Structure optimization of Cmpd-15 as negative allosteric modulators for the ß2-adrenergic receptor.

19 derivatives of 1-benzyl-3-arylpyrazole-5-carboxamides (H1-H19) and 5 derivatives of 1-benzyl-5-arylpyrazole-3-carboxamides (J1-J5) have been designed and synthesized as potential negative allosteric modulators (NAMs) for the ß2-adrenergic receptor (ß2AR). The new pyrazole derivatives were screened on the classic G-protein dependent signaling pathway at ß2AR. The majority of 1-benzyl-3-aryl-pyrazole-5-carboxamide derivatives show more potent allosteric antagonistic activity against ß2AR than Cmpd-15, the first reported ß2AR NAM. However, the 1-benzyl-5-arylpyrazole-3-carboxamide derivatives exhibit very poor or even no allosteric antagonistic activity for ß2AR. Furthermore, the active pyrazole derivatives have relative better drug-like profiles than Cmpd-15. Taken together, we discovered a series of derivatives of 1-benzyl-3-arylpyrazole-5-carboxamides as a novel scaffold of ß2AR NAM.

Implication of ß2-adrenergic receptor and miR-196a correlation in neurite outgrowth of LNCaP prostate cancer cells.

The ß2-adrenergic receptor has been shown to be involved in neuroendocrine differentiation and to contribute to the development of aggressive prostate cancer. In this study we have investigated whether miR-196a plays a role in the regulation of the ß2-adrenergic receptor in the LNCaP prostate cancer cell line. Our results show that the expression of miR-196a is elevated in LNCaP prostate cancer cells with reduced levels of ß2-adrenergic receptor after stably transfection with three different shRNAs. Furthermore, treatment with ß-blockers showed that this upregulation is strictly related to the low levels of ß2-adrenergic receptor and not to the inhibition of the receptor signaling activity. Finally, we found that the reduced ability of LNCaP cells with low levels of ß2-adrenergic receptor to initiate neuroendocrine differentiation under androgen depletion conditions is mediated by miR-196a. In conclusion, this study provides the rational for a role of miR-196a in the ß2-adrenergic receptor mediated neuroendocrine differentiation of LNCaP prostate cancer cells.

Suppression of beta 2 adrenergic receptor actions prevent UVB mediated cutaneous squamous cell tumorigenesis through inhibition of VEGF-A induced angiogenesis.

Although beta 2 adrenergic receptors (ß2 ADR) are present in the keratinocytes, their role in cutaneous squamous cell tumorigenesis needs to be ascertained. For the first time, we report here that selective ß2 ADR antagonists by inhibiting ß2 ADR actions significantly retarded the progression of ultraviolet B (UVB) induced premalignant cutaneous squamous cell lesions. These antagonists acted by inhibiting vascular endothelial growth factor-A (VEGF) mediated angiogenesis to prevent UVB radiation-induced squamous cell carcinoma of the skin.

Can Beta-2-Adrenergic Pathway Be a New Target to Combat SARS-CoV-2 Hyperinflammatory Syndrome?-Lessons Learned From Cancer.

SARS-CoV-2 infection is a new threat to global public health in the 21st century (2020), which has now rapidly spread around the globe causing severe pneumonia often linked to Acute Respiratory Distress Syndrome (ARDS) and hyperinflammatory syndrome. SARS-CoV-2 is highly contagious through saliva droplets. The structural analysis suggests that the virus enters human cells through the ligation of the spike protein to angiotensin-converting enzyme 2 (ACE2). The progression of Covid-19 has been divided into three main stages: stage I-viral response, stage II-pulmonary phase, and stage III-hyperinflammation phase. Once the patients enter stage III, it will likely need ventilation and it becomes difficult to manage. Thus, it will be of paramount importance to find therapies to prevent or slow down the progression of the disease toward stage III. The key event leading to hyperinflammation seems to be the activation of Th-17 immunity response and Cytokine storm. B2-adrenergic receptors (B2ARs) are expressed on airways and on all the immune cells such as macrophages, dendritic cells, B and T lymphocytes. Blocking (B2AR) has been proven, also in clinical settings, to reduce Th-17 response and negatively modulate inflammatory cytokines including IL-6 while increasing IFNγ. Non-selective beta-blockers are currently used to treat several diseases and have been proven to reduce stress-induced inflammation and reduce anxiety. For these reasons, we speculate that targeting B2AR in the early phase of Covid-19 might be beneficial to prevent hyperinflammation.

ß2-AR blockade potentiates MEK1/2 inhibitor effect on HNSCC by regulating the Nrf2-mediated defense mechanism.

The ß2-Adrenergic receptor (ß2-AR) is a G protein-coupled receptor (GPCR), involved in the development of many cancers, among which HNSCC. In this contest, ß2-AR signaling interacts with different pathways, such as PI3K and MAPK, commonly activated by TK receptors. For this reason, TK blockade is one of the most adopted therapeutic strategies in HNSCC patients. In our study we investigated the effects of the ß2-AR blocking in HNSCC cell lines, using the selective inhibitor ICI118,551 (ICI), in combination with the MAPK inhibitor U0126. We found that ICI leads to the blocking of p38 and NF-kB oncogenic pathways, strongly affecting also the ERK and PI3K pathways. Cotreatment with U0126 displays a synergic effect on cell viability and pathway alteration. Interestingly, we found that the ß2-AR blockade affects Nrf2-Keap1 stability and its nuclear translocation leading to a drastic ROS increase and oxidative stress. Our results are confirmed by a TCGA dataset analysis, showing that NFE2L2 gene is commonly overexpressed in HNSC, and correlated with a lower survival rate. In our system, the PI3K pathway inhibition culminated in the blocking of pro-survival autophagy, a mechanism normally adopted by cancer cells to became less responsive to the therapies. The mTOR expression, commonly upregulated in HNSC, was reduced in patients with disease-recurrence. It is well known that mTOR has a strong autophagy inhibition effect, therefore its downregulation promoted pro-survival autophagy, with a related increase recurrence rate. Our findings highlight for the first time the key role of ß2-AR and related pathway in HNSCC cell proliferation and drug resistance, proposing it as a valuable therapeutic molecular target.

The effects of ß1 and ß1+2 adrenergic receptor blockade on the exercise-induced mobilization and ex vivo expansion of virus-specific T cells: implications for cellular therapy and the anti-viral immune effects of exercise.

The adoptive transfer of donor-derived virus-specific T cells (VSTs) is an effective treatment for infections following allogeneic hematopoietic cell transplantation. Acute exercise mobilizes effector lymphocytes and VSTs to the circulation and augments the ex vivo manufacture of VSTs. This study determined if ß2 adrenergic receptor (AR) signaling precipitated the VST response to acute exercise. Healthy participants (n = 12) completed 30 min of steady-state cycling exercise after ingesting a placebo, a ß1 + 2 AR antagonist (nadolol) or a ß1 AR antagonist (bisoprolol). Circulating VSTs to cytomegalovirus (CMV), Epstein-Barr virus (EBV), and adenovirus (AdV) antigens were enumerated before and after exercise, and peripheral blood mononuclear cells were cultured with viral peptides for 8 days to expand multi-VSTs. Compared with placebo, nadolol blunted the exercise-induced mobilization of CMV-VSTs (Δ VSTs/100,000 CD3+ T cells = 93 ± 104 vs. 22 ± 91 for placebo and nadolol, respectively; p = 0.036), while bisoprolol did not, despite both drugs evoking similar reductions in exercising heart rate and blood pressure. Circulating AdV and EBV VSTs (VSTs/mL blood) only increased after exercise with placebo. Although not significant, nadolol partially mitigated exercise-induced increases in multi-VST expansion, particularly in participants that demonstrated an exercise-induced increase in VST expansion. We conclude that exercise-induced enhancements in VST mobilization and expansion are at least partially ß2 AR mediated, thus highlighting a role for the ß2 AR in targeted therapy for the augmentation of VST immune cell therapeutics in the allogeneic adoptive transfer setting. Moreover, long-term regular exercise may provide additional viral protection in the host through frequent ß2 AR-dependent mobilization and redistribution of VSTs cumulated with each bout of exercise.

The Effect of ß2-Adrenoceptor Agonists on Leucocyte-Endothelial Adhesion in a Rodent Model of Laparotomy and Endotoxemia.

Background: The ß2-adrenoceptor agonist dopexamine may possess anti-inflammatory actions which could reduce organ injury during endotoxemia and laparotomy. Related effects on leucocyte-endothelial adhesion remain unclear. Methods: Thirty anesthetized Wistar rats underwent laparotomy followed by induction of endotoxemia with lipopolysaccharide and peptidoglycan (n = 24) or sham (n = 6). Animals received dopexamine at 0.5 or 1 µg kg-1 min-1 (D0.5 and D1), salbutamol at 0.1 µg kg-1 min-1, or saline vehicle (Sham and Control) for 5 h. Intravital microscopy was performed in the ileum of the small intestine to assess leucocyteendothelial adhesion, arteriolar diameter, and functional capillary density. Global hemodynamics and biochemical indices of renal and hepatic function were also measured. Results: Endotoxemia was associated with an increase in adherent leucocytes in post-capillary venules, intestinal arteriolar vasoconstriction as well-reduced arterial pressure and relative cardiac index, but functional capillary density in the muscularis was not significantly altered. Dopexamine and salbutamol administration were associated with reduced leucocyte-endothelial adhesion in post-capillary venules compared to control animals. Arteriolar diameter, arterial pressure and relative cardiac index all remained similar between treated animals and controls. Functional capillary density was similar for all groups. Control group creatinine was significantly increased compared to sham and higher dose dopexamine. Conclusions: In a rodent model of laparotomy and endotoxemia, ß2-agonists were associated with reduced leucocyte-endothelial adhesion in post-capillary venules. This effect may explain some of the anti-inflammatory actions of these agents.

ß2-Adrenergic Receptor Stimulation Upregulates Cx43 Expression on Glioblastoma Multiforme and Olfactory Ensheathing Cells.

Glioblastoma multiforme (GBM) is described as an invasive astrocytic tumor in adults. Despite current standard treatment approaches, the outcome of GBM remains unfavorable. The downregulation of connexin 43 (Cx43) expression is one of the molecular transformations in GBM cells. The Cx43 levels and subsequently gap junctional intercellular communication (GJIC) have an important role in the efficient transfer of cytotoxic drugs to whole tumor cells. As shown in our previous study, the stimulation of the ß2-adrenergic receptor (ß2-AR) leads to the modulation of Cx43 expression level in the GBM cell line. Here we further examine the effect of clenbuterol hydrochloride as a selective ß2-AR agonist on the Cx43 expression in human GBM-derived astrocyte cells and human olfactory ensheathing cells (OECs) as a potent vector for future gene therapy. In this experiment, first we established a primary culture of astrocytes from GBM samples and verified the purity using immunocytofluorescent staining. Western blot analysis was performed to evaluate the Cx43 protein level. Our western blot findings reveal that clenbuterol hydrochloride upregulates the Cx43 protein level in both primary human astrocyte cells and human OECs. Conversely, ICI 118551 as a ß2-AR antagonist inhibits these effects. Moreover, clenbuterol hydrochloride increases the Cx43 expression in primary human astrocyte cells and OECs co-culture systems, and ICI 118551 reverses these effects. To confirm the western blot results, immunocytofluorescent staining was performed to evaluate the ß2-AR agonist effect on Cx43 expression. Our immunocytofluorescent results supported western blot analysis in primary human astrocyte cells and the OECs co-culture system. The results of this study suggest that the activation of ß2-AR with regard to Cx43 protein levels enhancement in GBM cells and OECs might be a promising approach for GBM treatment in the future.

The stress hormone norepinephrine promotes tumor progression through ß2-adrenoreceptors in oral cancer.

OBJECTIVE: Chronic stress hormone norepinephrine (NE) has been previously reported to play a role in the development of cancer, but the correlation between NE and oral squamous cell carcinoma (OSCC) progression is not well understood. METHOD: To address this, the expression of adrenergic receptors (ARs) in human OSCC cell lines and clinic OSCC samples was detected, and the role of NEin vivo and in vitro was further investigated. RESULTS: It was found that ß2-AR was the main AR of NE in OSCC. Stimulation of OSCC cells with NE significantly increased the OSCC proliferation and invasion, which was, however, blocked by ß2-AR inhibitor. NE could induce the phosphorylation of extracellular regulated protein kinases (ERK) and cAMP-response element binding protein (CREB). Inhibition of ERK and CREB pathway abrogated NE-induced OSCC invasion and proliferation. NE could enhance cancer stem cells (CSCs)-like phenotype and up-regulate the expression of stemness marker. In tumor-bearing nude mice, it was found that consecutive administration of NE significantly promoted the tumor growth, while daily injection of ß2-AR inhibitor blocked this phenomenon. CONCLUSIONS: Those findings indicated a critical role of the chronic stress hormone NE in OSCC progression. Inhibition of ß2-AR may serve as a potential therapeutic strategy for protecting OSCC patients from chronic stress related deleterious effect.

Sympathetic Enhancement of Memory T-Cell Homing and Hypertension Sensitization.

RATIONALE: Effector memory T lymphocytes (TEM cells) exacerbate hypertension in response to repeated hypertensive stimuli. These cells reside in the bone marrow for prolonged periods and can be reactivated on reexposure to the hypertensive stimulus. OBJECTIVE: Because hypertension is associated with increased sympathetic outflow to the bone marrow, we hypothesized that sympathetic nerves regulate accumulation and reactivation of bone marrow-residing hypertension-specific TEM cells. METHODS AND RESULTS: Using unilateral superior cervical ganglionectomy in wild-type C57BL/6 mice, we showed that sympathetic nerves create a bone marrow environment that supports residence of hypertension-specific CD8+ T cells. These cells, defined by their proliferative response on coculture with dendritic cells from Ang (angiotensin) II-infused mice, were reduced in denervated compared with innervated bone of Ang II-infused mice. Adoptively transferred CD8+ T cells from Ang II-infused mice preferentially homed to innervated compared with denervated bone. In contrast, ovalbumin responsive T cells from OT-I mice did not exhibit this preferential homing. Increasing superior cervical ganglion activity by activating Gq-coupled designer receptor exclusively activated by designer drug augmented CD8+ TEM bone marrow accumulation. Adoptive transfer studies using mice lacking ß2AR (ß2 adrenergic receptors) indicate that ß2AR in the bone marrow niche, rather than T-cell ß2AR is critical for TEM cell homing. Inhibition of global sympathetic outflow using Gi-coupled DREADD (designer receptor exclusively activated by designer drug) injected into the rostral ventrolateral medulla or treatment with a ß2AR antagonist reduced hypertension-specific CD8+ TEM cells in the bone marrow and reduced the hypertensive response to a subsequent response to low dose Ang II. CONCLUSIONS: Sympathetic nerves contribute to the homing and survival of hypertension-specific TEM cells in the bone marrow after they are formed in hypertension. Inhibition of sympathetic nerve activity and ß2AR blockade reduces these cells and prevents the blood pressure elevation and renal inflammation on reexposure to hypertension stimuli.

The ADRB1 (Adrenoceptor Beta 1) and ADRB2 genes significantly co-express with commonly mutated genes in prostate cancer.

BACKGROUND: Beta blockers act on the beta-adrenergic receptors ADRB1 and ADRB2 to reduce heart rate and blood pressure. Observational studies have revealed strong risk reductions in metastasis and cancer-specific mortality with the use of beta-blockers in patients with some cancers. But observational studies of prostate cancer have reported conflicting results. OBJECTIVES: We examined the relationship of ADRB1 (Adrenoceptor beta 1) gene expression and ADRB2 (Adrenoceptor beta 2) gene expression with Forkhead box protein A1 (FOXA1) gene expression in prostate cancer. We also analyzed survival data of solid tumor patients with respect to beta 1 (ADRB1) and beta 2 (ADRB2) adrenergic receptor gene expression. METHODS: We examined the genomics of prostate cancer and other solid primary tumors in the GDC TCGA Prostate Cancer (PRAD) data set. The Cancer Genome Atlas (TCGA) contains the analysis of over 11,000 tumors from 33 of the most prevalent forms of cancer. RESULTS: The presence of somatic mutations [Single nucleotide polymorphisms (SNPs) and small insertion/deletion polymorphism (INDELS)] in FOXA1 alters ADRB1 and ADRB2 gene expression. The correlation of FOXA1 gene expression with ADRB1 and ADRB2 gene expression is highly significant. Alterations in FOXA1 genes, ADRB1 genes, and ADRB2 genes are significantly co-occurrent, indicating that they may work in tandem to drive tumor formation and development. Increased ADRB1 and ADRB2 expressions reduce the overall survival of solid tumor patients in the GDC Pan Cancer set. CONCLUSIONS: FOXA1 signaling may regulate ADRB1 and ADRB2 expression, as well as androgen receptor expression. Analysis of these tumor mutations might indicate whether an individual prostate cancer patient will respond to beta blockers.

Development of covalent antagonists for ß1- and ß2-adrenergic receptors.

The selective covalent tethering of ligands to a specific GPCR binding site has attracted considerable interest in structural biology, molecular pharmacology and drug design. We recently reported on a covalently binding noradrenaline analog (FAUC37) facilitating crystallization of the ß2-adrenergic receptor (ß2ARH2.64C) in an active state. We herein present the stereospecific synthesis of covalently binding disulfide ligands based on the pharmacophores of adrenergic ß1- and ß2 receptor antagonists. Radioligand depletion experiments revealed that the disulfide-functionalized ligands were able to rapidly form a covalent bond with a specific cysteine residue of the receptor mutants ß1ARI2.64C and ß2ARH2.64C. The propranolol derivative (S)-1a induced nearly complete irreversible blockage of the ß2ARH2.64C within 30 min incubation. The CGP20712A-based ligand (S)-4 showed efficient covalent blocking of the ß2ARH2.64C at very low concentrations. The analog (S)-5a revealed extraordinary covalent cross-linking at the ß1ARI2.64C and ß2ARH2.64C mutant while retaining a 41-fold selectivity for the ß1AR wild type over ß2AR. These compounds may serve as valuable molecular tools for studying ß1/ß2 subtype selectivity or investigations on GPCR trafficking and dimerization.

Adrenergic Regulation of Macrophage-Mediated Innate/Inflammatory Responses in Obesity and Exercise in this Condition: Role of ß2 Adrenergic Receptors.

BACKGROUND: The effects of exercise on the innate/inflammatory immune responses are crucially mediated by catecholamines and adrenoreceptors; and mediations in both stimulatory and anti-inflammatory responses have been attributed to them. Obesity and metabolic syndrome are included among low-grade chronic inflammatory pathologies; particularly because patients have a dysregulation of the inflammatory and stress responses, which can lead to high levels of inflammatory cytokines that induce insulin resistance, contributing to the onset or exacerbation of type 2 diabetes. Macrophages play a crucial role in this obesity-induced inflammation. Although most of the antiinflammatory effects of catecholamines are mediated by ß adrenergic receptors (particularly ß2), it is not known whether in altered homeostatic conditions, such as obesity and during exercise, innate/ inflammatory responses of macrophages to ß2 adrenergic stimulation are similar to those in cells of healthy organisms at baseline. OBJECTIVE: This review aims to emphasize that there could be possible different responses to ß2 adrenergic stimulation in obesity, and exercise in this condition. METHODS: A revision of the literature based on the hypothesis that obesity affects ß2 adrenergic regulation of macrophage-mediated innate/inflammatory responses, as well as the effect of exercise in this context. CONCLUSION: The inflammatory responses mediated by ß2 adrenoreceptors are different in obese individuals with altered inflammatory states at baseline compared to healthy individuals, and exercise can also interfere with these responses. Nevertheless, it is clearly necessary to develop more studies that contribute to widening the knowledge of the neuroimmune regulation process in obesity, particularly in this context.

Methods of accelerating orthodontic tooth movement: A review of contemporary literature.

Technological progress and the introduction of modern therapeutic methods are constantly changing contemporary orthodontics. More and more orthodontic patients are working adults, who expect satisfactory therapeutic effects as soon as possible, increasing the importance of methods accelerating tooth movement. The aim of this study was to review the current literature regarding methods of accelerating tooth movement and reducing the duration of the active phase of therapy. The literature was collected from the PubMed and EBSCO databases using "accelerated orthodontic tooth movement" as the search key words. The methods described were categorized as conservative and surgical. The pharmacological agents used in conservative treatment, such as growth hormone, parathyroid hormone, thyroxine, and vitamin D, are especially worth mentioning. They stimulate osteoclasts to increase resorption through a variety of mechanisms. Effective methods also include physical stimuli, e.g., vibrations or photobiomodulation. Most studies describing the effects of pharmacological agents were based on animal subjects and they may therefore lack clinical relevancy. Corticotomy and its modifications based on the regional acceleratory phenomenon (RAP) might prove to be a useful augmentation of orthodontic treatment, especially in adults, including patients with periodontal disease.

Sympathetic Neuronal Activation Triggers Myeloid Progenitor Proliferation and Differentiation.

There is a growing body of research on the neural control of immunity and inflammation. However, it is not known whether the nervous system can regulate the production of inflammatory myeloid cells from hematopoietic progenitor cells in disease conditions. Myeloid cell numbers in diabetic patients were strongly correlated with plasma concentrations of norepinephrine, suggesting the role of sympathetic neuronal activation in myeloid cell production. The spleens of diabetic patients and mice contained higher numbers of tyrosine hydroxylase (TH)-expressing leukocytes that produced catecholamines. Granulocyte macrophage progenitors (GMPs) expressed the ß2 adrenergic receptor, a target of catecholamines. Ablation of splenic sympathetic neuronal signaling using surgical, chemical, and genetic approaches diminished GMP proliferation and myeloid cell development. Finally, mice lacking TH-producing leukocytes had reduced GMP proliferation, resulting in diminished myelopoiesis. Taken together, our study demonstrates that catecholamines produced by leukocytes and sympathetic nerve termini promote GMP proliferation and myeloid cell development.

Bone Marrow-Derived Mesenchymal Stem Cells Promoted Cutaneous Wound Healing by Regulating Keratinocyte Migration via ß2-Adrenergic Receptor Signaling.

Mesenchymal stem cells (MSCs) play an important role in cutaneous wound healing; however, the functional mechanisms involved in the healing process are poorly understood. A series of studies indicate that keratinocytes that migrate into the wound bed rely on an epithelial-mesenchymal transition (EMT)-like process to initiate re-epithelialization. We therefore examined whether bone marrow-derived MSCs (BMSCs) could affect biological behavior and induce EMT-like characteristics in the human epidermal keratinocytes (HEKs) and in the immortalized human keratinocyte cell line HaCaT cells, and we investigated the signaling pathways of BMSC-mediated phenotypic changes. By assessing the expression of EMT-related markers including E-cadherin, α-SMA, and Snail family transcription factors by ß2-adrenergic receptor (ß2-AR) blockage using ICI-118,551, a ß2-AR selective antagonist, or ß2-AR small interfering RNA (siRNA), we showed an involvement of ß2-AR signaling in the induction of EMT-like alterations in human keratinocytes in vitro. ß2-AR signaling also affected collective and individual cell migration in human keratinocyte cell lines, which was attenuated by administration of ICI-118,551. Treating the cells with BMSC-conditioned media (BMSC-CM) not only recapitulated the effect of isoproterenol (ISO) on cell migration but also induced the expression of ß2-AR and a panel of proteins associated with mesenchymal phenotype in HEKs and HaCaT cells. Similarly, a blockade of the ß2-AR by either ICI-118,551 or ß2-AR siRNAs reversed both responses of the epidermal keratinocyte cell lines relative to BMSC-CM exposure. These results were further verified in our vivo findings and indicated that the exogenous application of MSCs promoted cutaneous wound healing and endowed the keratinocytes surrounding the wound area with an increased migratory phenotype through activation of ß2-AR signaling. Our findings suggest a biochemical mechanism underlying the function of MSCs in wound re-epithelization, which provides a reliable theoretical basis for the wide application of MSCs in the treatment of chronic wounds.

Norepinephrine inhibits the cytotoxicity of NK92­MI cells via the ß2­adrenoceptor/cAMP/PKA/p­CREB signaling pathway.

Norepinephrine (NE) can regulate natural killer (NK) cell activity, but the mechanism remains unclear. In the present study the roles of adrenergic receptors (ARs) in inhibiting NK92­MI cells­mediated cytotoxicity by NE were investigated. To examine the effect of NE on NK92­MI cytotoxicity, a lactate dehydrogenase­release cytotoxicity assay was used to determine the cytotoxicity of NK92­MI cells against K562 cells. To evaluate the possible function of the α, ß1 and ß2 AR in mediating NE­induced effects, NK92­MI cells were pre­incubated with phenol­amine, CGP20712A and ICI118551 prior to stimulation by NE. To evaluate the role of cyclic adenosine monophosphate (cAMP)­protein kinase A (PKA) signaling pathway in the inhibitory effect on cytotoxicity of NK92­MI cell by NE, NK92­MI cells were pre­incubated with PKA inhibitor Rp­8­Br­cAMP prior to stimulation by NE. It was demonstrated that NE decreased cytotoxicity and downregulated the expression of perforin, granzyme B and interferon (IFN)­Î³ of NK92­MI cells in a dose­dependent manner. Blocking NE functional receptors by ARs antagonists, particularly of ß2 AR antagonist, suppressed the inhibitory effect of NE on cytotoxicity and expression of perforin, granzyme B, IFN­Î³ of NK92­MI cells significantly. Blockade of ß2 AR in NE treated NK92­MI cells resulted in a reduction of the expression of phosphorylated (p)­cAMP­responsive element­binding protein (CREB) and intracellular cAMP concentration. Inhibiting the activity of PKA by Rp­8­Br­cAMP in NE treated NK92­MI cells resulted in increased cytotoxicity. The results of the present study suggest that NE can inhibit cytotoxicity and expression of perforin, granzyme B, IFN­Î³ of NK92­MI cell mainly via the ß2­AR/cAMP/PKA/p­CREB signaling pathway.

Design, synthesis, and functional assessment of Cmpd-15 derivatives as negative allosteric modulators for the ß2-adrenergic receptor.

The ß2-adrenergic receptor (ß2AR), a G protein-coupled receptor, is an important therapeutic target. We recently described Cmpd-15, the first small molecule negative allosteric modulator (NAM) for the ß2AR. Herein we report in details the design, synthesis and structure-activity relationships (SAR) of seven Cmpd-15 derivatives. Furthermore, we provide in a dose-response paradigm, the details of the effects of these derivatives in modulating agonist-induced ß2AR activities (G-protein-mediated cAMP production and ß-arrestin recruitment to the receptor) as well as the binding affinity of an orthosteric agonist in radio-ligand competition binding assay. Our results show that some modifications, including removal of the formamide group in the para-formamido phenylalanine region and bromine in the meta-bromobenzyl methylbenzamide region caused dramatic reduction in the functional activity of Cmpd-15. These SAR results provide valuable insights into the mechanism of action of the NAM Cmpd-15 as well as the basis for future development of more potent and selective modulators for the ß2AR based on the chemical scaffold of Cmpd-15.

Blockade of Host ß2-Adrenergic Receptor Enhances Graft-versus-Tumor Effect through Modulating APCs.

Allogeneic hematopoietic cell transplantation is a potential curative therapy for hematologic malignancies. Host APCs are pivotal to the desired graft-versus-tumor (GVT) effect. Recent studies have shown that ß2-adrenergic receptor (ß2AR) signaling can have an important impact on immune cell function, including dendritic cells (DCs). In this article, we demonstrate that pretreatment of host mice with a ß2AR blocker significantly increases the GVT effect of donor CD8+ T cells by decreasing tumor burden without increasing graft-versus-host disease. ß2AR-deficient host mice have significantly increased effector memory and central memory CD8+ T cells and improved reconstitution of T cells, including CD4+Foxp3+ regulatory T cells. Notably, ß2AR deficiency induces increased CD11c+ DC development. Also, ß2AR-deficient bone marrow-derived DCs induce higher CD8+ T cell proliferation and improved tumor killing in vitro. Metabolic profiling shows that ß2AR deficiency renders DCs more immunogenic through upregulation of mTOR activity and reduction of STAT3 phosphorylation. Altogether, these findings demonstrate an important role for host ß2AR signaling in suppressing T cell reconstitution and GVT activity.