Isoquinoline small molecule ligands are agonists and probe-dependent allosteric modulators of the glucagon subfamily of GPCRs.

Class B1 G protein-coupled receptors (GPCRs) are peptide hormone receptors and well validated therapeutic targets, however development of non-peptide drugs targeting this class of receptors is challenging. Recently, a series of isoquinoline-based derivates were reported in the patent literature as allosteric ligands for the glucagon receptor subfamily, and two compounds, LSN3451217 and LSN3556672, were used to facilitate structural studies with the glucagon-like peptide-1 receptor (GLP-1R) and glucose dependent insulinotropic peptide receptor (GIPR) bound to orthosteric agonists. Here we pharmacologically characterized stereoisomers of LSN3451217 and LSN3556672, across the class B1 GPCR family. This revealed LSN3556672 isomers are agonists for the glucagon receptor (GCGR), GLP-1R, GIPR and the calcitonin receptor (CTR), albeit the degree of agonism varied at each receptor. In contrast, LSN3451217 isomers were more selective agonists at the GLP-1R, with lower potency at the GCGR and CTR and no activity at the GIPR. All compounds also modulated peptide-mediated cyclic adenosine monophosphate (cAMP) signaling at the GIPR, and to a lesser extent the GLP-1R, in a probe-dependent manner, with modest positive allosteric modulation observed for some peptides, and negligible effects observed with other peptides. In contrast neutral or weak negative/positive allosteric modulation was observed with peptides assessed at the GCGR and CTR. This study expands our knowledge on class B1 GPCR allosteric modulation and may have implications for future structural and drug discovery efforts targeting the class B1 GPCR subfamily.

Experimental verification of fiber coupling characteristics for FSO downlinks from the International Space Station.

Free-space optical (FSO) systems are compulsory to realize high capacity and interference-free communication links from low-Earth orbit (LEO) satellite constellations as well as spacecraft and space stations to the Earth. To be integrated with high-capacity ground networks, the collected portion of the incident beam should be coupled into an optical fiber. To accurately evaluate the signal-to-noise ratio (SNR) and bit-error rate (BER) performance metrics, the probability density function (PDF) of fiber coupling efficiency (CE) must be determined. Previous studies have experimentally verified the CE PDF for a single-mode fiber, however, there is no such investigation for the CE PDF of a multi-mode fiber (MMF) in a LEO-to-ground FSO downlink. In this paper, for the first time, the CE PDF for a 200-µm MMF is experimentally investigated using data from an FSO downlink from the Small Optical Link for International Space Station (SOLISS) terminal to a 40-cm sub-aperture optical ground station (OGS) supported by a fine-tracking system. An average CE of 5.45 dB was also achieved given that the alignment between SOLISS and OGS was not optimal. In addition, using the angle-of-arrival (AoA) and received power data, the statistical characteristics such as channel coherence time, power spectral density, spectrogram, and PDFs of AoA, beam misalignments, and atmospheric turbulence-induced fluctuations are revealed and compared with the state-of-the-art theoretical background.

Adenosine receptor signalling in Alzheimer's disease.

Alzheimer's disease (AD) is the most common dementia in the elderly and its increasing prevalence presents treatment challenges. Despite a better understanding of the disease, the current mainstay of treatment cannot modify pathogenesis or effectively address the associated cognitive and memory deficits. Emerging evidence suggests adenosine G protein-coupled receptors (GPCRs) are promising therapeutic targets for Alzheimer's disease. The adenosine A1 and A2A receptors are expressed in the human brain and have a proposed involvement in the pathogenesis of dementia. Targeting these receptors preclinically can mitigate pathogenic ß-amyloid and tau neurotoxicity whilst improving cognition and memory. In this review, we provide an accessible summary of the literature on Alzheimer's disease and the therapeutic potential of A1 and A2A receptors. Although there are no available medicines targeting these receptors approved for treating dementia, we provide insights into some novel strategies, including allosterism and the targeting of oligomers, which may increase drug discovery success and enhance the therapeutic response.

Development of Covalent, Clickable Probes for Adenosine A1 and A3 Receptors.

Adenosine receptors are attractive therapeutic targets for multiple conditions, including ischemia-reperfusion injury and neuropathic pain. Adenosine receptor drug discovery efforts would be facilitated by the development of appropriate tools to assist in target validation and direct receptor visualization in different native environments. We report the development of the first bifunctional (chemoreactive and clickable) ligands for the adenosine A1 receptor (A1R) and adenosine A3 receptor (A3R) based on an orthosteric antagonist xanthine-based scaffold and on an existing structure-activity relationship. Bifunctional ligands were functional antagonists with nanomolar affinity and irreversible binding at the A1R and A3R. In-depth pharmacological profiling of these bifunctional ligands showed moderate selectivity over A2A and A2B adenosine receptors. Once bound to the receptor, ligands were successfully "clicked" with a cyanine-5 fluorophore containing the complementary "click" partner, enabling receptor detection. These bifunctional ligands are expected to aid in the understanding of A1R and A3R localization and trafficking in native cells and living systems.

Development and Application of Subtype-Selective Fluorescent Antagonists for the Study of the Human Adenosine A1 Receptor in Living Cells.

The adenosine A1 receptor (A1AR) is a G-protein-coupled receptor (GPCR) that provides important therapeutic opportunities for a number of conditions including congestive heart failure, tachycardia, and neuropathic pain. The development of A1AR-selective fluorescent ligands will enhance our understanding of the subcellular mechanisms underlying A1AR pharmacology facilitating the development of more efficacious and selective therapies. Herein, we report the design, synthesis, and application of a novel series of A1AR-selective fluorescent probes based on 8-functionalized bicyclo[2.2.2]octylxanthine and 3-functionalized 8-(adamant-1-yl) xanthine scaffolds. These fluorescent conjugates allowed quantification of kinetic and equilibrium ligand binding parameters using NanoBRET and visualization of specific receptor distribution patterns in living cells by confocal imaging and total internal reflection fluorescence (TIRF) microscopy. As such, the novel A1AR-selective fluorescent antagonists described herein can be applied in conjunction with a series of fluorescence-based techniques to foster understanding of A1AR molecular pharmacology and signaling in living cells.

Visualising functional 5-HT3 receptors containing A and C subunits at or near the cell surface.

Five different subunits of the human serotonin 3 (5-hydroxytrptamine 3; 5-HT3) receptor exist and these are present in both central and peripheral systems. Different subunits alter the efficacy of 5-HT3 receptor antagonists used to treat diarrhoea predominant-irritable bowel syndrome, chemotherapy induced nausea and vomiting and depression. Cell surface arrangement of 5-HT3 receptor complexes and the contribution of C, D and E subunits to receptor function is poorly understood. Here, we examine interactions of A and C subunits using 5-HT3 receptor subunits containing fluorescent protein inserts between the 3rd and 4th transmembrane spanning region. HEK293T cells that do not normally express 5-HT3 receptor subunits, were transiently transfected with A or C or both subunits. Patch clamp experiments show that cells transfected with either fluorescent protein tagged A or A and C subunits generate whole cell currents in response to 5-HT. These findings correlate with the apparent distribution of fluorescent protein tagged A and C subunits at or near cell surfaces detected using TIRF microscopy. In co-transfected cells, the A and C subunits are associated forming AC heteromer complexes at or near the cell surface and a proportion can also form A or C homomers. In conclusion, it is likely that both A homomers and AC heteromers contribute to whole cell currents in response to 5-HT with minimal contribution from C homomers.

Identification of monellin as the first naturally derived proteinaceous allosteric agonist of metabotropic glutamate receptor 5.

Allosteric modulators bind sites distinct from orthosteric ligands, allowing for improved spatiotemporal control of receptors and greater subtype selectivity. However, we recently showed that allosteric ligands previously classified as selective for select Class C G protein-coupled receptors (GPCRs) had unappreciated activity at other off-target receptors, in some cases higher affinity, within the class. Here, we extended our investigation of off-target activity of "selective" allosteric ligands for the sweet taste receptor. Using metabotropic glutamate receptor 5 (mGlu5 ) as a representative of Class C GPCR, we assessed the sweet protein, monellin and the small-molecule artificial sweetener, NHDC. We found that monellin, but not NHDC, is an agonist for mGlu5 . Radioligand binding and functional assays performed in cells expressing N-terminally truncated mGlu5 demonstrated that monellin agonism was not mediated via the "common" allosteric binding site in the transmembrane domain but required the presence of the large extracellular N-terminal domain of mGlu5 . Monellin displayed neutral functional cooperativity with orthosteric ligands. However, monellin positively modulated the mGlu5 PAM-agonist, VU0424465, activity in intracellular calcium assays, but the interaction was neutral in inositol phosphate accumulation assays. Furthermore, monellin mGlu5 agonism was positively modulated by the mGlu5 pure PAM, VU0360172. Taken together, these data indicate that monellin is an allosteric agonist for mGlu5 , binding to an allosteric binding site on the N-terminus that is functionally linked to the common Class C GPCR allosteric site in a biased manner. This is the first evidence of a naturally derived proteinaceous allosteric ligand for the mGlu receptor family.

Biased agonism and allosteric modulation of metabotropic glutamate receptor 5.

Metabotropic glutamate receptors belong to class C G-protein-coupled receptors and consist of eight subtypes that are ubiquitously expressed throughout the central nervous system. In recent years, the metabotropic glutamate receptor subtype 5 (mGlu5) has emerged as a promising target for a broad range of psychiatric and neurological disorders. Drug discovery programs targetting mGlu5 are primarily focused on development of allosteric modulators that interact with sites distinct from the endogenous agonist glutamate. Significant efforts have seen mGlu5 allosteric modulators progress into clinical trials; however, recent failures due to lack of efficacy or adverse effects indicate a need for a better understanding of the functional consequences of mGlu5 allosteric modulation. Biased agonism is an interrelated phenomenon to allosterism, describing how different ligands acting through the same receptor can differentially influence signaling to distinct transducers and pathways. Emerging evidence demonstrates that allosteric modulators can induce biased pharmacology at the level of intrinsic agonism as well as through differential modulation of orthosteric agonist-signaling pathways. Here, we present key considerations in the discovery and development of mGlu5 allosteric modulators and the opportunities and pitfalls offered by biased agonism and modulation.

Secret key rates for intensity-modulated dual-threshold detection key distribution under individual beam splitting attacks.

We investigate the secret key rates for the recently proposed intensity-modulated dual-threshold key distribution [T. Ikuta and K. Inoue, New J. Phys. 18 (2016)] under beam splitting attacks. We show that previous assumptions on an eavesdropper that performs hard decision measurements on the channel, overestimates the secret key rate. We discuss the impact of an eavesdropper that can measure full soft information and give the secret key rates under forward and reverse reconciliation. Further, we perform simulations for different system assumptions and show the optimal modulation depths for these systems. We also outline an attack on this protocol based on photon counting that prohibits secret key generation.