Characterisation of endogenous A2A and A2B receptor-mediated cyclic AMP responses in HEK 293 cells using the GloSensor™ biosensor: evidence for an allosteric mechanism of action for the A2B-selective antagonist PSB 603

Endogenous adenosine A2B receptors (A2BAR) mediate cAMP accumulation in HEK 293 cells. Here we have used a biosensor to investigate the mechanism of action of the A2BAR antagonist PSB 603 in HEK 293 cells. The A2A agonist CGS 21680 elicited a small response in these cells (circa 20% of that obtained with NECA), suggesting that they also contain a small population of A2A receptors. The responses to NECA and adenosine were antagonised by PSB 603, but not by the selective A2AAR antagonist SCH 58261. In contrast, CGS 21680 responses were not antagonised by high concentrations of PSB 603, but were sensitive to inhibition by SCH 58261. Analysis of the effect of increasing concentrations of PSB 603 on the response to NECA indicated a non-competitive mode of action yielding a marked reduction in the NECA EMAX with no significant effect on EC50 values. Kinetics analysis of the effect of PSB 603 on the A2BAR-mediated NECA responses confirmed a saturable effect that was consistent with an allosteric mode of antagonism. The possibility that PSB 603 acts as a negative allosteric modulator of A2BAR suggests new approaches to the development of therapeutic agents to treat conditions where adenosine levels are high

Targeting Adenosine Receptors for the Treatment of Cardiac Fibrosis

Adenosine is a ubiquitous molecule with key regulatory and cytoprotective mechanisms at times of metabolic imbalance in the body. Among a plethora of physiological actions, adenosine has an important role in attenuating ischaemia-reperfusion injury and modulating the ensuing fibrosis and tissue remodeling following myocardial damage. Adenosine exerts these actions through interaction with four adenosine G protein-coupled receptors expressed in the heart. The adenosine A2B receptor (A2BAR) is the most abundant adenosine receptor (AR) in cardiac fibroblasts and is largely responsible for the influence of adenosine on cardiac fibrosis. In vitro and in vivo studies demonstrate that acute A2BAR stimulation can decrease fibrosis through the inhibition of fibroblast proliferation and reduction in collagen synthesis. However, in contrast, there is also evidence that chronic A2BAR antagonism reduces tissue fibrosis. This review explores the opposing pro- and anti-fibrotic activity attributed to the activation of cardiac ARs and investigates the therapeutic potential of targeting ARs for the treatment of cardiac fibrosis

Structural Basis for Binding of Allosteric Drug Leads in the Adenosine A1 Receptor

This work is licensed under a Creative Commons Attribution 4.0 International License.Despite intense interest in designing positive allosteric modulators (PAMs) as selective drugs of the adenosine A1 receptor (A1AR), structural binding modes of the receptor PAMs remain unknown. Using the first X-ray structure of the A1AR, we have performed all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) technique to determine binding modes of the A1AR allosteric drug leads. Two prototypical PAMs, PD81723 and VCP171, were selected. Each PAM was initially placed at least 20 Å away from the receptor. Extensive GaMD simulations using the AMBER and NAMD simulation packages at different acceleration levels captured spontaneous binding of PAMs to the A1AR. The simulations allowed us to identify low-energy binding modes of the PAMs at an allosteric site formed by the receptor extracellular loop 2 (ECL2), which are highly consistent with mutagenesis experimental data. Furthermore, the PAMs stabilized agonist binding in the receptor. In the absence of PAMs at the ECL2 allosteric site, the agonist sampled a significantly larger conformational space and even dissociated from the A1AR alone. In summary, the GaMD simulations elucidated structural binding modes of the PAMs and provided important insights into allostery in the A1AR, which will greatly facilitate the receptor structure-based drug design.Extreme Science and Engineering Discovery Environment award TG-MCB170129National Energy Research Scientific Computing Center project M2874American Heart Association (Award 17SDG33370094)College of Liberal Arts and Sciences at the University of KansasNHMRC Senior Principal Research FellowAustralian Heart Foundation Future Leader Fello

Negative cooperativity across 1-adrenoceptor homodimers provides insights into the nature of the secondary low-affinity CGP 12177 1-adrenoceptor binding conformation

At the β1-adrenoceptor, CGP 12177 potently antagonizes agonist responses at the primary high-affinity catecholamine conformation while also exerting agonist effects of its own through a secondary low-affinity conformation. A recent mutagenesis study identified transmembrane region (TM)4 of the β1-adrenoceptor as key for this low-affinity conformation. Others suggested that TM4 has a role in β1-adrenoceptor oligomerization. Here, assessment of the dissociation rate of a fluorescent analog of CGP 12177 [bordifluoropyrromethane-tetramethylrhodamine-(±)CGP 12177 (BODIPY-TMR-CGP)] at the human β1-adrenoceptor expressed in Chinese hamster ovary cells revealed negative cooperative interactions between 2 distinct β1-adrenoceptor conformations. The dissociation rate of 3 nM BODIPY-TMR-CGP was 0.09 ± 0.01 min−1 in the absence of competitor ligands, and this was enhanced 2.2- and 2.1-fold in the presence of 1 µM CGP 12177 and 1 µM propranolol, respectively. These effects on the BODIPY-TMR-CGP dissociation rate were markedly enhanced in β1-adrenoceptor homodimers constrained by bimolecular fluorescence complementation (9.8- and 9.9-fold for 1 µM CGP 12177 and 1 µM propranolol, respectively) and abolished in β1-adrenoceptors containing TM4 mutations vital for the second conformation pharmacology. This study suggests that negative cooperativity across a β1-adrenoceptor homodimer may be responsible for generating the low-affinity pharmacology of the secondary β1-adrenoceptor conformatio

Sustainability in the pharmacy and pharmaceutical science curriculum

BACKGROUND Sustainability is fast becoming a significant economic factor for the pharmaceutical industry and therefore important for future professionals in the sector. However, sustainability often plays only a marginal role in pharmaceutical science and pharmacy degrees. Sustainability in the pharmaceutical sector encompasses a wide range of environmental and socio-economic issues that require contributions from multiple science disciplines, complicating its introduction into curricula.   PLAN This curriculum initiative aims to identify suitable frameworks for embedding sustainability concepts and practices into the pharmaceutical science and pharmacy degrees at Monash University. We report here on preliminary insights from curriculum mapping, an analysis of literature frameworks and an evaluation of ‘pilot’ teaching activities addressing sustainability. Student perspectives will be investigated through assessment data and survey results (human ethics approval pending). ACTION AND EVALUATION Sustainability-focused teaching activities have recently been incorporated into the Monash University pharmaceutical science and pharmacy degrees. These activities draw on a range of frameworks and standards, including the UN sustainable development goals (United Nations, n.d.) and the ESG (environment, social, governance) framework. The AMEE consensus statement learning outcomes (Shaw et al., 2021) have been used for mapping sustainability content in the Monash Pharmacy degree. REFERENCES Shaw, E., Walpole, S., McLean, M., Alvarez-Nieto, C., Barna, S. et al. (2021) AMEE Consensus Statement: Planetary health and education for sustainable healthcare. Medical Teacher 43(3), 272-286, DOI: 10.1080/0142159X.2020.1860207 United Nations (n.d.), Sustainable development goals. Retrieved May 22, 2023 from https://sdgs.un.org/goal

Small molecule allosteric modulation of the adenosine A1 receptor

G protein-coupled receptors (GPCRs) represent the target for approximately a third of FDA-approved small molecule drugs. The adenosine A1 receptor (A1R), one of four adenosine GPCR subtypes, has important (patho)physiological roles in humans. A1R has well-established roles in the regulation of the cardiovascular and nervous systems, where it has been identified as a potential therapeutic target for a number of conditions, including cardiac ischemia-reperfusion injury, cognition, epilepsy, and neuropathic pain. A1R small molecule drugs, typically orthosteric ligands, have undergone clinical trials. To date, none have progressed into the clinic, predominantly due to dose-limiting unwanted effects. The development of A1R allosteric modulators that target a topographically distinct binding site represent a promising approach to overcome current limitations. Pharmacological parameters of allosteric ligands, including affinity, efficacy and cooperativity, can be optimized to regulate A1R activity with high subtype, spatial and temporal selectivity. This review aims to offer insights into the A1R as a potential therapeutic target and highlight recent advances in the structural understanding of A1R allosteric modulation

Ligand-Independent Adenosine A 2B Receptor Constitutive Activity as a Promoter of Prostate Cancer Cell Proliferation s

ABSTRACT Aberrant ligand-independent G protein-coupled receptor constitutive activity has been implicated in the pathophysiology of a number of cancers. The adenosine A 2B receptor (A 2B AR) is dynamically upregulated under pathologic conditions associated with a hypoxic microenvironment, including solid tumors. This, in turn, may amplify ligand-independent A 2B AR signal transduction. The contribution of A 2B AR constitutive activity to disease progression is currently unknown yet of fundamental importance, as the preferred therapeutic modality for drugs designed to reduce A 2B AR constitutive activity would be inverse agonism as opposed to neutral antagonism. The current study investigated A 2B AR constitutive activity in a heterologous expression system and a native 22Rv1 human prostate cancer cell line exposed to hypoxic conditions (2% O 2 ). (4-chlorophenyl)piperazide-1-sulfonyl)phenyl)-1-propylxanthine), mediated a concentration-dependent decrease in baseline cAMP levels in both cellular systems. Proliferation of multiple prostate cancer cell lines was also attenuated in the presence of PSB-603. Importantly, both the decrease in baseline cAMP accumulation and the reduction of proliferation were not influenced by the addition of adenosine deaminase, demonstrating that these effects are not dependent on stimulation of A 2B ARs by the endogenous agonist adenosine. Our study is the first to reveal that wild-type human A 2B ARs have high constitutive activity in both model and native cells. Furthermore, our findings demonstrate that this ligand-independent A 2B AR constitutive activity is sufficient to promote prostate cancer cell proliferation in vitro. More broadly, A 2B AR constitutive activity may have wider, currently unappreciated implications in pathologic conditions associated with a hypoxic microenvironment

Spatial Guilds in the Serengeti Food Web Revealed by a Bayesian Group Model

Food webs, networks of feeding relationships among organisms, provide fundamental insights into mechanisms that determine ecosystem stability and persistence. Despite long-standing interest in the compartmental structure of food webs, past network analyses of food webs have been constrained by a standard definition of compartments, or modules, that requires many links within compartments and few links between them. Empirical analyses have been further limited by low-resolution data for primary producers. In this paper, we present a Bayesian computational method for identifying group structure in food webs using a flexible definition of a group that can describe both functional roles and standard compartments. The Serengeti ecosystem provides an opportunity to examine structure in a newly compiled food web that includes species-level resolution among plants, allowing us to address whether groups in the food web correspond to tightly-connected compartments or functional groups, and whether network structure reflects spatial or trophic organization, or a combination of the two. We have compiled the major mammalian and plant components of the Serengeti food web from published literature, and we infer its group structure using our method. We find that network structure corresponds to spatially distinct plant groups coupled at higher trophic levels by groups of herbivores, which are in turn coupled by carnivore groups. Thus the group structure of the Serengeti web represents a mixture of trophic guild structure and spatial patterns, in contrast to the standard compartments typically identified in ecological networks. From data consisting only of nodes and links, the group structure that emerges supports recent ideas on spatial coupling and energy channels in ecosystems that have been proposed as important for persistence.Comment: 28 pages, 6 figures (+ 3 supporting), 2 tables (+ 4 supporting

Assessing Vaccination Sentiments with Online Social Media: Implications for Infectious Disease Dynamics and Control

There is great interest in the dynamics of health behaviors in social networks and how they affect collective public health outcomes, but measuring population health behaviors over time and space requires substantial resources. Here, we use publicly available data from 101,853 users of online social media collected over a time period of almost six months to measure the spatio-temporal sentiment towards a new vaccine. We validated our approach by identifying a strong correlation between sentiments expressed online and CDC- estimated vaccination rates by region. Analysis of the network of opinionated users showed that information flows more often between users who share the same sentiments - and less often between users who do not share the same sentiments - than expected by chance alone. We also found that most communities are dominated by either positive or negative sentiments towards the novel vaccine. Simulations of infectious disease transmission show that if clusters of negative vaccine sentiments lead to clusters of unprotected individuals, the likelihood of disease outbreaks are greatly increased. Online social media provide unprecedented access to data allowing for inexpensive and efficient tools to identify target areas for intervention efforts and to evaluate their effectiveness.Comment: Accepted for publication in PLoS Computational Biolog