Danny J. Scholten

Pharmacological modulation of chemokine receptor function

G protein‐coupled chemokine receptors and their peptidergic ligands are interesting therapeutic targets due to their involvement in various immune‐related diseases, including rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, chronic obstructive pulmonary disease, HIV‐1 infection and cancer. To tackle these diseases, a lot of effort has been focused on discovery and development of small‐molecule chemokine receptor antagonists. This has been rewarded by the market approval of two novel chemokine receptor inhibitors, AMD3100 (CXCR4) and Maraviroc (CCR5) for stem cell mobilization and treatment of HIV‐1 infection respectively. The recent GPCR crystal structures together with mutagenesis and pharmacological studies have aided in understanding how small‐molecule ligands interact with chemokine receptors. Many of these ligands display behaviour deviating from simple competition and do not interact with the chemokine binding site, providing evidence for an allosteric mode of action. This review aims to give an overview of the evidence supporting modulation of this intriguing receptor family by a range of ligands, including small molecules, peptides and antibodies. Moreover, the computer‐assisted modelling of chemokine receptor–ligand interactions is discussed in view of GPCR crystal structures. Finally, the implications of concepts such as functional selectivity and chemokine receptor dimerization are considered.

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 Gαi-proteins. CXCR7 has recently been shown to interact with β-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 β-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.

Noncompetitive Antagonism and Inverse Agonism as Mechanism of Action of Nonpeptidergic Antagonists at Primate and Rodent CXCR3 Chemokine Receptors

The chemokine receptor CXCR3 is involved in various inflammatory diseases, such as rheumatoid arthritis, multiple sclerosis, psoriasis, and allograft rejection in transplantation patients. The CXCR3 ligands CXCL9, CXCL10, and CXCL11 are expressed at sites of inflammation, and they attract CXCR3-bearing lymphocytes, thus contributing to the inflammatory process. In this study, we characterize five nonpeptidergic compounds of different chemical classes that block the action of CXCL10 and CXCL11 at the human CXCR3, i.e., the 3H-pyrido[2,3-d]pyrimidin-4-one derivatives N-1R-[3-(4-ethoxy-phenyl)-4-oxo-3,4-dihydro-pyrido[2,3-d]pyrimidin-2-yl]-ethyl-N-pyridin-3-ylmethyl-2-(4-fluoro-3-trifluoromethyl-phenyl)-acetamide (VUF10472/NBI-74330) and N-1R-[3-(4-ethoxy-phenyl)-4-oxo-3,4-dihydro-pyrido[2,3-d]pyrimidin-2-yl]-ethyl-N-pyridin-3-ylmethyl-2-(4-trifluoromethoxy-phenyl)-acetamide (VUF10085/AMG-487), the 3H-quinazolin-4-one decanoic acid {1-[3-(4-cyano-phenyl)-4-oxo-3,4-dihydro-quinazolin-2-yl]-ethyl}-(2-dimethylamino-ethyl)-amide (VUF5834), the imidazolium compound 1,3-bis-[2-(3,4-dichloro-phenyl)-2-oxo-ethyl]-3H-imidazol-1-ium bromide (VUF10132), and the quaternary ammonium anilide N,N-dimethyl-N-[4-[[[2-(4-methylphenyl)-6,7-dihydro-5H-benzocyclohepten-8-yl]-carbonyl]amino]benzyl] tetrahydro-2H-pyran-4-aminium chloride (TAK-779). To understand the action of these CXCR3 antagonists in various animal models of disease, the compounds were also tested at rat and mouse CXCR3, as well as at CXCR3 from rhesus macaque, which was cloned and characterized for the first time in this study. Except for TAK-779, all compounds show slightly lower affinity for rodent CXCR3 than for primate CXCR3. In addition, we have characterized the molecular mechanism of action of the various antagonists at the human CXCR3 receptor. All tested compounds act as noncompetitive antagonists at CXCR3. Moreover, this noncompetitive behavior is accompanied by inverse agonistic properties of all five compounds as determined on an identified constitutively active mutant of CXCR3, CXCR3 N3.35A. It is interesting to note that all compounds except TAK-779 act as full inverse agonists at CXCR3 N3.35A. TAK-779 shows weak partial inverse agonism at CXCR3 N3.35A, and it probably has a different mode of interaction with CXCR3 than the other two classes of small-molecule inverse agonists.

Pharmacological characterization of a small‐molecule agonist for the chemokine receptor CXCR3

The chemokine receptor CXCR3 is a GPCR found predominantly on activated T cells. CXCR3 is activated by three endogenous peptides; CXCL9, CXCL10 and CXCL11. Recently, a small‐molecule agonist, VUF10661, has been reported in the literature and synthesized in our laboratory. The aim of the present study was to provide a detailed pharmacological characterization of VUF10661 by comparing its effects with those of CXCL11.

Synthesis, modeling and functional activity of substituted styrene-amides as small-molecule CXCR7 agonists.

The chemokine receptor CXCR7 is an atypical G protein-coupled receptor as it preferentially signals through the β-arrestin pathway rather than through G proteins. CXCR7 is thought to be of importance in cancer and the development of CXCR7-targeting ligands is of huge importance to further elucidate the pharmacology and the therapeutic potential of CXCR7. In the present study, we synthesized 24 derivatives based on a compound scaffold patented by Chemocentryx and obtained CXCR7 ligands with pK(i) values ranging from 5.3 to 8.1. SAR studies were supported by computational 3D Fingerprint studies, revealing several important affinity descriptors. Two key compounds (29 and 30, VUF11207 and VUF11403) were found to be high-potency ligands that induce recruitment of β-arrestin2 and subsequent internalization of CXCR7, making them important tool compounds in future CXCR7 research.

Identification of Overlapping but Differential Binding Sites for the High-Affinity CXCR3 Antagonists NBI-74330 and VUF11211

CXC chemokine receptor CXCR3 and/or its main three ligands CXCL9, CXCL10, and CXCL11 are highly upregulated in a variety of diseases. As such, considerable efforts have been made to develop small-molecule receptor CXCR3 antagonists, yielding distinct chemical classes of antagonists blocking binding and/or function of CXCR3 chemokines. Although it is suggested that these compounds bind in an allosteric fashion, thus far no evidence has been provided regarding the molecular details of their interaction with CXCR3. Using site-directed mutagenesis complemented with in silico homology modeling, we report the binding modes of two high-affinity CXCR3 antagonists of distinct chemotypes: VUF11211 [(S)-5-chloro-6-(4-(1-(4-chlorobenzyl)piperidin-4-yl)-3-ethylpiperazin-1-yl)-N-ethylnicotinamide] (piperazinyl-piperidine) with a rigid elongated structure containing two basic groups and NBI-74330 [(R)-N-(1-(3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl)ethyl)-2-(4-fluoro-3-(trifluoromethyl)phenyl)-N-(pyridin-3-ylmethyl)acetamide] (8-azaquinazolinone) without any basic group. Here we show that NBI-74330 is anchored in the transmembrane minor pocket lined by helices 2 (W2.60, D2.63), 3 (F3.32), and 7 (S7.39, Y7.43), whereas VUF11211 extends from the minor pocket into the major pocket of the transmembrane domains, located between residues in helices 1 (Y1.39), 2 (W2.60), 3 (F3.32), 4 (D4.60), 6 (Y6.51), and 7 (S7.39, Y7.43). Mutation of these residues did not affect CXCL11 binding significantly, confirming the allosteric nature of the interaction of these small molecules with CXCR3. Moreover, the model derived from our in silico–guided studies fits well with the already published structure–activity relationship data on these ligands. Altogether, in this study, we show overlapping, yet different binding sites for two high-affinity CXCR3 antagonists, which offer new opportunities for the structure-based design of allosteric modulators for CXCR3.

Label-free impedance responses of endogenous and synthetic chemokine receptor CXCR3 agonists correlate with G i -protein pathway activation

The chemokine receptor CXCR3 is a G-protein-coupled receptor that signals through the Gα(i) class of heterotrimeric G-proteins. CXCR3 is highly expressed on activated T cells and has been proposed to be a therapeutic target in autoimmune disease. CXCR3 is activated by the chemokines CXCL9, CXCL10 and CXCL11. CXCR3 signaling properties in response to CXCL10, CXCL11 and the synthetic agonist VUF10661 have previously been evaluated using conventional endpoint assays. In the present study, label-free impedance measurements were used to characterize holistic responses of CXCR3-expressing cells to stimulation with chemokines and VUF10661 in real time and to compare these responses with both G-protein and non-G-protein (β-arrestin2) mediated responses. Differences in response kinetics were apparent between the chemokines and VUF10661. Moreover, CXCR3-independent effects could be distinguished from CXCR3-specific responses with the use of the selective CXCR3 antagonist NBI-74330 and the Gα(i) inhibitor pertussis toxin. By comparing the various responses, we observed that CXCL9 is a biased CXCR3 agonist, stimulating solely G-protein-dependent pathways. Moreover, CXCR3-mediated changes in cellular impedance correlated with G-protein signaling, but not β-arrestin2 recruitment.

Therapeutic targeting of chemokine receptors by small molecules

Targeting of chemokine receptors by small molecules has been widely pursued. This review highlights recent illustrative disclosures of clinical relevance that could further shape our appreciation, and add to our understanding, of the therapeutic value of chemokine receptor targeting. Disclosures include new structures, announcements of new trials, or results of conducted trials (including setbacks). This review shows how most of the discussed disclosures seem to be concentrated on selected receptors, for example, CCR1, CCR2, CCR5, CCR9, CXCR2 and CXCR4, with a wide variety of associated ligand chemotypes and diseases. With two approved antagonist drugs and several in Phase III trials, as well as new antagonist chemotypes entering the pipeline, the chemokine receptor field proves dynamic and upcoming results will further fuel the field.

Chemical Subtleties in Small-Molecule Modulation of Peptide Receptor Function: The Case of CXCR3 Biaryl-Type Ligands

The G protein-coupled chemokine receptor CXCR3 plays a role in numerous inflammatory events. The endogenous ligands for the chemokine receptors are peptides, but in this study we disclose small-molecule ligands that are able to activate CXCR3. A class of biaryl-type compounds that is assembled by convenient synthetic routes is described as a new class of CXCR3 agonists. Intriguingly, structure-activity relationship and structure-function relationship studies reveal that subtle chemical modifications on the outer aryl ring (e.g., either the size or position of a halogen atom) result in a full spectrum of agonist efficacies on CXCR3. Quantum mechanics calculations and nuclear Overhauser effect spectroscopy NMR studies suggest that the biaryl dihedral angle and the electronic nature of ortho-substituents play an important role in determining agonist efficacies. Compounds 38 (VUF11222) and 39 (VUF11418) are the first reported nonpeptidomimetic agonists on CXCR3, rendering them highly useful chemical tools for detailed assessment of CXCR3 activation as well as for studying downstream CXCR3 signaling.

C(X)CR in silico: Computer-aided prediction of chemokine receptor-ligand interactions.

This review will focus on the construction, refinement, and validation of chemokine receptor models for the purpose of structure-based virtual screening and ligand design. The review will present a comparative analysis of ligand binding pockets in chemokine receptors, including a review of the recently released CXCR4 X-ray structures, and their implication on chemokine receptor (homology) modeling. The recommended protein-ligand modeling procedure as well as the use of experimental anchors to steer the modeling procedure is discussed and an overview of several successful structure-based ligand discovery and design studies is provided. This review shows that receptor models, despite structural inaccuracies, can be efficiently used to find novel ligands for chemokine receptors.: