain region Ile819-Glu837 plays a key role in CaR activation by Ca2+. The implications of our finding that NPS 2143 corrects the molecular defect of ADH mutations for treatment of this disease are also discussed.

A2B and A3 Adenosine Receptors Modulate Vascular Endothelial Growth Factor and Interleukin-8 Expression in Human Melanoma Cells Treated with Etoposide and Doxorubicin

Insulin requires A2B adenosine receptors to modulate the L-arginine/nitric oxide signalling in the human fetoplacental vascular endothelium from late-onset preeclampsia

Objective To investigate expression of adenosine receptors in uveal melanocytes and uveal melanoma cell, and the effect of adenosine on cell proliferation on these two types of cells. Methods Adenosine receptors (A1, A2A, A2B, A3) mRNA expression were detected in cultured uveal melanocytes cell U95 and uveal melanoma cell S65 by Real-time PCR. After U95 and S65 cells were treated with 0, 0.1, 1, 10 or 100 μLmol/L adenosine for 24 h or 48 h, cell viability of each group was measured by MTT. Results Four subtypes of adenosine receptors (A1, A2A, A2B and A3) mRNA were expressed in U95 and S65 cells. The expression of adenosine receptors A1 and A2B is similar to each other but higher than that of A2A and A3 in U95 cells (A1: 1.32±0.47; A2A: 0.28± 0.05; A2B: 1.03±0.10; A3: 0.20±0.07; F=15.90, P<0.01). The mRNA expression of adenosine receptor A2B was much higher than the three other subtypes of adenosine receptors in S65 cells (A1: 1.03±0.27; A2A: 2.23±0.35; A2B: 127.34±18.69; A3: 0.17±0.07; F:163.20, P<0.01). Adenosine receptor A2B and A2A mRNA expression was higher in S65 cells than in U95 cells (A2A: t=9.35, P< 0.01; A2B: t=11.43, P<0.01). After treated with 0, 0.1, 1, 10 and 100 μmol/L adenosine for 24 h and 48 h, S65 cells growth inhibition ratio were 3.47%, 14.93%, 19.79%, 26.04% at 24 h and 11.43%, 23.10%, 34.76%, 46.67% at 48 h. However different concentrations of adenosine had no effect on the proliferation of U95 cells. Conclusion The adenosine receptor subtypes express differently in uveal melanocytes and uveal melanoma cells. Adenosine is able to inhibit uveal melanoma cells proliferation, but has no effect on uveal melanocytes cells, which may be because of different adenosine receptor subtypes expression in these two types of cells. Key words: Adenosine; Uvea; Melanoma,uveal neoplasms; Cell proliferation

Metabotropic glutamate receptors as drug targets for the treatment of absence epilepsy

Introduction: The adenosine pathways via type A2a and A2b receptors are known to suppress immunity by regulating the functions of immune cells, such as macrophages, dendritic cells, MDSC, T cells and NK cells in tumor microenvironment. Also, the adenosine pathway through an A1 receptor induces tumor growth by promoting cancer cell proliferation. On the other hand, it has been recently reported that the downregulation of A1R induces expression of tumor PD-L1. Herein, we introduce a novel antagonist of adenosine receptor, HM87277, which has potent antagonistic activity against three types of adenosine receptors A1R, A2aR and A2bR, and present preclinical results that the triple antagonist HM87277 may be more effective in treating cancer patients through appropriate combination with immune checkpoint blockers (ICBs). Materials and Methods: Receptor binding affinities were assessed using human adenosine receptors of type A1, A2a, A2b and A3. Cellular functional activity tests such as cAMP production. To identify adenosine receptor downstream signaling in cancer cells, p-ERK and p-JNK were measured by western blotting in SK-BR3 cells. PD-L1 induction by adenosine receptor antagonist was determined by flow cytometry using Canto II (BD) in PD-L1 expressing SK-MEL-28 cells. For cytotoxicity assay of T cell, isolated human CD8+ T cells were co-cultured with A1R expressing cancer cells in the presence of a high concentration of adenosine. Antitumor activity of HM87277 alone and in combination with ICBs was assessed in syngeneic mouse models using murine cancer cell lines. Results: HM87277 showed strong binding affinity toward human adenosine receptors of type A1, A2a and A2b, and potently inhibited the NECA-mediated production of intracellular cAMP in HEK293-A2aR cells. HM87277 significantly increased the proliferation of effector T cells. HM87277 potently inhibited the p-ERK and p-JNK, and induced the expression of PD-L1 in A1R-expressing cancer cells. As well, the combination of HM87277 and anti-PD-L1 mAb was observed to have synergistic inhibitory effect on tumor cell growth in co-culture with SK-MEL-28 and CD8 + T cells. Furthermore, in the mouse syngeneic model using murine cancer cell lines, HM87277 effectively inhibited tumor growth alone or in combination with anti-PD-L1 mAb. Conclusion: These results indicate that a triple antagonist HM87277, which simultaneously antagonizes type A1, A2a and A2b adenosine receptors, may not only stimulate immune cells such as cytotoxic CD8+ T cell but also inhibit the growth of cancer cells themselves through inhibition of ERK and JNK pathway. In addition, it showed a strong combination effect by enhancing the sensitivity to ICBs through the increase of PD-L1 in tumor cells. Therefore, it is considered that the triple antagonist HM87277 is worth developing as a potential anticancer agent. Citation Format: Sunyoung Jang, Seunah Jun, Hosun Lee, Yongtaek Lee, Joo-Yun Byun, Junghwa Park, Yu-Yon Kim, Young Gil Ahn, YoungHoon Kim, Kwee Hyun Suh. Discovery and characterization of a novel triple A1/A2a/A2b adenosine receptor antagonist for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1704.

Analgesic, anti-inflammatory and joint protective effects of ACD137, a selective negative allosteric modulator of TrkA, in models of chemotherapy-induced peripheral neuropathy and osteoarthritis.

Negative regulatory mechanism of immune responses is essential to prevent excessive inflammatory tissue injury. Adenosine involves in physiological down-regulation of inflammation via A2A adenosine receptor (A2AR). In this study, we provide evidence that A2B adenosine receptor (A2BR) is also important in the physiological control of hepatic inflammation. Acute hepatitis induced by intravenous concanavalin A (Con A) injection was significantly more severe in A2BR-/- mice than in wild-type mice. The exaggerated inflammation in A2BR-/- mice accompanied higher levels of IL-4 from NKT cells suggesting A2BR-mediated suppression of NKT cell activation. In vitro activation of NKT cells by α-galactosylceramide (α-GalCer) showed that stimulation of A2BR is inhibitory to NKT cell activation. Furthermore, injection of α-GalCer induced higher serum cytokine levels and stronger liver damage in A2BR-/- mice. These results show that the lack of A2BR diminished negative regulatory mechanism of NKT cells by endogenously produced adenosine and, therefore, caused more severe acute hepatitis. Indeed, transfer of A2BR-/- NKT cells into RAG2-/- mice resulted in stronger Con A-induced liver injury than wild-type NKT cells. There have been controversial reports regarding the role of A2BR in inflammation. Our results demonstrate that NKT cell-mediated immune responses are under the physiological control of extracellular adenosine through A2BR signaling.

Osteoblast differentiation and survival: A role for A2B adenosine receptor allosteric modulators

Adenosine mediates its actions through four G protein-coupled receptors, A1, A2a, A2b and A3. The A1 receptor (A1R) is dominant in adipocytes where it mediates many actions that include inhibition of lipolysis, stimulation of leptin secretion and protection against obesity-related insulin resistance. The objective of this study is to investigate whether induced expression of A1Rs stimulates adipogenesis, or whether A1R expression is a consequence of cells having an adipocyte phenotype. Human A1R and A2b receptors (A2bRs) were stably transfected into a murine osteoblast precursor cell line, 7F2. Adipogenesis was determined by lipid accumulation and expression of adipocyte and osteoblast marker molecules. Adenosine receptor expression and activation of associated signal molecules were also evaluated as 7F2 cells were induced to differentiate to adipocytes. 7F2 cells transfected with the A1R showed increased adipocyte marker mRNA expression; lipoprotein lipase and glycerol-3-phosphate dehydrogenase were both upregulated, whereas the osteoblast marker alkaline phosphatase (ALP) was downregulated. When cultured in adipocyte differentiating media, such cells also showed increased adipogenesis as judged by lipid accumulation. Conversely, A2bR transfection stimulated osteocalcin and ALP expression, and in addition, adipogenesis was inhibited in the presence of adipocyte differentiation media. Adipogenic differentiation of naive 7F2 cells also resulted in increased expression of the A1R and reduced or modified expression of the A2a and A2bR. The loss of A2 receptors after adipogenic differentiation was accompanied by a loss of cyclic adenosine monophosphate and ERK1/2 signalling. These data show that expression of A1Rs induced adipocyte differentiation, whereas A2bR expression inhibited adipogenesis and stimulated an osteoblastic phenotype. These data suggest that targeting A1 and A2bR could be considered in the management of obesity and diabetes. Targeting adenosine signal pathways may be useful in treatment strategies for diseases in which there is an imbalance between osteoblasts and adipocytes.