Bilayer lipids modulate ligand binding to atypical chemokine receptor 3.

Chemokine receptors belong to the large class of G protein-coupled receptors (GPCRs) and are involved in a number of (patho)physiological processes. Previous studies highlighted the importance of membrane lipids for modulating GPCR structure and function. However, the underlying mechanisms of how lipids regulate GPCRs are often poorly understood. Here, we report that anionic lipid bilayers increase the binding affinity of the chemokine CXCL12 for the atypical chemokine receptor 3 (ACKR3) by modulating the CXCL12 binding kinetics. Notably, the anionic bilayer favors CXCL12 over the more positively charged chemokine CXCL11, which we explained by bilayer interactions orienting CXCL12 but not CXCL11 for productive ACKR3 binding. Furthermore, our data suggest a stabilization of active ACKR3 conformations in anionic bilayers. Taken together, the described regulation of chemokine selectivity of ACKR3 by the lipid bilayer proposes an extended version of the classical model of chemokine binding including the lipid environment of the receptor.

Structural basis for selectivity and antagonism in extracellular GPCR-nanobodies.

G protein-coupled receptors (GPCRs) are pivotal therapeutic targets, but their complex structure poses challenges for effective drug design. Nanobodies, or single-domain antibodies, have emerged as a promising therapeutic strategy to target GPCRs, offering advantages over traditional small molecules and antibodies. However, an incomplete understanding of the structural features enabling GPCR-nanobody interactions has limited their development. In this study, we investigate VUN701, a nanobody antagonist targeting the atypical chemokine receptor 3 (ACKR3). We determine that an extended CDR3 loop is required for ACKR3 binding. Uncommon in most nanobodies, an extended CDR3 is prevalent in GPCR-targeting nanobodies. Combining experimental and computational approaches, we map an inhibitory ACKR3-VUN701 interface and define a distinct conformational mechanism for GPCR inactivation. Our results provide insights into class A GPCR-nanobody selectivity and suggest a strategy for the development of these new therapeutic tools.

Structural snapshot of a ß-arrestin-biased receptor.

Atypical chemokine receptor subtype 3 (ACKR3), a chemokine receptor, couples selectively to ß-arrestins (ßarrs) but not to G proteins despite having seven transmembrane (7TM) helix architecture. Yen et al. present cryogenic-electron microscopy (cryo-EM) structures of agonist-bound ACKR3, elucidating a distinct chemokine-binding mechanism, and offering a structural template to probe the transducer-coupling bias at this receptor.

Conformational selection guides ß-arrestin recruitment at a biased G protein-coupled receptor.

G protein-coupled receptors (GPCRs) recruit ß-arrestins to coordinate diverse cellular processes, but the structural dynamics driving this process are poorly understood. Atypical chemokine receptors (ACKRs) are intrinsically biased GPCRs that engage ß-arrestins but not G proteins, making them a model system for investigating the structural basis of ß-arrestin recruitment. Here, we performed nuclear magnetic resonance (NMR) experiments on 13CH3-ε-methionine-labeled ACKR3, revealing that ß-arrestin recruitment is associated with conformational exchange at key regions of the extracellular ligand-binding pocket and intracellular ß-arrestin-coupling region. NMR studies of ACKR3 mutants defective in ß-arrestin recruitment identified an allosteric hub in the receptor core that coordinates transitions among heterogeneously populated and selected conformational states. Our data suggest that conformational selection guides ß-arrestin recruitment by tuning receptor dynamics at intracellular and extracellular regions.

Stimulation of the atypical chemokine receptor 3 (ACKR3) by a small-molecule agonist attenuates fibrosis in a preclinical liver but not lung injury model.

Atypical chemokine receptor 3 (ACKR3, formerly CXC chemokine receptor 7) is a G protein-coupled receptor that recruits ß-arrestins, but is devoid of functional G protein signaling after receptor stimulation. In preclinical models of liver and lung fibrosis, ACKR3 was previously shown to be upregulated after acute injury in liver sinusoidal and pulmonary capillary endothelial cells, respectively. This upregulation was linked with a pro-regenerative and anti-fibrotic role for ACKR3. A recently described ACKR3-targeting small molecule agonist protected mice from isoproterenol-induced cardiac fibrosis. Here, we aimed to evaluate its protective role in preclinical models of liver and lung fibrosis. After confirming its in vitro pharmacological activity (i.e., ACKR3-mediated ß-arrestin recruitment and receptor binding), in vivo administration of this ACKR3 agonist led to increased mouse CXCL12 plasma levels, indicating in vivo interaction of the agonist with ACKR3. Whereas twice daily in vivo administration of the ACKR3 agonist lacked inhibitory effect on bleomycin-induced lung fibrosis, it had a modest, but significant anti-fibrotic effect in the carbon tetrachloride (CCl4)-induced liver fibrosis model. In the latter model, ACKR3 stimulation affected the expression of several fibrosis-related genes and led to reduced collagen content as determined by picro-sirius red staining and hydroxyproline quantification. These data confirm that ACKR3 agonism, at least to some extent, attenuates fibrosis, although this effect is rather modest and heterogeneous across various tissue types. Stimulating ACKR3 alone without intervening in other signaling pathways involved in the multicellular crosstalk leading to fibrosis will, therefore, most likely not be sufficient to deliver a satisfactory clinical outcome.

Class A G protein-coupled receptors assemble into functional higher-order hetero-oligomers.

Although class A seven-transmembrane helix (7TM) receptor hetero-oligomers have been proposed, information on the assembly and function of such higher-order hetero-oligomers is not available. Utilizing bioluminescence resonance energy transfer (BRET), bimolecular luminescence/fluorescence complementation (BiLC/BiFC), and BiLC/BiFC BRET in HEK293T cells, we provide evidence that chemokine (C-X-C motif) receptor 4, atypical chemokine receptor 3, α1a -adrenoceptor, and arginine vasopressin receptor 1A form hetero-oligomers composed of 2-4 different protomers. We show that hetero-oligomerization per se and ligand binding to individual protomers regulate agonist-induced coupling to the signaling transducers of interacting receptor partners. Our findings support the concept that receptor hetero-oligomers form supramolecular machineries with molecular signaling properties distinct from the individual protomers. These findings provide a mechanism for the phenomenon of context-dependent receptor function.

The evolution and functional characterization of CXC chemokines and receptors in lamprey.

Chemokines are a large family of soluble peptides guiding cell migration in development and immune defense. They interact with chemokine receptors and are essential for the coordination of cell migration in diverse physiological processes. The CXC subfamily is one of the largest groups in the chemokine family and consists of multiple members. In this study, we identified homologues of three chemokine ligands (CXCL8, CXCL_F5 and CXCL12) and two CXC receptor like molecules (CXCR_L1 and CXCR_L2) in lamprey. Sequence analysis revealed that they share the same genomic organization with their counterparts in jawed vertebrates but synteny was not conserved. Lamprey CXCL8 and CXCL12 have four conserved cysteine residues whilst the CXCL_F5 has two additional cysteine residues. In addition, CXCL_F5 is evolutionarily related to the fish specific CXC chemokine groups previously identified and contains multiple cationic aa residues in the extended C- terminal region. The two CXCRs possess seven transmembrane domains and conserved structural elements for receptor activation and signaling, including the DRYXXI(V)Y motif in TM2, the disulphide bond connecting ECL2 and TM3, the WXP motif in TM6 and NPXXY motif in TM7. The identified CXC chemokines and receptors were constitutively expressed in tissues including the liver, kidney, intestine, heart, gills, supraneural body and primary leukocytes, but exhibited distinct expression patterns. Relatively high expression was detected in the gills for CXCL8, CXCL_F5 and CXCR_L1 and in the supraneural body for CXCL12 and CXCR_L2. All the genes except CXCL12 were upregulated by stimulation with LPS, pokeweed and bacterial infection, and the CXCL8 and CXCL_F5 was induced by poly (I:C). Functional analysis showed that the CXCL8 and CXCL_F5 specifically interacted with CXCR_L1 and CXCR_L2, respectively. Our results demonstrate that the CXC chemokine system had diversified in jawless fish.

Application of 2,2'-dipyridyl disulfide-mediated thiazolidine ring-opening reaction to glycoprotein synthesis: Total chemical synthesis of evasin-3.

Thiazolidine ring-opening reaction is one of the key steps in protein chemical synthesis via sequential native chemical ligation strategy. We recently developed a novel thiazolidine ring-opening reaction with 2,2'-dipyridyl disulfide (DPDS). In order to investigate the applicability of this reaction to glycoprotein synthesis, we synthesized evasin-3, a cysteine-rich glycoprotein with chemokine-binding ability originally found in tick saliva. The sequence of evasin-3 was divided into three segments, and these segments were separately synthesized with the ordinary solid-phase peptide synthesis method. After the first ligation of middle and C-terminal segments, thiazolidine used as a protecting group of Cys residue at the N-terminus of the middle segment was converted to Cys with DPDS. In this thiazolidine ring-opening reaction, DPDS treatment did not affect the N-linked glycan moiety. After the second ligation with the N-terminal segment and the refolding reaction, evasin-3 could be obtained in good yield. The synthetic evasin-3 showed the binding ability specifically to CXCL chemokines. These results clearly indicate that this DPDS method is useful for glycoprotein synthesis.

The chemokine X-factor: Structure-function analysis of the CXC motif at CXCR4 and ACKR3.

The human chemokine family consists of 46 protein ligands that induce chemotactic cell migration by activating a family of 23 G protein-coupled receptors. The two major chemokine subfamilies, CC and CXC, bind distinct receptor subsets. A sequence motif defining these families, the X position in the CXC motif, is not predicted to make significant contacts with the receptor, but instead links structural elements associated with binding and activation. Here, we use comparative analysis of chemokine NMR structures, structural modeling, and molecular dynamic simulations that suggested the X position reorients the chemokine N terminus. Using CXCL12 as a model CXC chemokine, deletion of the X residue (Pro-10) had little to no impact on the folded chemokine structure but diminished CXCR4 agonist activity as measured by ERK phosphorylation, chemotaxis, and Gi/o-mediated cAMP inhibition. Functional impairment was attributed to over 100-fold loss of CXCR4 binding affinity. Binding to the other CXCL12 receptor, ACKR3, was diminished nearly 500-fold. Deletion of Pro-10 had little effect on CXCL12 binding to the CXCR4 N terminus, a major component of the chemokine-GPCR interface. Replacement of the X residue with the most frequent amino acid at this position (P10Q) had an intermediate effect between WT and P10del in each assay, with ACKR3 having a higher tolerance for this mutation. This work shows that the X residue helps to position the CXCL12 N terminus for optimal docking into the orthosteric pocket of CXCR4 and suggests that the CC/CXC motif contributes directly to receptor selectivity by orienting the chemokine N terminus in a subfamily-specific direction.

Crystal Structure of ß-Arrestin 2 in Complex with CXCR7 Phosphopeptide.

ß-Arrestins (ßarrs) critically regulate G-protein-coupled receptor (GPCR) signaling and trafficking. ßarrs have two isoforms, ßarr1 and ßarr2. Receptor phosphorylation is a key determinant for the binding of ßarrs, and understanding the intricate details of receptor-ßarr interaction is the next frontier in GPCR structural biology. The high-resolution structure of active ßarr1 in complex with a phosphopeptide derived from GPCR has been revealed, but that of ßarr2 remains elusive. Here, we present a 2.3-Å crystal structure of ßarr2 in complex with a phosphopeptide (C7pp) derived from the carboxyl terminus of CXCR7. The structural analysis of C7pp-bound ßarr2 reveals key differences from the previously determined active conformation of ßarr1. One of the key differences is that C7pp-bound ßarr2 shows a relatively small inter-domain rotation. Antibody-fragment-based conformational sensor and hydrogen/deuterium exchange experiments further corroborated the structural features of ßarr2 and suggested that ßarr2 adopts a range of inter-domain rotations.

Differential activity and selectivity of N-terminal modified CXCL12 chemokines at the CXCR4 and ACKR3 receptors.

Chemokines play critical roles in numerous physiologic and pathologic processes through their action on seven-transmembrane (TM) receptors. The N-terminal domain of chemokines, which is a key determinant of signaling via its binding within a pocket formed by receptors' TM helices, can be the target of proteolytic processing. An illustrative case of this regulatory mechanism is the natural processing of CXCL12 that generates chemokine variants lacking the first two N-terminal residues. Whereas such truncated variants behave as antagonists of CXCR4, the canonical G protein-coupled receptor of CXCL12, they are agonists of the atypical chemokine receptor 3 (ACKR3/CXCR7), suggesting the implication of different structural determinants in the complexes formed between CXCL12 and its two receptors. Recent analyses have suggested that the CXCL12 N-terminus first engages the TM helices of ACKR3 followed by the receptor N-terminus wrapping around the chemokine core. Here we investigated the first stage of ACKR3-CXCL12 interactions by comparing the activity of substituted or N-terminally truncated variants of CXCL12 toward CXCR4 and ACKR3. We showed that modification of the first two N-terminal residues of the chemokine (K1R or P2G) does not alter the ability of CXCL12 to activate ACKR3. Our results also identified the K1R variant as a G protein-biased agonist of CXCR4. Comparative molecular dynamics simulations of the complexes formed by ACKR3 either with CXCL12 or with the P2G variant identified interactions between the N-terminal 2-4 residues of CXCL12 and a pocket formed by receptor's TM helices 2, 6, and 7 as critical determinants for ACKR3 activation.

Kinetics of CXCL12 binding to atypical chemokine receptor 3 reveal a role for the receptor N terminus in chemokine binding.

Chemokines bind to membrane-spanning chemokine receptors, which signal through G proteins and promote cell migration. However, atypical chemokine receptor 3 (ACKR3) does not appear to couple to G proteins, and instead of directly promoting cell migration, it regulates the extracellular concentration of chemokines that it shares with the G protein-coupled receptors (GPCRs) CXCR3 and CXCR4, thereby influencing the responses of these receptors. Understanding how these receptors bind their ligands is important for understanding these different processes. Here, we applied association and dissociation kinetic measurements coupled to ß-arrestin recruitment assays to investigate ACKR3:chemokine interactions. Our results showed that CXCL12 binding is unusually slow and driven by the interplay between multiple binding epitopes. We also found that the amino terminus of the receptor played a key role in chemokine binding and activation by preventing chemokine dissociation. It was thought that chemokines initially bind receptors through interactions between the globular domain of the chemokine and the receptor amino terminus, which then guides the chemokine amino terminus into the transmembrane pocket of the receptor to initiate signaling. On the basis of our kinetic data, we propose an alternative mechanism in which the amino terminus of the chemokine initially forms interactions with the extracellular loops and transmembrane pocket of the receptor, which is followed by the receptor amino terminus wrapping around the core of the chemokine to prolong its residence time. These data provide insight into how ACKR3 competes and cooperates with canonical GPCRs in its function as a scavenger receptor.

Decreased ACKR3 (CXCR7) function causes oculomotor synkinesis in mice and humans.

Oculomotor synkinesis is the involuntary movement of the eyes or eyelids with a voluntary attempt at a different movement. The chemokine receptor CXCR4 and its ligand CXCL12 regulate oculomotor nerve development; mice with loss of either molecule have oculomotor synkinesis. In a consanguineous family with congenital ptosis and elevation of the ptotic eyelid with ipsilateral abduction, we identified a co-segregating homozygous missense variant (c.772G>A) in ACKR3, which encodes an atypical chemokine receptor that binds CXCL12 and functions as a scavenger receptor, regulating levels of CXCL12 available for CXCR4 signaling. The mutant protein (p.V258M) is expressed and traffics to the cell surface but has a lower binding affinity for CXCL12. Mice with loss of Ackr3 have variable phenotypes that include misrouting of the oculomotor and abducens nerves. All embryos show oculomotor nerve misrouting, ranging from complete misprojection in the midbrain, to aberrant peripheral branching, to a thin nerve, which aberrantly innervates the lateral rectus (as seen in Duane syndrome). The abducens nerve phenotype ranges from complete absence, to aberrant projections within the orbit, to a normal trajectory. Loss of ACKR3 in the midbrain leads to downregulation of CXCR4 protein, consistent with reports that excess CXCL12 causes ligand-induced degradation of CXCR4. Correspondingly, excess CXCL12 applied to ex vivo oculomotor slices causes axon misrouting, similar to inhibition of CXCR4. Thus, ACKR3, through its regulation of CXCL12 levels, is an important regulator of axon guidance in the oculomotor system; complete loss causes oculomotor synkinesis in mice, while reduced function causes oculomotor synkinesis in humans.

Discovery of Amantamide, a Selective CXCR7 Agonist from Marine Cyanobacteria.

CXCR7 plays an emerging role in several physiological processes. A linear peptide, amantamide (1), was isolated from marine cyanobacteria, and the structure was determined by NMR and mass spectrometry. The total synthesis was achieved by solid-phase method. After screening two biological target libraries, 1 was identified as a selective CXCR7 agonist. The selective activation of CXCR7 by 1 could provide the basis for developing CXCR7-targeted therapeutics and deciphering the role of CXCR7 in different diseases.

CXCR7/CXCR4 heterodimer-induced histone demethylation: a new mechanism of colorectal tumorigenesis.

Both chemokine receptors (CXCRs) 7 and 4 can facilitate immune cell migration and mediate a vast array of physiological and pathological events. Herein we report, in both human and animal studies, that these two CXCRs can form heterodimers in vivo and promote colorectal tumorigenesis through histone demethylation. Compared with adjacent non-neoplastic tissue, human colorectal cancer (CRC) tissue showed a significant higher expression of CXCR4 and CXCR7, which was colocalized in the cancer cell epithelium. The CXCR/CXCR4 heterodimerization was associated with increased histone demethylase JMJD2A. Villin-CXCR7-CXCR4 transgenic mice demonstrated a greater degree of exacerbated colitis and tumorigenesis than villin-CXCR7 and villin-CXCR4 mice. The CXCR7/CXCR4 heterodimerization also promoted APC mutation-driven colorectal tumorigenesis in APCMin/+/villin-CXCR7-CXCR4 mice. Further analysis showed that the CXCR7/CXCR4 heterodimer induced nuclear ßarr1 recruitment and histone demethylase JMJD2A, leading to histone demethylation and resulting in transcription of inflammatory factors and oncogenes. This study uncovered a novel mechanism of colorectal tumorigenesis through the CXCR7/CXCR4 heterodimer-induced histone demethylation. Inhibition of CXCR7/CXCR4 heterodimer-induced histone demethylation could be an effective strategy for the prevention and treatment of colorectal cancer.

Molecular characterization and expression analysis of Asian swamp eel (Monopterus albus) CXC chemokine receptor (CXCR) 1a, CXCR1b, CXCR2, CXCR3a, CXCR3b, and CXCR4 after bacteria and poly I:C challenge.

The CXC chemokine receptors (CXCRs) play critical roles in innate and adaptive immune systems. In this study, six Asian swamp eel (Monopterus albus) CXCRs (MaCXCR1-4) were identified and their molecular characterization and expression patterns were analyzed. The open reading frames (ORFs) of MaCXCR1a, MaCXCR1b, MaCXCR2, MaCXCR3a, MaCXCR3b, and MaCXCR4 were 1074 bp (base pairs), 1080 bp, 1125 bp, 1146 bp, 1083 bp, and 1140 bp, and encoded proteins of 357 aa (amino acids), 359 aa, 374 aa, 381 aa, 360 aa, and 379 aa, respectively. All these CXCRs have seven conserved transmembrane domains and four cysteines (with the exception of MaCXCR3b). Multiple sequence alignment revealed that the MaCXCRs possess a typical G-protein receptor family 1 signature and a DRY motif. There are also one to four potential N-glycosylation sites in the extracellular regions of the MaCXCRs, mainly distributed in the N-terminus and extracellular hydrophilic loop (ECL) 2 region. Phylogenetic analysis demonstrated that the MaCXCRs were clustered together with homologous proteins from other fish. Taken together with the amino acid identity and similarity analysis, these results suggested that the MaCXCRs are conserved with other homologous genes, in which CXCR4 is more conserved than CXCR1-3. The MaCXCRs loci showed conserved synteny among teleost fish, and we found that human CXCR1 shares a common ancestor with fish CXCR1a. MaCXCRs were constitutively expressed in a wide range of tissues (especially in immune-related tissues) with different expression levels, suggesting that the MaCXCRs have different roles in un-stimulated tissues, and may play vital roles under normal conditions. MaCXCRs showed different fold changes in the spleen after Aeromonas veronii and polyinosinic-polycytidylic acid (poly I:C) challenge, which suggested that MaCXCR1a and MaCXCR3a have longer antiviral activities compared with their antibacterial functions, and that MaCXCR1b possesses stronger antiviral than antibacterial activity. MaCXCR4 may play vital roles during bacterial and viral infection; however, MaCXCR2 has relatively small effect in antibacterial and antiviral responses. The differential responses of these genes to bacteria and poly I:C implied the differences in the mechanisms of defense against viruses and bacteria.

How do chemokines navigate neutrophils to the target site: Dissecting the structural mechanisms and signaling pathways.

Chemokines play crucial roles in combating microbial infection and initiating tissue repair by recruiting neutrophils in a timely and coordinated manner. In humans, no less than seven chemokines (CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and CXCL8) and two receptors (CXCR1 and CXCR2) mediate neutrophil functions but in a context dependent manner. Neutrophil-activating chemokines reversibly exist as monomers and dimers, and their receptor binding triggers conformational changes that are coupled to G-protein and ß-arrestin signaling pathways. G-protein signaling activates a variety of effectors including Ca2+ channels and phospholipase C. ß-arrestin serves as a multifunctional adaptor and is coupled to several signaling hubs including MAP kinase and tyrosine kinase pathways. Both G-protein and ß-arrestin signaling pathways play important non-overlapping roles in neutrophil trafficking and activation. Functional studies have established many similarities but distinct differences for a given chemokine and between chemokines at the level of monomer vs. dimer, CXCR1 vs. CXCR2 activation, and G-protein vs. ß-arrestin pathways. We propose that two forms of the ligand binding two receptors and activating two signaling pathways enables fine-tuned neutrophil function compared to a single form, a single receptor, or a single pathway. We summarize the current knowledge on the molecular mechanisms by which chemokine monomers/dimers activate CXCR1/CXCR2 and how these interactions trigger G-protein/ß-arrestin-coupled signaling pathways. We also discuss current challenges and knowledge gaps, and likely advances in the near future that will lead to a better understanding of the relationship between the chemokine-CXCR1/CXCR2-G-protein/ß-arrestin axis and neutrophil function.

Partial agonist activity of α1-adrenergic receptor antagonists for chemokine (C-X-C motif) receptor 4 and atypical chemokine receptor 3.

We observed in PRESTO-Tango ß-arrestin recruitment assays that the α1-adrenergic receptor (AR) antagonist prazosin activates chemokine (C-X-C motif) receptor (CXCR)4. This prompted us to further examine this unexpected pharmacological behavior. We screened a panel of 14 α1/2- and ß1/2/3-AR antagonists for CXCR4 and atypical chemokine receptor (ACKR)3 agonist activity in PRESTO-Tango assays against the cognate agonist CXCL12. We observed that multiple α1-AR antagonists activate CXCR4 (CXCL12 = prazosin = cyclazosin > doxazosin) and ACKR3 (CXCL12 = prazosin = cyclazosin > alfuzosin = doxazosin = phentolamine > terazosin = silodosin = tamsulosin). The two strongest CXCR4/ACKR3 activators, prazosin and cyclazosin, were selected for a more detailed evaluation. We found that the drugs dose-dependently activate both receptors in ß-arrestin recruitment assays, stimulate ERK1/2 phosphorylation in HEK293 cells overexpressing each receptor, and that their effects on CXCR4 could be inhibited with AMD3100. Both α1-AR antagonists induced significant chemical shift changes in the 1H-13C-heteronuclear single quantum correlation spectrum of CXCR4 and ACKR3 in membranes, suggesting receptor binding. Furthermore, prazosin and cyclazosin induced internalization of endogenous CXCR4/ACKR3 in human vascular smooth muscle cells (hVSMC). While these drugs did not in induce chemotaxis in hVSMC, they inhibited CXCL12-induced chemotaxis with high efficacy and potency (IC50: prazosin-4.5 nM, cyclazosin 11.6 pM). Our findings reveal unexpected pharmacological properties of prazosin, cyclazosin, and likely other α1-AR antagonists. The results of the present study imply that prazosin and cyclazosin are biased or partial CXCR4/ACKR3 agonists, which function as potent CXCL12 antagonists. Our findings could provide a mechanistic basis for previously observed anti-cancer properties of α1-AR antagonists and support the concept that prazosin could be re-purposed for the treatment of disease processes in which CXCR4 and ACKR3 are thought to play significant pathophysiological roles, such as cancer metastases or various autoimmune pathologies.

Structure-Activity Relationship Study of Cyclic Pentapeptide Ligands for Atypical Chemokine Receptor 3 (ACKR3).

The atypical chemokine receptor 3 (ACKR3)/CXC chemokine receptor 7 (CXCR7) recognizes stromal cell-derived factor 1 (SDF-1)/CXCL12 and is involved in a number of physiological and pathological processes. Here, we investigated the SAR of the component amino acids in an ACKR3-selective ligand, FC313 [ cyclo(-d-Tyr-l-Arg-l-MeArg-l-Nal(2)-l-Pro-)], for the development of highly active ACKR3 ligands. Notably, modification at the l-Pro position with a bulky hydrophobic side chain led to improved bioactivity toward ACKR3.

Biological/pathological functions of the CXCL12/CXCR4/CXCR7 axes in the pathogenesis of bladder cancer.

CXC chemokine ligand 12 (CXCL12) is an important member of the CXC subfamily of chemokines, and has been extensively studied in various human body organs and systems, both in physiological and clinical states. Ligation of CXCL12 to CXCR4 and CXCR7 as its receptors on peripheral immune cells gives rise to pleiotropic activities. CXCL12 itself is a highly effective chemoattractant which conservatively attracts lymphocytes and monocytes, whereas there exists no evidence to show attraction for neutrophils. CXCL12 regulates inflammation, neo-vascularization, metastasis, and tumor growth, phenomena which are all pivotally involved in cancer development and further metastasis. Generation and secretion of CXCL12 by stromal cells facilitate attraction of cancer cells, acting through its cognate receptor, CXCR4, which is expressed by both hematopoietic and non-hematopoietic tumor cells. CXCR4 stimulates tumor progression by different mechanisms and is required for metastatic spread to organs where CXCL12 is expressed, thereby allowing tumor cells to access cellular niches, such as the marrow, which favor tumor cell survival and proliferation. It has also been demonstrated that CXCL12 binds to another seven-transmembrane G-protein receptor or G-protein-coupled receptor, namely CXCR7. These studies indicated critical roles for CXCR4 and CXCR7 mediation of tumor metastasis in several types of cancers, suggesting their contributions as biomarkers of tumor behavior as well as potential therapeutic targets. Furthermore, CXCL12 itself has the capability to stimulate survival and growth of neoplastic cells in a paracrine fashion. CXCL12 is a supportive chemokine for tumor neovascularization via attracting endothelial cells to the tumor microenvironment. It has been suggested that elevated protein and mRNA levels of CXCL12/CXCR4/CXCR7 are associated with human bladder cancer (BC). Taken together, mounting evidence suggests a role for CXCR4, CXCR7, and their ligand CXCL12 during the genesis of BC and its further development. However, a better understanding is still required before exploring CXCL12/CXCR4/CXCR7 targeting in the clinic.