Olav Larsen

Biased and g protein-independent signaling of chemokine receptors.

Biased signaling or functional selectivity occurs when a 7TM-receptor preferentially activates one of several available pathways. It can be divided into three distinct forms: ligand bias, receptor bias, and tissue or cell bias, where it is mediated by different ligands (on the same receptor), different receptors (with the same ligand), or different tissues or cells (for the same ligand–receptor pair). Most often biased signaling is differentiated into G protein-dependent and β-arrestin-dependent signaling. Yet, it may also cover signaling differences within these groups. Moreover, it may not be absolute, i.e., full versus no activation. Here we discuss biased signaling in the chemokine system, including the structural basis for biased signaling in chemokine receptors, as well as in class A 7TM receptors in general. This includes overall helical movements and the contributions of micro-switches based on recently published 7TM crystals and molecular dynamics studies. All three forms of biased signaling are abundant in the chemokine system. This challenges our understanding of “classic” redundancy inevitably ascribed to this system, where multiple chemokines bind to the same receptor and where a single chemokine may bind to several receptors – in both cases with the same functional outcome. The ubiquitous biased signaling confers a hitherto unknown specificity to the chemokine system with a complex interaction pattern that is better described as promiscuous with context-defined roles and different functional outcomes in a ligand-, receptor-, or cell/tissue-defined manner. As the low number of successful drug development plans implies, there are great difficulties in targeting chemokine receptors; in particular with regard to receptor antagonists as anti-inflammatory drugs. Un-defined and putative non-selective targeting of the complete cellular signaling system could be the underlying cause of lack of success. Therefore, biased ligands could be the solution.

Rationally designed chemokine-based toxin targeting the viral G protein-coupled receptor US28 potently inhibits cytomegalovirus infection in vivo

Significance All drugs currently used for the clinical treatment of human cytomegalovirus (HCMV) infection are associated with considerable adverse side effects and with the development of drug resistance that results in therapy failure. Here we describe a novel, rationally designed fusion toxin protein (FTP)-based strategy to target HCMV on the basis of its virally expressed G protein-coupled receptor (US28) and cognate chemokine ligand. Viral G protein-coupled receptors are expressed by a number of other clinically important viruses. We suggest that FTP-based molecules targeting virally expressed 7TM receptors may represent a new class of drugs amenable for development against complex viral pathogens. The use of receptor–ligand interactions to direct toxins to kill diseased cells selectively has shown considerable promise for treatment of a number of cancers and, more recently, autoimmune disease. Here we move the fusion toxin protein (FTP) technology beyond cancer/autoimmune therapeutics to target the human viral pathogen, human cytomegalovirus (HCMV), on the basis of its expression of the 7TM G protein-coupled chemokine receptor US28. The virus origin of US28 provides an exceptional chemokine-binding profile with high selectivity and improved binding for the CX3C chemokine, CX3CL1. Moreover, US28 is constitutively internalizing by nature, providing highly effective FTP delivery. We designed a synthetic CX3CL1 variant engineered to have ultra-high affinity for US28 and greater specificity for US28 than the natural sole receptor for CX3CL1, CX3CR1, and we fused the synthetic variant with the cytotoxic domain of Pseudomonas Exotoxin A. This novel strategy of a rationally designed FTP provided unparalleled anti-HCMV efficacy and potency in vitro and in vivo.

Natural nitration of CXCL12 reduces its signaling capacity and chemotactic activity in vitro and abrogates intra-articular lymphocyte recruitment in vivo

The chemokine CXCL12/stromal cell-derived factor-1 is important for leukocyte migration to lymphoid organs and inflamed tissues and stimulates tumor development. In vitro, CXCL12 activity through CXCR4 is abolished by proteolytic processing. However, limited information is available on in vivo effects of posttranslationally modified CXCL12. Natural CXCL12 was purified from the coculture supernatant of stromal cells stimulated with leukocytes and inflammatory agents. In this conditioned medium, CXCL12 with a nitration on Tyr7, designated [3-NT7]CXCL12, was discovered via Edman degradation. CXCL12 and [3-NT7]CXCL12 were chemically synthesized to evaluate the biological effects of this modification. [3-NT7]CXCL12 recruited β-arrestin 2 and phosphorylated the Akt kinase similar to CXCL12 in receptor-transfected cells. Also the affinity of CXCL12 and [3-NT7]CXCL12 for glycosaminoglycans, the G protein-coupled chemokine receptor CXCR4 and the atypical chemokine receptor ACKR3 were comparable. However, [3-NT7]CXCL12 showed a reduced ability to enhance intracellular calcium concentrations, to generate inositol triphosphate, to phosphorylate ERK1/2 and to induce monocyte and lymphocyte chemotaxis in vitro. Moreover, nitrated CXCL12 failed to induce in vivo extravasation of lymphocytes to the joint. In summary, nitration on Tyr7 under inflammatory conditions is a novel natural posttranslational regulatory mechanism of CXCL12 which may downregulate the CXCR4-mediated inflammatory and tumor-promoting activities of CXCL12.

Truncation of CXCL12 by CD26 reduces its CXC chemokine receptor 4- and atypical chemokine receptor 3-dependent activity on endothelial cells and lymphocytes

Graphical abstract Figure. No Caption available. ABSTRACT The chemokine CXCL12 or stromal cell‐derived factor 1/SDF‐1 attracts hematopoietic progenitor cells and mature leukocytes through the G protein‐coupled CXC chemokine receptor 4 (CXCR4). In addition, it interacts with atypical chemokine receptor 3 (ACKR3 or CXCR7) and glycosaminoglycans. CXCL12 activity is regulated through posttranslational cleavage by CD26/dipeptidyl peptidase 4 that removes two NH2‐terminal amino acids. CD26‐truncated CXCL12 does not induce calcium signaling or chemotaxis of mononuclear cells. CXCL12(3–68) was chemically synthesized de novo for detailed biological characterization. Compared to unmodified CXCL12, CXCL12(3–68) was no longer able to signal through CXCR4 via inositol trisphosphate (IP3), Akt or extracellular signal‐regulated kinases 1 and 2 (ERK1/2). Interestingly, the recruitment of &bgr;‐arrestin 2 to the cell membrane via CXCR4 by CXCL12(3–68) was abolished, whereas a weakened but significant &bgr;‐arrestin recruitment remained via ACKR3. CXCL12‐induced endothelial cell migration and signal transduction was completely abrogated by CD26. Intact CXCL12 hardly induced lymphocyte migration upon intra‐articular injection in mice. In contrast, oral treatment of mice with the CD26 inhibitor sitagliptin reduced CD26 activity and CXCL12 cleavage in blood plasma. The potential of CXCL12 to induce intra‐articular lymphocyte infiltration was significantly increased in sitagliptin‐treated mice and CXCL12(3–68) failed to induce migration under both CD26‐inhibiting and non‐inhibiting conditions. In conclusion, CD26‐cleavage skews CXCL12 towards &bgr;‐arrestin dependent recruitment through ACKR3 and destroys the CXCR4‐mediated lymphocyte chemoattractant properties of CXCL12 in vivo. Hence, pharmacological CD26‐blockade in tissues may enhance CXCL12‐induced inflammation.

Biased signaling of lipids and allosteric actions of synthetic molecules for GPR119

GPR119 is a Gαs-coupled lipid-sensor in the gut, where it mediates release of incretin hormones from the enteroendocrine cells and in pancreatic α-cells, where it releases insulin. Naturally occurring lipids such as monoacylglycerols (MAGs) and N-acylethanolamines (NAEs), like oleoylethanolamide (OEA), activate GPR119, and multiple synthetic ligands have been described. Here, we extend the GPR119 signaling profile to Gαq and Gαi in addition to β-arrestin recruitment and the downstream transcription factors CRE (cAMP response element), SRE (serum response element) and NFAT (nuclear factor of activated T cells). The endogenous OEA and the synthetic AR231453 were full agonists in all pathways except for NFAT, where no ligand-modulation was observed. The potency of AR231453 varied <16-fold (EC50 from 6 to 95nM) across the different signaling pathways, whereas that of OEA varied >175-fold (from 85nM to 15μM) indicating a biased signaling for OEA. The degree of constitutive activity was 1-10%, 10-30% and 30-70% of OEA-induced Emax in Gαi, Gαq and Gαs-driven pathways, respectively. This coincided with the lowest and highest OEA potency observed in Gαi and Gαs-driven pathways, respectively. Incubation for 2h with the 2-MAG-lipase inhibitor JZL84 doubled the constitutive activity, indicating that endogenous lipids contribute to the apparent constitutive activity. Finally, besides being an agonist, AR231453 acted as a positive allosteric modulator of OEA and increased its potency by 54-fold at 100nM AR231453. Our studies uncovering broad and biased signaling, masked constitutive activity by endogenous MAGs, and ago-allosteric properties of synthetic ligands may explain why many GPR119 drug-discovery programs have failed so far.

Discovery and Characterization of Biased Allosteric Agonists of the Chemokine Receptor CXCR3

In this work we report a design, synthesis, and detailed functional characterization of unique strongly biased allosteric agonists of CXCR3 that contain tetrahydroisoquinoline carboxamide cores. Compound 11 (FAUC1036) is the first strongly biased allosteric agonist of CXCR3 that selectively induces weak chemotaxis and leads to receptor internalization and the β-arrestin 2 recruitment with potency comparable to that of the chemokine CXCL11 without any activation of G proteins. A subtle structural change (addition of a methoxy group, 14 (FAUC1104)) led to a contrasting biased allosteric partial agonist that activated solely G proteins, induced chemotaxis, but failed to induce receptor internalization or β-arrestin 2 recruitment. Concomitant structure-activity relationship studies indicated very steep structure-activity relationships, which steer the ligand bias between the β-arrestin 2 and G protein pathway. Overall, the information presented provides a powerful platform for further development and rational design of strongly biased allosteric agonists of CXCR3.

Differential CCR7 Targeting in Dendritic Cells by Three Naturally Occurring CC-Chemokines

The CCR7 ligands CCL19 and CCL21 are increasingly recognized as functionally different (biased). Using mature human dendritic cells (DCs), we show that CCL19 is more potent than CCL21 in inducing 3D chemotaxis. Intriguingly, CCL21 induces prolonged and more efficient ERK1/2 activation compared with CCL19 and a C-terminal truncated (tailless) CCL21 in DCs. In contrast, tailless-CCL21 displays increased potency in DC chemotaxis compared with native CCL21. Using a CCL21-specific antibody, we show that CCL21, but not tailless-CCL21, accumulates at the cell surface. In addition, removal of sialic acid from the cell surface by neuraminidase treatment impairs ERK1/2 activation by CCL21, but not by CCL19 or tailless-CCL21. Using standard laboratory cell lines, we observe low potency of both CCL21 and tailless-CCL21 in G protein activation and β-arrestin recruitment compared with CCL19, indicating that the tail itself does not improve receptor interaction. Chemokines interact with their receptors in a stepwise manner with ultimate docking of their N-terminus into the main binding pocket. Employing site-directed mutagenesis we identify residues in this pocket of selective CCL21 importance. We also identify a molecular switch in the top of TM7 important for keeping CCR7 in an inactive conformation (Tyr312), as introduction of the chemokine receptor-conserved Glu (or Ala) induces high constitutive activity. Summarized, we show that the interaction of the tail of CCL21 with polysialic acid is needed for strong ERK signaling, whereas it impairs CCL21-mediated chemotaxis and has no impact on receptor docking consistent with the current model of chemokine:receptor interaction. This indicates that future selective pharmacological targeting of CCL19 versus CCL21 should focus on a differential targeting of the main receptor pocket, while selective targeting of tailless-CCL21 versus CCL21 and CCL19 requires targeting of the glycosaminoglycan (GAG) interaction.

Bile acids are important direct and indirect regulators of the secretion of appetite- and metabolism-regulating hormones from the gut and pancreas

Objective Bile acids (BAs) facilitate fat absorption and may play a role in glucose and metabolism regulation, stimulating the secretion of gut hormones. The relative importance and mechanisms involved in BA-stimulated secretion of appetite and metabolism regulating hormones from the gut and pancreas is not well described and was the purpose of this study. Methods The effects of bile acids on the secretion of gut and pancreatic hormones was studied in rats and compared to the most well described nutritional secretagogue: glucose. The molecular mechanisms that underlie the secretion was studied by isolated perfused rat and mouse small intestine and pancreas preparations and supported by immunohistochemistry, expression analysis, and pharmacological studies. Results Bile acids robustly stimulate secretion of not only the incretin hormones, glucose-dependent insulinotropic peptide (GIP), and glucagon-like peptide-1 (GLP-1), but also glucagon and insulin in vivo, to levels comparable to those resulting from glucose stimulation. The mechanisms of GLP-1, neurotensin, and peptide YY (PYY) secretion was secondary to intestinal absorption and depended on activation of basolateral membrane Takeda G-protein receptor 5 (TGR5) receptors on the L-cells in the following order of potency: Lithocholic acid (LCA) >Deoxycholicacid (DCA)>Chenodeoxycholicacid (CDCA)> Cholic acid (CA). Thus BAs did not stimulate secretion of GLP-1 and PYY from perfused small intestine in TGR5 KO mice but stimulated robust responses in wild type littermates. TGR5 is not expressed on α-cells or β-cells, and BAs had no direct effects on glucagon or insulin secretion from the perfused pancreas. Conclusion BAs should be considered not only as fat emulsifiers but also as important regulators of appetite- and metabolism-regulating hormones by activation of basolateral intestinal TGR5.

Protein engineering of the chemokine CCL20 prevents psoriasiform dermatitis in an IL-23–dependent murine model

Significance Psoriasis is a chronic skin disease characterized by the infiltration of inflammatory T cells to the skin in response to injury. When inflammatory T cells and dendritic cells are recruited to the skin by CCL20 and other chemokines, they release cytokines that contribute to psoriatic inflammation. We engineered a molecule derived from the natural CCL20 protein that adopts a unique dimeric structure, partially activates its G-protein receptor, blocks T cell homing, and prevents the signs of psoriasis in a mouse model of this common human skin disease. Our remarkable findings reveal the potential of engineered-CCL20 molecules as therapeutic agents for psoriasis and the general utility of chemokine engineering for treating inflammatory diseases. Psoriasis is a chronic inflammatory skin disease characterized by the infiltration of T cell and other immune cells to the skin in response to injury or autoantigens. Conventional, as well as unconventional, γδ T cells are recruited to the dermis and epidermis by CCL20 and other chemokines. Together with its receptor CCR6, CCL20 plays a critical role in the development of psoriasiform dermatitis in mouse models. We screened a panel of CCL20 variants designed to form dimers stabilized by intermolecular disulfide bonds. A single-atom substitution yielded a CCL20 variant (CCL20 S64C) that acted as a partial agonist for the chemokine receptor CCR6. CCL20 S64C bound CCR6 and induced intracellular calcium release, consistent with G-protein activation, but exhibited minimal chemotactic activity. Instead, CCL20 S64C inhibited CCR6-mediated T cell migration with nominal impact on other chemokine receptor signaling. When given in an IL-23–dependent mouse model for psoriasis, CCL20 S64C prevented psoriatic inflammation and the up-regulation of IL-17A and IL-22. Our results validate CCR6 as a tractable therapeutic target for psoriasis and demonstrate the value of CCL20 S64C as a lead compound.

Corrigendum: Differential CCR7 Targeting in Dendritic Cells by Three Naturally Occurring CC-Chemokines

[This corrects the article on p. 568 in vol. 7, PMID: 28018341.].