BRET-Based Biosensors to Measure Agonist Efficacies in Histamine H1 Receptor-Mediated G Protein Activation, Signaling and Interactions with GRKs and ß-Arrestins.

The histamine H1 receptor (H1R) 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, H1R is a well-established therapeutic target for antihistamines that relieve allergy symptoms. H1R signals via heterotrimeric Gq proteins and is phosphorylated by GPCR kinase (GRK) subtypes 2, 5, and 6, consequently facilitating the subsequent recruitment of ß-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 H1R, bioluminescence resonance energy transfer (BRET)-based biosensors were applied to measure H1R signaling through heterotrimeric Gq proteins, second messengers (inositol 1,4,5-triphosphate and Ca2+), and receptor-protein interactions (GRKs and ß-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 ß-arrestin2 to H1R over Gq biosensor activation.

A toolbox of molecular photoswitches to modulate the CXCR3 chemokine receptor with light.

We report a detailed structure-activity relationship for the scaffold of VUF16216, a compound we have previously communicated as a small-molecule efficacy photoswitch for the peptidergic chemokine GPCR CXCR3. A series of photoswitchable azobenzene ligands was prepared through various synthetic strategies and multistep syntheses. Photochemical and pharmacological properties were used to guide the design iterations. Investigations of positional and substituent effects reveal that halogen substituents on the ortho-position of the outer ring are preferred for conferring partial agonism on the cis form of the ligands. This effect could be expanded by an electron-donating group on the para-position of the central ring. A variety of efficacy differences between the trans and cis forms emerges from these compounds. Tool compounds VUF15888 (4d) and VUF16620 (6e) represent more subtle efficacy switches, while VUF16216 (6f) displays the largest efficacy switch, from antagonism to full agonism. The compound class disclosed here can aid in new photopharmacology studies of CXCR3 signaling.

Inhibition of autotransporter biogenesis by small molecules.

Disarming pathogens by targeting virulence factors is a promising alternative to classic antibiotics. Many virulence factors in Gram-negative bacteria are secreted via the autotransporter (AT) pathway, also known as Type 5 secretion. These factors are secreted with the assistance of two membrane-based protein complexes: Sec and Bam. To identify inhibitors of the AT pathway, we used transcriptomics analysis to develop a fluorescence-based high-throughput assay that reports on the stress induced by the model AT hemoglobin protease (Hbp) when its secretion across the outer membrane is inhibited. Screening a library of 1600 fragments yielded the compound VUF15259 that provokes cell envelope stress and secretion inhibition of the ATs Hbp and Antigen-43. VUF15259 also impairs ß-barrel folding activity of various outer membrane proteins. Furthermore, we found that mutants that are compromised in outer membrane protein biogenesis are more susceptible to VUF15259. Finally, VUF15259 induces the release of vesicles that appear to assemble in short chains. Taken together, VUF15259 is the first reported compound that inhibits AT secretion and our data are mostly consistent with VUF15259 interfering with the Bam-complex as potential mode of action. The validation of the presented assay incites its use to screen larger compound libraries with drug-like compounds.

Photoswitching the Efficacy of a Small-Molecule Ligand for a Peptidergic GPCR: from Antagonism to Agonism.

For optical control of GPCR function, we set out to develop small-molecule ligands with photoswitchable efficacy in which both configurations bind the target protein but exert distinct pharmacological effects, that is, stimulate or antagonize GPCR activation. Our design was based on a previously identified efficacy hotspot for the peptidergic chemokine receptor CXCR3 and resulted in the synthesis and characterization of five new azobenzene-containing CXCR3 ligands. G protein activation assays and real-time electrophysiology experiments demonstrated photoswitching from antagonism to partial agonism and even to full agonism (compound VUF16216). SAR evaluation suggests that the size and electron-donating properties of the substituents on the inner aromatic ring are important for the efficacy photoswitching. These compounds are the first GPCR azo ligands with a nearly full efficacy photoswitch and may become valuable pharmacological tools for the optical control of peptidergic GPCR signaling.

The constitutive activity of the virally encoded chemokine receptor US28 accelerates glioblastoma growth.

Glioblastoma (GBM) is the most aggressive and an incurable type of brain cancer. Human cytomegalovirus (HCMV) DNA and encoded proteins, including the chemokine receptor US28, have been detected in GBM tumors. US28 displays constitutive activity and is able to bind several human chemokines, leading to the activation of various proliferative and inflammatory signaling pathways. Here we show that HCMV, through the expression of US28, significantly enhanced the growth of 3D spheroids of U251- and neurospheres of primary glioblastoma cells. Moreover, US28 expression accelerated the growth of glioblastoma cells in an orthotopic intracranial GBM-model in mice. We developed highly potent and selective US28-targeting nanobodies, which bind to the extracellular domain of US28 and detect US28 in GBM tissue. The nanobodies inhibited chemokine binding and reduced the constitutive US28-mediated signaling with nanomolar potencies and significantly impaired HCMV/US28-mediated tumor growth in vitro and in vivo. This study emphasizes the oncomodulatory role of HCMV-encoded US28 and provides a potential therapeutic approach for HCMV-positive tumors using the nanobody technology.

The viral G protein-coupled receptor ORF74 unmasks phospholipase C signaling of the receptor tyrosine kinase IGF-1R.

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes the constitutively active G protein-coupled receptor ORF74, which is expressed on the surface of infected host cells and has been linked to the development of the angioproliferative tumor Kaposi's sarcoma. Furthermore, the insulin-like growth factor (IGF)-1 receptor, a receptor tyrosine kinase, also plays an essential role in Kaposi's sarcoma growth and survival. In this study we examined the effect of the constitutively active viral receptor ORF74 on human IGF-1R signaling. Constitutive and CXCL1-induced ORF74 signaling did not transactivate IGF-1R. In contrast, IGF-1 stimulated phospholipase C (PLC) activation in an ORF74-dependent manner without affecting chemokine binding to ORF74. Inhibition of constitutive ORF74 activity by mutagenesis or the inverse agonist CXCL10, or neutralizing IGF-1R with an antibody or silencing IGF-1R expression using siRNA inhibited PLC activation by IGF-1. Transactivation of ORF74 in response to IGF-1 occurred independently of Src, PI3K, and secreted ORF74 ligands. Furthermore, tyrosine residues in the carboxyl-terminus and intracellular loop 2 of ORF74 are not essential for IGF-1-induced PLC activation. Interestingly, PLC activation in response to IGF-1 is specific for ORF74 as IGF-1 was unable to activate PLC in cells expressing the constitutively active human cytomegalovirus (HCMV)-encoded GPCR US28. Interestingly, IGF-1 does not induce ß-arrestin recruitment to ORF74. The proximity ligation assay revealed close proximity between ORF74 and IGF-1R on the cell surface, but a physical interaction was not confirmed by co-immunoprecipitation. Unmasking IGF-1R signaling to PLC in response to IGF-1 is a previously unrecognized action of ORF74.

Molecular Pharmacology of Chemokine Receptors.

Chemokine receptors are involved in various pathologies such as inflammatory diseases, cancer, and HIV infection. Small molecule and antibody-based antagonists have been developed to inhibit chemokine-induced receptor activity. Currently two small molecule inhibitors targeting CXCR4 and CCR5 are on the market for stem cell mobilization and the treatment of HIV infection, respectively. Antibody fragments (e.g., nanobodies) targeting chemokine receptors are primarily orthosteric ligands, competing for the chemokine binding site. This is opposed by most small molecules, which act as allosteric modulators and bind to the receptor at a topographically distinct site as compared to chemokines. Allosteric modulators can be distinguished from orthosteric ligands by unique features, such as a saturable effect and probe dependency. For successful drug development, it is essential to determine pharmacological parameters (i.e., affinity, potency, and efficacy) and the mode of action of potential drugs during early stages of research in order to predict the biological effect of chemokine receptor targeting drugs in the clinic. This chapter explains how the pharmacological profile of chemokine receptor targeting ligands can be determined and quantified using binding and functional experiments.

The Viral G Protein-Coupled Receptor ORF74 Hijacks ß-Arrestins for Endocytic Trafficking in Response to Human Chemokines.

Kaposi's sarcoma-associated herpesvirus-infected cells express the virally encoded G protein-coupled receptor ORF74. Although ORF74 is constitutively active, it binds human CXC chemokines that modulate this basal activity. ORF74-induced signaling has been demonstrated to underlie the development of the angioproliferative tumor Kaposi's sarcoma. Whereas G protein-dependent signaling of ORF74 has been the subject of several studies, the interaction of this viral GPCR with ß-arrestins has hitherto not been investigated. Bioluminescence resonance energy transfer experiments demonstrate that ORF74 recruits ß-arrestins and subsequently internalizes in response to human CXCL1 and CXCL8, but not CXCL10. Internalized ORF74 traffics via early endosomes to recycling and late endosomes. Site-directed mutagenesis and homology modeling identified four serine and threonine residues at the distal end of the intracellular carboxyl-terminal of ORF74 that are required for ß-arrestin recruitment and subsequent endocytic trafficking. Hijacking of the human endocytic trafficking machinery is a previously unrecognized action of ORF74.

Modulation of cellular signaling by herpesvirus-encoded G protein-coupled receptors.

Human herpesviruses (HHVs) are widespread infectious pathogens that have been associated with proliferative and inflammatory diseases. During viral evolution, HHVs have pirated genes encoding viral G protein-coupled receptors (vGPCRs), which are expressed on infected host cells. These vGPCRs show highest homology to human chemokine receptors, which play a key role in the immune system. Importantly, vGPCRs have acquired unique properties such as constitutive activity and the ability to bind a broad range of human chemokines. This allows vGPCRs to hijack human proteins and modulate cellular signaling for the benefit of the virus, ultimately resulting in immune evasion and viral dissemination to establish a widespread and lifelong infection. Knowledge on the mechanisms by which herpesviruses reprogram cellular signaling might provide insight in the contribution of vGPCRs to viral survival and herpesvirus-associated pathologies.

Constitutive ß-catenin signaling by the viral chemokine receptor US28.

Chronic activation of Wnt/ß-catenin signaling is found in a variety of human malignancies including melanoma, colorectal and hepatocellular carcinomas. Interestingly, expression of the HCMV-encoded chemokine receptor US28 in intestinal epithelial cells promotes intestinal neoplasia in transgenic mice, which is associated with increased nuclear accumulation of ß-catenin. In this study we show that this viral receptor constitutively activates ß-catenin and enhances ß-catenin-dependent transcription. Our data illustrate that this viral receptor does not activate ß-catenin via the classical Wnt/Frizzled signaling pathway. Analysis of US28 mediated signaling indicates the involvement of the Rho-Rho kinase (ROCK) pathway in the activation of ß-catenin. Moreover, cells infected with HCMV show significant increases in ß-catenin stabilization and signaling, which is mediated to a large extent by expression of US28. The modulation of the ß-catenin signal transduction pathway by a viral chemokine receptor provides alternative regulation of this pathway, with potential relevance for the development of colon cancer and virus-associated diseases.

Ubiquitination of CXCR7 controls receptor trafficking.

The chemokine receptor CXCR7 binds CXCL11 and CXCL12 with high affinity, chemokines that were previously thought to bind exclusively to CXCR4 and CXCR3, respectively. Expression of CXCR7 has been associated with cardiac development as well as with tumor growth and progression. Despite having all the canonical features of G protein-coupled receptors (GPCRs), the signalling pathways following CXCR7 activation remain controversial, since unlike typical chemokine receptors, CXCR7 fails to activate Gα(i)-proteins. CXCR7 has recently been shown to interact with ß-arrestins and such interaction has been suggested to be responsible for G protein-independent signals through ERK-1/2 phosphorylation. Signal transduction by CXCR7 is controlled at the membrane by the process of GPCR trafficking. In the present study we investigated the regulatory processes triggered by CXCR7 activation as well as the molecular interactions that participate in such processes. We show that, CXCR7 internalizes and recycles back to the cell surface after agonist exposure, and that internalization is not only ß-arrestin-mediated but also dependent on the Serine/Threonine residues at the C-terminus of the receptor. Furthermore we describe, for the first time, the constitutive ubiquitination of CXCR7. Such ubiquitination is a key modification responsible for the correct trafficking of CXCR7 from and to the plasma membrane. Moreover, we found that CXCR7 is reversibly de-ubiquitinated upon treatment with CXCL12. Finally, we have also identified the Lysine residues at the C-terminus of CXCR7 to be essential for receptor cell surface delivery. Together these data demonstrate the differential regulation of CXCR7 compared to the related CXCR3 and CXCR4 receptors, and highlight the importance of understanding the molecular determinants responsible for this process.

Agonist activation of the G protein-coupled receptor GPR35 involves transmembrane domain III and is transduced via Gα13 and ß-arrestin-2.

BACKGROUND AND PURPOSE: GPR35 is a poorly characterized G protein-coupled receptor at which kynurenic acid has been suggested to be the endogenous ligand. We wished to test this and develop assays appropriate for the study of this receptor. EXPERIMENTAL APPROACH: Human and rat orthologues of GPR35 were engineered and expressed and assays developed to assess interaction with ß-arrestin-2, activation of Gα13 and agonist-induced internalization. KEY RESULTS: GPR35-ß-arrestin-2 interaction assays confirmed that both the endogenous tryptophan metabolite kynurenic acid and the synthetic ligand zaprinast had agonist action at each orthologue. Zaprinast was substantially more potent than kynurenic acid at each and both agonists displayed substantially greater potency at rat GPR35. Two novel thiazolidinediones also displayed agonism and displayed similar potency at each GPR35 orthologue. The three ligand classes acted orthosterically with respect to each other, suggesting overlapping binding sites and, consistent with this, mutation to alanine of the conserved arginine at position 3.36 or tyrosine 3.32 in transmembrane domain III abolished ß-arrestin-2 recruitment in response to each ligand at each orthologue. CONCLUSIONS AND IMPLICATIONS: These studies indicate that ß-arrestin-2 interaction assays are highly appropriate to explore the pharmacology of GPR35 and that Gα13 activation is an alternative avenue of signal generation from GPR35. Arginine and tyrosine residues in transmembrane domain III are integral to agonist recognition and function of this receptor. The potency of kynurenic acid at human GPR35 is sufficiently low, however, to question whether it is likely to be the true endogenous ligand for this receptor.