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The G protein-coupled receptor (GPCR) US28 encoded by the human cytomegalovirus (HCMV) is associated with accelerated progression of glioblastomas, aggressive brain tumors with a generally poor prognosis. Here, we showed that US28 increased the malignancy of U251 glioblastoma cells by enhancing signaling mediated by sphingosine-1-phosphate (S1P), a bioactive lipid that stimulates oncogenic pathways in glioblastoma. US28 expression increased the abundance of the key components of the S1P signaling axis, including an enzyme that generates S1P [sphingosine kinase 1 (SK1)], an S1P receptor [S1P receptor 1 (S1P 1 )], and S1P itself. Enhanced S1P signaling promoted glioblastoma cell proliferation and survival by activating the kinases AKT and CHK1 and the transcriptional regulators cMYC and STAT3 and by increasing the abundance of cancerous inhibitor of PP2A (CIP2A), driving several feed-forward signaling loops. Inhibition of S1P signaling abrogated the proliferative and anti-apoptotic effects of US28. US28 also activated the S1P signaling axis in HCMV-infected cells. This study uncovers central roles for S1P and CIP2A in feed-forward signaling that contributes to the US28-mediated exacerbation of glioblastoma.

The therapeutic potential of ligands targeting disease-associated membrane proteins is predicted by ligand-receptor binding constants, which can be determined using NanoLuciferase (NanoLuc)-based bioluminescence resonance energy transfer (NanoBRET) methods. However, the broad applicability of these methods is hampered by the restricted availability of fluorescent probes. We describe the use of antibody fragments, like nanobodies, as universal building blocks for fluorescent probes for use in NanoBRET. Our nanobody-NanoBRET (NanoB 2 ) workflow starts with the generation of NanoLuc-tagged receptors and fluorescent nanobodies, enabling homogeneous, real-time monitoring of nanobody-receptor binding. Moreover, NanoB 2 facilitates the assessment of receptor binding of unlabeled ligands in competition binding experiments. The broad significance is illustrated by the successful application of NanoB 2 to different drug targets (e.g., multiple G protein-coupled receptors [GPCRs] and a receptor tyrosine kinase [RTK]) at distinct therapeutically relevant binding sites (i.e., extracellular and intracellular)., Competing Interests: R.H. and S.M.A. are employees of QVQ Holding B.V. R.H. and M.J.S. are inventors of patent WO2019151865A1 describing VUN100., (© 2023 The Authors.)

The airway epithelium represents the main barrier between inhaled air and the tissues of the respiratory tract and is therefore an important point of contact with xenobiotic substances into the human body. Several studies have recently shown that in vitro models of the airway grown at an air-liquid interface (ALI) can be particularly useful to obtain mechanistic information about the toxicity of chemical compounds. However, such methods are not very amenable to high throughput since the primary cells cannot be expanded indefinitely in culture to obtain a sustainable number of cells. Induced pluripotent stem cells (iPSCs) have become a popular option in the recent years for modelling the airways of the lung, but despite progress in the field, such models have so far not been assessed for their ability to metabolise xenobiotic compounds and how they compare to the primary bronchial airway model (pBAE). Here, we report a comparative analysis by TempoSeq (oligo-directed sequencing) of an iPSC-derived airway model (iBAE) with a primary bronchial airway model (pBAE). The iBAE and pBAE were differentiated at an ALI and then evaluated in a 5-compound screen with exposure to a sub-lethal concentration of each compound for 24 h. We found that despite lower expression of xenobiotic metabolism genes, the iBAE similarly predicted the toxic pathways when compared to the pBAE model. Our results show that iPSC airway models at ALI show promise for inhalation toxicity assessments with further development., (© 2022. The Author(s).)

The histamine H 1 receptor (H 1 R) is a G protein-coupled receptor (GPCR) and plays a key role in allergic reactions upon activation by histamine which is locally released from mast cells and basophils. Consequently, H 1 R is a well-established therapeutic target for antihistamines that relieve allergy symptoms. H 1 R signals via heterotrimeric G q proteins and is phosphorylated by GPCR kinase (GRK) subtypes 2, 5, and 6, consequently facilitating the subsequent recruitment of β-arrestin1 and/or 2. Stimulation of a GPCR with structurally different agonists can result in preferential engagement of one or more of these intracellular signaling molecules. To evaluate this so-called biased agonism for H 1 R, bioluminescence resonance energy transfer (BRET)-based biosensors were applied to measure H 1 R signaling through heterotrimeric G q proteins, second messengers (inositol 1,4,5-triphosphate and Ca 2+ ), and receptor-protein interactions (GRKs and β-arrestins) in response to histamine, 2-phenylhistamines, and histaprodifens in a similar cellular background. Although differences in efficacy were observed for these agonists between some functional readouts as compared to reference agonist histamine, subsequent data analysis using an operational model of agonism revealed only signaling bias of the agonist Br-phHA-HA in recruiting β-arrestin2 to H 1 R over G q biosensor activation.

As one of the primary points of entry of xenobiotic substances and infectious agents into the body, the lungs are subject to a range of dysfunctions and diseases that together account for a significant number of patient deaths. In view of this, there is an outstanding need for in vitro systems in which to assess the impact of both infectious agents and xenobiotic substances of the lungs. To address this issue, we have developed a protocol to generate airway epithelial basal-like cells from induced pluripotent stem cells, which simplifies the manufacture of cellular models of the human upper airways. Basal-like cells generated in this study were cultured on transwell inserts to allow formation of a confluent monolayer and then exposed to an air-liquid interface to induce differentiation into a pseudostratified epithelial construct with a marked similarity to the upper airway epithelium in vivo. These constructs contain the component cell types required of an epithelial model system, produce mucus and functional cilia, and can support SARS-CoV-2 infection/replication and the secretion of cytokines in a manner similar to that of in vivo airways. This method offers a readily accessible and highly scalable protocol for the manufacture of upper airway models that could find applications in development of therapies for respiratory viral infections and the assessment of drug toxicity on the human lungs., (© 2021 The Authors. STEM CELLS published by Wiley Periodicals LLC on behalf of AlphaMed Press.)

Background: Emphysematous COPD is characterized by aberrant alveolar repair. Macrophage migration inhibitory factor (MIF) contributes to alveolar repair, but for its structural and functional homolog D-dopachrome tautomerase (DDT) this is unknown. MIF mediates its effects through CD74 and/or C-X-C chemokine receptors 2 (CXCR2), 4(CXCR4), and possibly 7 (ACKR3). DDT can also signal through CD74, but interactions with other receptors have not been described yet. We therefore aimed at investigating if and how DDT contributes to epithelial repair in COPD., Methods: We studied effects of recombinant DDT on cell proliferation and survival by clonogenic assay and annexin V-PI staining respectively. DDT-induced signaling was investigated by Western blot. Effects on epithelial growth and differentiation was studied using lung organoid cultures with primary murine or human epithelial cells and incubating with DDT or an ACKR3-blocking nanobody. DDT-ACKR3 interactions were identified by ELISA and co-immunoprecipitation., Findings: We found that DDT promoted proliferation of and prevented staurosporine-induced apoptosis in A549 lung epithelial cells. Importantly, DDT also stimulated growth of primary alveolar epithelial cells as DDT treatment resulted in significantly more and larger murine and human alveolar organoids compared to untreated controls. The anti-apoptotic effect of DDT and DDT-induced organoid growth were inhibited in the presence of an ACKR3-blocking nanobody. Furthermore, ELISA assay and co-immunoprecipitation suggested DDT complexes with ACKR3. DDT could activate the PI3K-Akt pathway and this activation was enhanced in ACKR3-overexpressing cells., Interpretation: In conclusion, DDT contributes to alveolar epithelial repair via ACKR3 and may thus augment lung epithelial repair in COPD., Funding: As stated in the Acknowledgments., Competing Interests: Declaration of Competing Interest Dr. Gosens reports grants from Chiesi, grants from Aquilo, grants from Boehringer Ingelheim, grants from Novartis, outside the submitted work. Dr. Melgert reports grants from Boehringer Ingelheim, outside the submitted work. The other authors have no competing interests to declare, financial or otherwise., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

There is an increasing interest in guiding hit optimization by considering the target binding kinetics of ligands. However, compared to conventional structure-activity relationships, structure-kinetics relationships have not been as thoroughly explored, even for well-studied archetypical drug targets such as the histamine H 1 receptor (H 1 R), a member of the family A G-protein coupled receptor. In this study, we show that the binding kinetics of H 1 R antagonists at the H 1 R is dependent on the cyclicity of both the aromatic head group and the amine moiety of H 1 R ligands, the chemotypes that are characteristic for the first-generation H 1 R antagonists. Fusing the two aromatic rings of H 1 R ligands into one tricyclic aromatic head group prolongs the H 1 R residence time for benchmark H 1 R ligands as well as for tailored synthetic analogues. The effect of constraining the aromatic rings and the basic amines is systematically explored, leading to a coherent series and detailed discussions of structure-kinetics relationships. This study shows that cyclicity has a pronounced effect on the binding kinetics., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

Background : The atypical chemokine receptor 3 (ACKR3) belongs to the superfamily of G protein-coupled receptors (GPCRs). Unlike classical GPCRs, this receptor does not activate G proteins in most cell types but recruits β-arrestins upon activation. ACKR3 plays an important role in cancer and vascular diseases. As recruitment of β-arrestins is triggered by phosphorylation of the C-terminal tail of GPCRs, we studied the role of different potential phosphorylation sites within the ACKR3 C-tail to further delineate the molecular mechanism of internalization and trafficking of this GPCR. Methods : We used various bioluminescence and fluorescence resonance energy transfer-based sensors and techniques in Human Embryonic Kidney (HEK) 293T cells expressing WT or phosphorylation site mutants of ACKR3 to measure CXCL12-induced recruitment of β-arrestins and G-protein-coupled receptor kinases (GRKs), receptor internalization and trafficking. Results : Upon CXCL12 stimulation, ACKR3 recruits both β-arrestin 1 and 2 with equivalent kinetic profiles. We identified interactions with GRK2, 3 and 5, with GRK2 and 3 being important for β-arrestin recruitment. Upon activation, ACKR3 internalizes and recycles back to the cell membrane. We demonstrate that β-arrestin recruitment to the receptor is mainly determined by a single cluster of phosphorylated residues on the C-tail of ACKR3, and that residue T 352 and in part S 355 are important residues for β-arrestin1 recruitment. Phosphorylation of the C-tail appears essential for ligand-induced internalization and important for differential β-arrestin recruitment. GRK2 and 3 play a key role in receptor internalization. Moreover, ACKR3 can still internalize when β-arrestin recruitment is impaired or in the absence of β-arrestins, using alternative internalization pathways. Our data indicate that distinct residues within the C-tail of ACKR3 differentially regulate CXCL12-induced β-arrestin recruitment, ACKR3 trafficking and internalization.

Elevated expression of the chemokine receptors CXCR4 and ACKR3 and of their cognate ligand CXCL12 is detected in a wide range of tumors and the tumor microenvironment (TME). Yet, the molecular mechanisms by which the CXCL12/CXCR4/ACKR3 axis contributes to the pathogenesis are complex and not fully understood. To dissect the role of this axis in cancer, we discuss its ability to impinge on canonical and less conventional signaling networks in different cancer cell types; its bidirectional crosstalk, notably with receptor tyrosine kinase (RTK) and other factors present in the TME; and the infiltration of immune cells that supporttumor progression. We discuss current and emerging avenues that target the CXCL12/CXCR4/ACKR3 axis. Coordinately targeting both RTKs and CXCR4/ACKR3 and/or CXCL12 is an attractive approach to consider in multitargeted cancer therapies. In addition, inhibiting infiltrating immune cells or reactivating the immune system along with modulating the CXCL12/CXCR4/ACKR3 axis in the TME has therapeutic promise.

Human cytomegalovirus (HCMV) encodes four G protein-coupled receptor (GPCR) homologs. Three of these receptors, UL78, US27 and US28, are known for their roles in HCMV dissemination and latency. Despite importance of its rodent orthologs for viral replication and pathogenesis, such a function is not reported for the HCMV-encoded GPCR UL33. Using the clinical HCMV strain Merlin, we show that UL33 facilitates both cell-associated and cell-free virus transmission. A UL33-deficient virus derivative revealed retarded virus spread, formation of less and smaller plaques, and reduced extracellular progeny during multi-cycle growth analysis in fibroblast cultures compared to parental virus. The growth of UL33-revertant, US28-deficient, and US28-revertant viruses were similar to parental virus under multistep growth conditions. UL33- and US28-deficient Merlin viruses impaired cell-associated virus spread to a similar degree. Thus, the growth defect displayed by the UL33-deficient virus but not the US28-deficient virus reflects UL33's contribution to extracellular transmission. In conclusion, UL33 facilitates cell-associated and cell-free spread of the clinical HCMV strain Merlin in fibroblast cultures.

Recent reports regarding the significance of chemokine receptors in disease have put a spotlight on atypical chemokine receptor 3 (ACKR3). This atypical chemokine receptor is overexpressed in numerous cancer types and has been involved in the modulation of tumor cell proliferation and migration, tumor angiogenesis, or resistance to drugs, thus contributing to cancer progression and metastasis occurrence. Here, we focus on the clinical significance and potential mechanisms underlying the pathologic role of ACKR3 in breast, lung, and brain cancer and discuss its possible relevance as a prognostic factor and potential therapeutic target in these contexts., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Drug-target binding kinetics are an important predictor of in vivo drug efficacy, yet the relationship between ligand structures and their binding kinetics is often poorly understood. We show that both rupatadine ( 1 ) and desloratadine ( 2 ) have a long residence time at the histamine H 1 receptor (H 1 R). Through development of a [ 3 H]levocetirizine radiolabel, we find that the residence time of 1 exceeds that of 2 more than 10-fold. This was further explored with 22 synthesized rupatadine and desloratadine analogues. Methylene-linked cycloaliphatic or β-branched substitutions of desloratadine increase the residence time at the H 1 R, conveying a longer duration of receptor antagonism. However, cycloaliphatic substituents directly attached to the piperidine amine (i.e., lacking the spacer) have decreased binding affinity and residence time compared to their methylene-linked structural analogues. Guided by docking studies, steric constraints within the binding pocket are hypothesized to explain the observed differences in affinity and binding kinetics between analogues.

Drug-target binding kinetics has recently attracted considerable interest in view of the potential predictive power for in vivo drug efficacy. The recently introduced antihistamine bilastine has a long duration of in vivo drug action, which outlasts pharmacological active bilastine concentrations in blood. To provide a molecular basis for the long duration of action, we explored the kinetics of bilastine binding to the human histamine H 1 receptor using [ 3 H]mepyramine binding studies and compared its pharmacodynamics properties to the reference compounds fexofenadine and diphenhydramine, which have a long (60 ± 20 min) and short (0.41 ± 0.1 min) residence time, respectively. Bilastine shows a long drug-target residence time at the H 1 receptor (73 ± 5 min) and this results in a prolonged H 1 receptor antagonism in vitro (Ca 2+ mobilization in Fluo-4 loaded HeLa cells), following a washout of unbound antagonist. Hence, the long residence time of bilastine can explain the observed long duration of drug action in vivo., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)