CXCR1 and CXCR2 Chemokine Receptor Agonists Produced by Tumors Induce Neutrophil Extracellular Traps that Interfere with Immune Cytotoxicity.

Neutrophils are expanded and abundant in cancer-bearing hosts. Under the influence of CXCR1 and CXCR2 chemokine receptor agonists and other chemotactic factors produced by tumors, neutrophils, and granulocytic myeloid-derived suppressor cells (MDSCs) from cancer patients extrude their neutrophil extracellular traps (NETs). In our hands, CXCR1 and CXCR2 agonists proved to be the major mediators of cancer-promoted NETosis. NETs wrap and coat tumor cells and shield them from cytotoxicity, as mediated by CD8+ T cells and natural killer (NK) cells, by obstructing contact between immune cells and the surrounding target cells. Tumor cells protected from cytotoxicity by NETs underlie successful cancer metastases in mice and the immunotherapeutic synergy of protein arginine deiminase 4 (PAD4) inhibitors, which curtail NETosis with immune checkpoint inhibitors. Intravital microscopy provides evidence of neutrophil NETs interfering cytolytic cytotoxic T lymphocytes (CTLs) and NK cell contacts with tumor cells.

Identification and mechanism of G protein-biased ligands for chemokine receptor CCR1.

Biased signaling of G protein-coupled receptors describes an ability of different ligands that preferentially activate an alternative downstream signaling pathway. In this work, we identified and characterized different N-terminal truncations of endogenous chemokine CCL15 as balanced or biased agonists targeting CCR1, and presented three cryogenic-electron microscopy structures of the CCR1-Gi complex in the ligand-free form or bound to different CCL15 truncations with a resolution of 2.6-2.9 Å, illustrating the structural basis of natural biased signaling that initiates an inflammation response. Complemented with pharmacological and computational studies, these structures revealed it was the conformational change of Tyr291 (Y2917.43) in CCR1 that triggered its polar network rearrangement in the orthosteric binding pocket and allosterically regulated the activation of ß-arrestin signaling. Our structure of CCL15-bound CCR1 also exhibited a critical site for ligand binding distinct from many other chemokine-receptor complexes, providing new insights into the mode of chemokine recognition.

Resolvin E1 Attenuates Chronic Pain-Induced Depression-Like Behavior in Mice: Possible Involvement of Chemerin Receptor ChemR23.

The antidepressant effect of eicosapentaenoic acid-derived bioactive lipid, resolvin E1 (RvE1), was examined in a murine model of chronic pain-induced depression using a tail suspension test. Because RvE1 reportedly possesses agonistic activity on a chemerin receptor ChemR23, we also examined the antidepressant effect of chemerin. Two weeks after surgery for unilateral spared nerve injury to prepare neuropathic pain model mice, immobility time was measured in a tail suspension test. Chronic pain significantly increased immobility time, and this depression-like behavior was attenuated by intracerebroventricular injection of RvE1 (1 ng) or chemerin (500 ng). These results demonstrate that RvE1 exerts an antidepressant effect in a murine model of chronic pain-induced depression, which is likely to be via ChemR23. RvE1 and its receptor may be promising targets to develop novel antidepressants.

International Union of Basic and Clinical Pharmacology CIII: Chemerin Receptors CMKLR1 (Chemerin1) and GPR1 (Chemerin2) Nomenclature, Pharmacology, and Function.

Chemerin, a chemoattractant protein and adipokine, has been identified as the endogenous ligand for a G protein-coupled receptor encoded by the gene CMKLR1 (also known as ChemR23), and as a consequence the receptor protein was renamed the chemerin receptor in 2013. Since then, chemerin has been identified as the endogenous ligand for a second G protein-coupled receptor, encoded by the gene GPR1 Therefore, the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification recommends that the official name of the receptor protein for chemokine-like receptor 1 (CMKLR1) is chemerin receptor 1, and G protein-coupled receptor 1 is chemerin receptor 2 to follow the convention of naming the receptor protein after the endogenous ligand. Chemerin receptor 1 and chemerin receptor 2 can be abbreviated to Chemerin1 and Chemerin2, respectively. Chemerin requires C-terminal processing for activity, and human chemerin21-157 is reported to be the most active form, with peptide fragments derived from the C terminus biologically active at both receptors. Small-molecule antagonist, CCX832, selectively blocks CMKLR1, and resolvin E1 activation of CMKLR1 is discussed. Activation of both receptors by chemerin is via coupling to Gi/o, causing inhibition of adenylyl cyclase and increased Ca2+ flux. Receptors and ligand are widely expressed in humans, rats, and mice, and both receptors share ∼80% identity across these species. CMKLR1 knockout mice highlight the role of this receptor in inflammation and obesity, and similarly, GPR1 knockout mice exhibit glucose intolerance. In addition, the chemerin receptors have been implicated in cardiovascular disease, cancer, steroidogenesis, human immunodeficiency virus replication, and neurogenerative disease.

GPCR Pharmacological Profiling of Aaptamine from the Philippine Sponge Stylissa sp. Extends Its Therapeutic Potential for Noncommunicable Diseases.

We report the first isolation of the alkaloid aaptamine from the Philippine marine sponge Stylissa sp. Aaptamine possessed weak antiproliferative activity against HCT116 colon cancer cells and inhibited the proteasome in vitro at 50 µM. These activities may be functionally linked. Due to its known, more potent activity on certain G-protein coupled receptors (GPCRs), including α-adrenergic and δ-opioid receptors, the compound was profiled more broadly at sub-growth inhibitory concentrations against a panel of 168 GPCRs to potentially reveal additional targets and therapeutic opportunities. GPCRs represent the largest class of drug targets. The primary screen at 20 µM using the ß-arrestin functional assay identified the antagonist, agonist, and potentiators of agonist activity of aaptamine. Dose-response analysis validated the α-adrenoreceptor antagonist activity of aaptamine (ADRA2C, IC50 11.9 µM) and revealed the even more potent antagonism of the ß-adrenoreceptor (ADRB2, IC50 0.20 µM) and dopamine receptor D4 (DRD4, IC50 6.9 µM). Additionally, aaptamine showed agonist activity on selected chemokine receptors, by itself (CXCR7, EC50 6.2 µM; CCR1, EC50 11.8 µM) or as a potentiator of agonist activity (CXCR3, EC50 31.8 µM; CCR3, EC50 16.2 µM). These GPCRs play a critical role in the treatment of cardiovascular disease, diabetes, cancer, and neurological disorders. The results of this study may thus provide novel preventive and therapeutic strategies for noncommunicable diseases (NCDs).

E6130, a Novel CX3C Chemokine Receptor 1 (CX3CR1) Modulator, Attenuates Mucosal Inflammation and Reduces CX3CR1+ Leukocyte Trafficking in Mice with Colitis.

The chemokine fractalkine (CX3C chemokine ligand 1; CX3CL1) and its receptor CX3CR1 are involved in the pathogenesis of several diseases, including inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, rheumatoid arthritis, hepatitis, myositis, multiple sclerosis, renal ischemia, and atherosclerosis. There are no orally available agents that modulate the fractalkine/CX3CR1 axis. [(3S,4R)-1-[2-Chloro-6-(trifluoromethyl)benzyl]-3-{[1-(cyclohex-1-en-1-ylmethyl)piperidin-4-yl]carbamoyl}-4-methylpyrrolidin-3-yl]acetic acid (2S)-hydroxy(phenyl)acetate (E6130) is an orally available highly selective modulator of CX3CR1 that may be effective for treatment of inflammatory bowel disease. We found that E6130 inhibited the fractalkine-induced chemotaxis of human peripheral blood natural killer cells (IC50 4.9 nM), most likely via E6130-induced down-regulation of CX3CR1 on the cell surface. E6130 had agonistic activity via CX3CR1 with respect to guanosine 5'-3-O-(thio)triphosphate binding in CX3CR1-expressing Chinese hamster ovary K1 (CHO-K1) membrane and had no antagonistic activity. Orally administered E6130 ameliorated several inflammatory bowel disease-related parameters in a murine CD4+CD45RBhigh T-cell-transfer colitis model and a murine oxazolone-induced colitis model. In the CD4+CD45RBhigh T-cell transfer model, E6130 inhibited the migration of CX3CR1+ immune cells and decreased the number of these cells in the gut mucosal membrane. These results suggest that E6130 is a promising therapeutic agent for treatment of inflammatory bowel disease.

Development of a membrane-anchored chemerin receptor agonist as a novel modulator of allergic airway inflammation and neuropathic pain.

The chemerin receptor (CMKLR1) is a G protein-coupled receptor found on select immune, epithelial, and dorsal root ganglion/spinal cord neuronal cells. CMKLR1 is primarily coupled to the inhibitory G protein, Gαi, and has been shown to modulate the resolution of inflammation and neuropathic pain. CMKLR1 is activated by both lipid and peptide agonists, resolvin E1 and chemerin, respectively. Notably, these ligands have short half-lives. To expedite the development of long acting, stable chemerin analogs as candidate therapeutics, we used membrane-tethered ligand technology. Membrane-tethered ligands are recombinant proteins comprised of an extracellular peptide ligand, a linker sequence, and an anchoring transmembrane domain. Using this technology, we established that a 9-amino acid-tethered chemerin fragment (amino acids 149-157) activates both mouse and human CMKLR1 with efficacy exceeding that of the full-length peptide (amino acids 21-157). To enable in vivo delivery of a corresponding soluble membrane anchored ligand, we generated lipidated analogs of the 9-amino acid fragment. Pharmacological assessment revealed high potency and wash resistance (an index of membrane anchoring). When tested in vivo, a chemerin SMAL decreased allergic airway inflammation and attenuated neuropathic pain in mice. This compound provides a prototype membrane-anchored peptide for the treatment of inflammatory disease. A parallel approach may be applied to developing therapeutics targeting other peptide hormone G protein-coupled receptors.

Biased agonism as a mechanism for differential signaling by chemokine receptors.

Chemokines display considerable promiscuity with multiple ligands and receptors shared in common, a phenomenon that is thought to underlie their biochemical "redundancy." Their receptors are part of a larger seven-transmembrane receptor superfamily, commonly referred to as G protein-coupled receptors, which have been demonstrated to be able to signal with different efficacies to their multiple downstream signaling pathways, a phenomenon referred to as biased agonism. Biased agonism has been primarily reported as a phenomenon of synthetic ligands, and the biologic prevalence and importance of such signaling are unclear. Here, to assess the presence of biased agonism that may underlie differential signaling by chemokines targeting the same receptor, we performed a detailed pharmacologic analysis of a set of chemokine receptors with multiple endogenous ligands using assays for G protein signaling, ß-arrestin recruitment, and receptor internalization. We found that chemokines targeting the same receptor can display marked differences in their efficacies for G protein- or ß-arrestin-mediated signaling or receptor internalization. This ligand bias correlates with changes in leukocyte migration, consistent with different mechanisms underlying the signaling downstream of these receptors induced by their ligands. These findings demonstrate that biased agonism is a common and likely evolutionarily conserved biological mechanism for generating qualitatively distinct patterns of signaling via the same receptor in response to different endogenous ligands.

Allosteric modulation of the G protein-coupled US28 receptor of human cytomegalovirus: are the small-weight inverse agonist of US28 'camouflaged' agonists?

The highly constitutively active G protein-coupled receptor US28 of human cytomegalovirus (HCMV) is thought to camouflage agonism by mediating constitutive endocytosis. With the use of the US28Δ300 mutant, which is largely devoid of constitutive internalization, I have demonstrated that the coupling of the receptor to its downstream signaling partners is responsible for the inverse agonism to agonism efficacy switch in some small-weight ligands of US28.

Chemokine N-terminal-derived peptides differentially regulate signaling by the receptors CCR1 and CCR5.

Inflammatory chemokines are often elevated in disease settings, where the largest group of CC-chemokines are the macrophage inflammatory proteins (MIP), which are promiscuous for the receptors CCR1 and CCR5. MIP chemokines, such as CCL3 and CCL5 are processed at the N terminus, which influences signaling in a highly diverse manner. Here, we investigate the signaling capacity of peptides corresponding to truncated N termini. These 3-10-residue peptides displayed weak potency but, surprisingly, retained their signaling on CCR1. In contrast, none of the peptides generated a signal on CCR5, but a CCL3-derived tetrapeptide was a positive modulator boosting the signal of several chemokine variants on CCR5. In conclusion, chemokine N termini can be mimicked to produce small CCR1-selective agonists, as well as CCR5-selective modulators.

Identification of novel allosteric modulators for the G-protein coupled US28 receptor of human cytomegalovirus.

The highly constitutively active G-protein coupled receptor US28 of human cytomegalovirus (HCMV) is an interesting pharmacological target because of its implication on viral dissemination, cardiovascular diseases and tumorigenesis. We found that dihydroisoquinolinone and tetrahydroisoquinoline scaffolds may be promising lead structures for novel US28 allosteric inverse agonists. These scaffolds were rapidly synthesized by radical carboamination reactions followed by non-radical transformations. Our novel US28 allosteric modulators provide valuable scaffolds for further ligand optimization and may be helpful chemical tools to investigate molecular mechanisms of US28 constitutive signaling and its role in pathogenesis.

Cyclic Analogues of the Chemerin C-Terminus Mimic a Loop Conformation Essential for Activating the Chemokine-like Receptor 1.

The chemokine-like receptor 1 (CMKLR1) is a promising target for treating autoinflammatory diseases, cancer, and reproductive disorders. However, the interaction between CMKLR1 and its protein-ligand chemerin remains uncharacterized, and no drugs targeting this interaction have passed clinical trials. Here, we identify the binding mode of chemerin-9, the C-terminus of chemerin, at the receptor by combining complementary mutagenesis with structure-based modeling. Incorporating our experimental data, we present a detailed model of this binding site, including experimentally confirmed pairwise interactions for the most critical ligand residues: Chemerin-9 residue F8 binds to a hydrophobic pocket in CMKLR1 formed by the extracellular loop (ECL) 2, while F6 interacts with Y2.68, suggesting a turn-like structure. On the basis of this model, we created the first cyclic peptide with nanomolar activity, confirming the overall binding conformation. This constrained agonist mimics the loop conformation adopted by the natural ligand and can serve as a lead compound for future drug design.

Natural proteolytic processing of hemofiltrate CC chemokine 1 generates a potent CC chemokine receptor (CCR)1 and CCR5 agonist with anti-HIV properties.

Hemofiltrate CC chemokine (HCC)-1 is a recently described human chemokine that is constitutively expressed in numerous tissues and is present at high concentrations in normal plasma. Using a cell line expressing CC chemokine receptor (CCR)5 as a bioassay, we isolated from human hemofiltrate an HCC-1 variant lacking the first eight amino acids. HCC-1[9-74] was a potent agonist of CCR1, CCR3, and CCR5 and promoted calcium flux and chemotaxis of T lymphoblasts, monocytes, and eosinophils. It also blocked entry of HIV-1 strains using CCR5 as coreceptor. Limited tryptic digestion of HCC-1 generated the active variant. Conditioned media from several tumor cell lines activated HCC-1 with a high efficiency, and this activity could be inhibited by serine protease inhibitors. Our results indicate that HCC-1 represents a nonfunctional precursor that can be rapidly converted to the active chemokine by proteolytic processing. This process represents an additional mechanism by which tumor cells might generate chemoattractant molecules and recruit inflammatory cells. It might also affect HIV-1 replication in infected individuals and play an important role in AIDS pathogenesis.

Identification of novel allosteric nonpeptidergic inhibitors of the human cytomegalovirus-encoded chemokine receptor US28.

Human cytomegalovirus (HCMV) is a widespread human pathogen, possessing onco-modulatory properties. Constitutive signaling of the HCMV-encoded chemokine receptor US28 and its ability to bind a broad spectrum of chemokines might facilitate HCMV-associated tumor progression. Novel nonpeptidergic chemotypes were identified as neutral antagonists or inverse agonists on US28, that allosterically inhibit chemokine binding to US28.

Kaposi's sarcoma-associated herpesvirus-G protein-coupled receptor-expressing endothelial cells exhibit reduced migration and stimulated chemotaxis by chemokine inverse agonists.

A constitutively active G protein-coupled receptor (GPCR) encoded by Kaposi's sarcoma-associated herpesvirus (human herpesvirus-8) (KSHV) is expressed in endothelial (spindle) cells of Kaposi's sarcoma lesions. In this study, we report novel effects of basal signaling by this receptor and of inverse agonist chemokines on migration of KSHV-GPCR-expressing mouse lung endothelial cells. We show that basal signaling by KSHV-GPCR inhibits migration of endothelial cells in two systems, movement through porous filters and in vitro wound closure. Naturally occurring chemokines, interferon gamma-inducible protein-10 and stromal-derived factor-1, which act as inverse agonists at KSHV-GPCR, abrogate the inhibition of migration and stimulate directed migration (or chemotaxis) of these cells. Thus, the expression of KSHV-GPCR may allow infected endothelial cells in situ to remain in a localized environment or to directionally migrate along a gradient of specific chemokines that are inverse agonists at KSHV-GPCR.

6-C-kine (SLC), a lymphocyte adhesion-triggering chemokine expressed by high endothelium, is an agonist for the MIP-3beta receptor CCR7.

The beta chemokine known as 6-C-kine, secondary lymphoid-tissue chemokine (SLC), TCA4, or Exodus-2 (herein referred to as 6CK/SLC) can trigger rapid integrin-dependent arrest of lymphocytes rolling under physiological shear and is highly expressed by high endothelial venules, specialized vessels involved in lymphocyte homing from the blood into lymph nodes and Peyer's patches. We show that 6CK/SLC is an agonist for the lymphocyte chemoattractant receptor, CCR7 (EBI-1, BLR-2), previously described as a receptor for the related beta chemokine MIP-3beta (ELC or Exodus-3). Moreover, 6CK/SLC and MIP-3beta attract the same major populations of circulating lymphocytes, including naive and memory T cells > B cells (but not natural killer cells); desensitization to MIP-3beta inhibits lymphocyte chemotaxis to 6CK/SLC but not to the alpha chemokine SDF-1 (stromal cell-derived factor); and 6CK/SLC competes for MIP-3beta binding to resting mouse lymphocytes. The findings suggest that the majority of circulating lymphocytes respond to 6CK/SLC and MIP-3beta in large part through their common receptor CCR7 and that these molecules may be important mediators of physiological lymphocyte recirculation in vivo.

An apparent paradox: chemokine receptor agonists can be used for anti-inflammatory therapy.

Inflammation plays an important role in a wide range of human diseases. Chemokines are a group of proteins which control the migration and activation of the immune cells involved in all aspects of the inflammatory response. Chemokines bind to specific receptors of the seven-transmembrane spanning type on target leukocytes and also bind to cell-surface glycosaminoglycans (GAG). Leukocytes express a range of chemokine receptors which can cross-desensitise each other, potentially allowing a single chemokine receptor agonist to desensitise all the chemokine receptors on a cell. If an appropriate single receptor agonist is engineered to be non-chemotactic itself, then a treated cell will lose the potential to migrate in response to chemokines towards any developing site of inflammation. A non-GAG-binding but receptor agonistic form of the chemokine CCL7 can inhibit leukocyte recruitment in response to a diverse range of chemokines in vitro and in vivo. We hypothesise that this modified chemokine mediates its effect by inducing homologous and heterologous receptor desensitisation and further propose that other suitable candidates could include agonistic chemokine receptor-specific antibodies or small molecule chemokine receptor agonists. Hence, an appropriate chemokine receptor agonist could be used to inhibit multiple chemokine receptors, thereby producing a powerful and robust anti-inflammatory effect. This review considers the mechanisms leading to chemokine receptor desensitisation and discusses the potential to develop a new class of anti-inflammatory agents based on targeted stimulation of chemokine receptors.

Ligand-selective small molecule modulators of the constitutively active vGPCR US28.

US28 is a broad-spectrum constitutively active G protein-coupled receptor encoded by the human cytomegalovirus (HCMV). It binds and scavenges multiple CC-chemokines as well as CX3CL1 (fractalkine) by constitutive receptor endocytosis to escape immune surveillance. We herein report the design and characterization of a novel library of US28-acting commercially available ligands based on the molecular descriptors of two previously reported US28-acting structures. Among these, we identify compounds capable of selectively recognizing CCL2-and CCL4-, but not CX3CL1-induced receptor conformations. Moreover, we find a direct correlation between the binding properties of small molecule ligands to CCL-induced conformations at the wild-type receptor and functional activity at the C-terminal truncated US28Δ300. As US28Δ300 is devoid of arrestin-recruitment and endocytosis, this highlights the potential usefulness of this construct in future drug discovery efforts aimed at specific US28 conformations. The new scaffolds identified herein represent valuable starting points for the generation of novel anti-HCMV therapies targeting the virus-encoded chemokine receptor US28 in a conformational-selective manner.

Viral GPCR US28 can signal in response to chemokine agonists of nearly unlimited structural degeneracy.

Human cytomegalovirus has hijacked and evolved a human G-protein-coupled receptor into US28, which functions as a promiscuous chemokine 'sink' to facilitate evasion of host immune responses. To probe the molecular basis of US28's unique ligand cross-reactivity, we deep-sequenced CX3CL1 chemokine libraries selected on 'molecular casts' of the US28 active-state and find that US28 can engage thousands of distinct chemokine sequences, many of which elicit diverse signaling outcomes. The structure of a G-protein-biased CX3CL1-variant in complex with US28 revealed an entirely unique chemokine amino terminal peptide conformation and remodeled constellation of receptor-ligand interactions. Receptor signaling, however, is remarkably robust to mutational disruption of these interactions. Thus, US28 accommodates and functionally discriminates amongst highly degenerate chemokine sequences by sensing the steric bulk of the ligands, which distort both receptor extracellular loops and the walls of the ligand binding pocket to varying degrees, rather than requiring sequence-specific bonding chemistries for recognition and signaling.

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.