Ligand‐Biased and Probe‐Dependent Modulation of Chemokine Receptor CXCR3 Signaling by Negative Allosteric Modulators

Over the last decade, functional selectivity (or ligand bias) has evolved from being a peculiar phenomenon to being recognized as an essential feature of synthetic ligands that target G protein‐coupled receptors (GPCRs). The CXC chemokine receptor 3 (CXCR3) is an outstanding platform to study various aspects of biased signaling, because nature itself uses functional selectivity to manipulate receptor signaling. At the same time, CXCR3 is an attractive therapeutic target in the treatment of autoimmune diseases and cancer. Herein we report the discovery of an 8‐azaquinazolinone derivative (N‐{1‐[3‐(4‐ethoxyphenyl)‐4‐oxo‐3,4‐dihydropyrido[2,3‐d]pyrimidin‐2‐yl]ethyl}‐4‐(4‐fluorobutoxy)‐N‐[(1‐methylpiperidin‐4‐yl)methyl]butanamide, 1 b ) that can inhibit CXC chemokine 11 (CXCL11)‐dependent G protein activation over β‐arrestin recruitment with 187‐fold selectivity. This compound also demonstrates probe‐dependent activity, that is, it inhibits CXCL11‐ over CXCL10‐mediated G protein activation with 12‐fold selectivity. Together with a previously reported biased negative allosteric modulator from our group, the present study provides additional information on the molecular requirements for allosteric modulation of CXCR3.     

Discovery of Biased Mu-Opioid Receptor Agonists for the Treatment of Pain

G protein-biased mu-opioid receptor (MOR) agonists have been developed as promising new potent analgesic drugs with fewer adverse side effects than standard MOR agonists. PZM21 represents a unique chemotype unrelated to known opioids, which makes it a desirable lead for modification to find analgesics with new chemical entities. In the present study, we synthesized and tested novel PZM21 derivatives as potent biased MOR agonists by introducing a benzodioxolane group to replace the hydroxybenzene of PZM21. The new compounds displayed more potent analgesic activities in vivo and greater bias toward G protein signaling in vitro than did PZM21. These results suggest that the benzodioxolane group is essential for the maintenance of bias. Compounds 7 i ((S)-1-(3-(benzo[d][1,3]dioxol-4-yl)-2-(dimethylamino)propyl)-3-phenethylurea) and 7 j ((S)-1-(3-(benzo[d][1,3]dioxol-4-yl)-2-(dimethylamino)propyl)-3-benzylurea) could serve as new leads for further modifications to find novel biased MOR agonists with greater G protein signaling potency and less β-arrestin-2 recruitment.     

Characterization of Potency of the P2Y13 Receptor Agonists: A Meta-Analysis

P2Y13 is an ADP-stimulated G-protein coupled receptor implicated in many physiological processes, including neurotransmission, metabolism, pain, and bone homeostasis. Quantitative understanding of P2Y13 activation dynamics is important for translational studies. We systematically identified PubMed annotated studies that characterized concentration-dependence of P2Y13 responses to natural and synthetic agonists. Since the comparison of the efficacy (maximum response) is difficult for studies performed in different systems, we normalized the data and conducted a meta-analysis of EC₅₀ (concentration at half-maximum response) and Hill coefficient (slope) of P2Y13-mediated responses to different agonists. For signaling events induced by heterologously expressed P2Y13, EC₅₀ of ADP-like agonists was 17.2 nM (95% CI: 7.7–38.5), with Hills coefficient of 4.4 (95% CI: 3.3–5.4), while ATP-like agonists had EC₅₀ of 0.45 μM (95% CI: 0.06–3.15). For functional responses of endogenously expressed P2Y13, EC₅₀ of ADP-like agonists was 1.76 μM (95% CI: 0.3–10.06). The EC₅₀ of ADP-like agonists was lower for the brain P2Y13 than the blood P2Y13. ADP-like agonists were also more potent for human P2Y13 compared to rodent P2Y13. Thus, P2Y13 appears to be the most ADP-sensitive receptor characterized to date. The detailed understanding of tissue- and species-related differences in the P2Y13 response to ADP will improve the selectivity and specificity of future pharmacological compounds.     

The Amino Acid Transporter Mct10/Tat1 Is Important to Maintain the TSH Receptor at Its Canonical Basolateral Localization and Assures Regular Turnover of Thyroid Follicle Cells in Male Mice

Cathepsin K-mediated thyroglobulin proteolysis contributes to thyroid hormone (TH) liberation, while TH transporters like Mct8 and Mct10 ensure TH release from thyroid follicles into the blood circulation. Thus, thyroid stimulating hormone (TSH) released upon TH demand binds to TSH receptors of thyrocytes, where it triggers Gα_q-mediated short-term effects like cathepsin-mediated thyroglobulin utilization, and Gα_s-mediated long-term signaling responses like thyroglobulin biosynthesis and thyrocyte proliferation. As reported recently, mice lacking Mct8 and Mct10 on a cathepsin K-deficient background exhibit excessive thyroglobulin proteolysis hinting towards altered TSH receptor signaling. Indeed, a combination of canonical basolateral and non-canonical vesicular TSH receptor localization was observed in Ctsk^−/−/Mct8^−/y/Mct10^−/− mice, which implies prolonged Gα_s-mediated signaling since endo-lysosomal down-regulation of the TSH receptor was not detected. Inspection of single knockout genotypes revealed that the TSH receptor localizes basolaterally in Ctsk^−/− and Mct8^−/y mice, whereas its localization is restricted to vesicles in Mct10^−/− thyrocytes. The additional lack of cathepsin K reverses this effect, because Ctsk^−/−/Mct10^−/− mice display TSH receptors basolaterally, thereby indicating that cathepsin K and Mct10 contribute to TSH receptor homeostasis by maintaining its canonical localization in thyrocytes. Moreover, Mct10^−/− mice displayed reduced numbers of dead thyrocytes, while their thyroid gland morphology was comparable to wild-type controls. In contrast, Mct8^−/y, Mct8^−/y/Mct10^−/−, and Ctsk^−/−/Mct8^−/y/Mct10^−/− mice showed enlarged thyroid follicles and increased cell death, indicating that Mct8 deficiency results in altered thyroid morphology. We conclude that vesicular TSH receptor localization does not result in different thyroid tissue architecture; however, Mct10 deficiency possibly modulates TSH receptor signaling for regulating thyrocyte survival.