The European Research Network on Signal Transduction (ERNEST): Toward a Multidimensional Holistic Understanding of G Protein-Coupled Receptor Signaling.

G protein-coupled receptors (GPCRs) are intensively studied due to their therapeutic potential as drug targets. Members of this large family of transmembrane receptor proteins mediate signal transduction in diverse cell types and play key roles in human physiology and health. In 2013 the research consortium GLISTEN (COST Action CM1207) was founded with the goal of harnessing the substantial growth in knowledge of GPCR structure and dynamics to push forward the development of molecular modulators of GPCR function. The success of GLISTEN, coupled with new findings and paradigm shifts in the field, led in 2019 to the creation of a related consortium called ERNEST (COST Action CA18133). ERNEST broadens focus to entire signaling cascades, based on emerging ideas of how complexity and specificity in signal transduction are not determined by receptor-ligand interactions alone. A holistic approach that unites the diverse data and perspectives of the research community into a single multidimensional map holds great promise for improved drug design and therapeutic targeting.

Enteroendocrine K Cells Exert Complementary Effects to Control Bone Quality and Mass in Mice.

The involvement of a gut-bone axis in controlling bone physiology has been long suspected, although the exact mechanisms are unclear. We explored whether glucose-dependent insulinotropic polypeptide (GIP)-producing enteroendocrine K cells were involved in this process. The bone phenotype of transgenic mouse models lacking GIP secretion (GIP-GFP-KI) or enteroendocrine K cells (GIP-DT) was investigated. Mice deficient in GIP secretion exhibited lower bone strength, trabecular bone mass, trabecular number, and cortical thickness, notably due to higher bone resorption. Alterations of microstructure, modifications of bone compositional parameters, represented by lower collagen cross-linking, were also apparent. None of these alterations were observed in GIP-DT mice lacking enteroendocrine K cells, suggesting that another K-cell secretory product acts to counteract GIP action. To assess this, stable analogues of the known K-cell peptide hormones, xenin and GIP, were administered to mature NIH Swiss male mice. Both were capable of modulating bone strength mostly by altering bone microstructure, bone gene expression, and bone compositional parameters. However, the two molecules exhibited opposite actions on bone physiology, with evidence that xenin effects are mediated indirectly, possibly via neural networks. Our data highlight a previously unknown interaction between GIP and xenin, which both moderate gut-bone connectivity. © 2020 American Society for Bone and Mineral Research.

Structure-based discovery of novel US28 small molecule ligands with different modes of action.

The human cytomegalovirus-encoded G protein-coupled receptor US28 is a constitutively active receptor, which can recognize various chemokines. Despite the recent determination of its 2.9 Å crystal structure, potent and US28-specific tool compounds are still scarce. Here, we used structural information from a refined US28:VUF2274 complex for virtual screening of >12 million commercially available small molecule compounds. Using a combined receptor- and ligand-based approach, we tested 98 of the top 0.1% ranked compounds, revealing novel chemotypes as compared to the ~1.45 million known ligands in the ChEMBL database. Two compounds were confirmed as agonist and inverse agonist, respectively, in both IP accumulation and Ca2+ mobilization assays. The screening setup presented in this work is computationally inexpensive and therefore particularly useful in an academic setting as it enables simultaneous testing in binding as well as in different functional assays and/or species without actual chemical synthesis.

Pharmacokinetics of high-dose intravenous melatonin in humans.

This crossover study investigated the pharmacokinetics and adverse effects of high-dose intravenous melatonin. Volunteers participated in 3 identical study sessions, receiving an intravenous bolus of 10 mg melatonin, 100 mg melatonin, and placebo. Blood samples were collected at baseline and 0, 60, 120, 180, 240, 300, 360, and 420 minutes after the bolus. Quantitative determination of plasma melatonin concentrations was performed using a radioimmunoassay technique. Pharmacokinetic parameters were estimated by a compartmental pharmacokinetic analysis. Adverse effects included assessments of sedation and registration of other symptoms. Sedation, evaluated as simple reaction times, was measured at baseline and 120, 180, 300, and 420 minutes after the bolus. Twelve male volunteers completed the study. Median (IQR) Cmax after the bolus injections of 10 mg and 100 mg of melatonin were 221,500.0 (185,637.5-326,175.0) pg/mL and 1,251,500.0 (864,375.0-1,770,500.0) pg/mL, respectively; mean (SD) t1/2 was 42.3 (5.6) minutes and 46.2 (6.2) minutes; mean (SD) Vd was 1.6 (0.9) L/kg and 2.0 (0.8) L/kg; mean (SD) CL was 0.0253 (0.0096) L/min · kg and 0.0300 (0.0120) L/min · kg; and median (IQR) AUC0- ∞ , 8,997,633.0 (6,071,696.2-11,602,811.9) pg · min/mL and 54,685,979.4 (36,028,638.6-105,779,612.0) pg · min/mL. High-dose intravenous melatonin did not induce sedation, evaluated as simple reaction times. No adverse effects were reported in the study.

High content screening for G protein-coupled receptors using cell-based protein translocation assays.

G protein-coupled receptors (GPCRs) have been one of the most productive classes of drug targets for several decades, and new technologies for GPCR-based discovery promise to keep this field active for years to come. While molecular screens for GPCR receptor agonist- and antagonist-based drugs will continue to be valuable discovery tools, the most exciting developments in the field involve cell-based assays for GPCR function. Some cell-based discovery strategies, such as the use of beta-arrestin as a surrogate marker for GPCR function, have already been reduced to practice, and have been used as valuable discovery tools for several years. The application of high content cell-based screening to GPCR discovery has opened up additional possibilities, such as direct tracking of GPCRs, G proteins and other signaling pathway components using intracellular translocation assays. These assays provide the capability to probe GPCR function at the cellular level with better resolution than has previously been possible, and offer practical strategies for more definitive selectivity evaluation and counter-screening in the early stages of drug discovery. The potential of cell-based translocation assays for GPCR discovery is described, and proof-of-concept data from a pilot screen with a CXCR4 assay are presented. This chemokine receptor is a highly relevant drug target which plays an important role in the pathogenesis of inflammatory disease and also has been shown to be a co-receptor for entry of HIV into cells as well as to play a role in metastasis of certain cancer cells.

The CXC chemokine receptor encoded by herpesvirus saimiri, ECRF3, shows ligand-regulated signaling through Gi, Gq, and G12/13 proteins but constitutive signaling only through Gi and G12/13 proteins.

Open reading frame 74 (ORF74) of many gamma(2)-herpesviruses encodes a CXC chemokine receptor. The molecular pharmacological profile of ORF74 from herpesvirus saimiri, ECRF3, is characterized here and compared with that of the well known ORF74 from human herpesvirus 8 (HHV8). The ECRF3 receptor bound the so-called ELR (Glu-Leu-Arg) CXC chemokines (125)I-CXCL1/GRO alpha, (125)I-CXCL6/GCP-2, and (125)I-CXCL8/interleukin-8 with high affinity; but in contrast to ORF74 from HHV8, it did not bind the non-ELR CXC chemokine (125)I-CXCL10/IP10. Interestingly, the B(max) value for CXCL6/GCP-2 was 3-fold higher than the capacity for maximal binding of CXCL1/GRO alpha to ECRF3 and 85-fold higher than that of CXCL8/interleukin-8, despite similar affinities. Like ORF74 from HHV8, ECRF3 activated a broad range of pathways (G(q), G(i), and G(12/13) as well as the cAMP response element-binding protein, NF-kappa B, NFAT, and serum response element transcription factors) in a ligand-regulated manner, with CXCL6/GCP-2 being the most potent and efficacious agonist. ECRF3 signaled constitutively through G(i) and G(12/13), but surprisingly not through G(q). At the level of transcription factor activation, the serum response element was activated constitutively by ECRF3, whereas cAMP response element-binding protein, NFAT, and NF-kappa B were only ligand-regulated. The maximal signaling capacities were similar for the two receptors; however, the ligand-regulated signaling was responsible for the major part of the total ECRF3 signaling and only for a minor part of the total HHV8 ORF74 signaling. The activation pattern of ECRF3 with constitutive activation of some (but not all) of the employed pathways has not been seen before in endogenous or virus-encoded chemokine receptors. The results suggest that the unique ligand selectivity of ECRF3 among ORF74 receptors could reflect differences in the cellular tropism of the gamma(2)-herpesviruses.