Fluorescent Ligands Targeting the Intracellular Allosteric Binding Site of the Chemokine Receptor CCR2.

Fluorescently labeled ligands are versatile molecular tools to study G protein-coupled receptors (GPCRs) and can be used for a range of different applications, including bioluminescence resonance energy transfer (BRET) assays. Here, we report the structure-based development of fluorescent ligands targeting the intracellular allosteric binding site (IABS) of the CC chemokine receptor 2 (CCR2), a class A GPCR that has been pursued as a drug target in oncology and inflammation. Starting from previously reported intracellular CCR2 antagonists, several tetramethylrhodamine (TAMRA)-labeled CCR2 ligands were designed, synthesized, and tested for their suitability as fluorescent reporters to probe binding to the IABS of CCR2. By means of these studies, we developed 14 as a fluorescent CCR2 ligand, enabling cell-free as well as cellular NanoBRET-based binding studies in a non-isotopic and high-throughput manner. Further, we show that 14 can be used as a tool for fragment-based screening approaches. Thus, our small-molecule-based fluorescent CCR2 ligand 14 represents a promising tool for future studies of CCR2 pharmacology.

A cell-based multiplex immunoassay platform using fluorescent protein-barcoded reporter cell lines.

Multiplex immunoassays with acellular antigens are well-established based on solid-phase platforms such as the Luminex® technology. Cell barcoding by amine-reactive fluorescent dyes enables analogous cell-based multiplex assays, but requires multiple labeling reactions and quality checks prior to every assay. Here we describe generation of stable, fluorescent protein-barcoded reporter cell lines suitable for multiplex screening of antibody to membrane proteins. The utility of this cell-based system, with the potential of a 256-plex cell panel, is demonstrated by flow cytometry deconvolution of barcoded cell panels expressing influenza A hemagglutinin trimers, or native human CCR2 or CCR5 multi-span proteins and their epitope-defining mutants. This platform will prove useful for characterizing immunity and discovering antibodies to membrane-associated proteins.

The molecular structure and role of CCL2 (MCP-1) and C-C chemokine receptor CCR2 in skeletal biology and diseases.

Monocyte chemoattractant protein-1, also called chemokine (C-C motif) ligand 2 (CCL2) or small inducible cytokine A2, is an inflammatory mediator capable of recruiting monocytes, memory T cells, and dendritic cells. CCL2 is a member of the CC chemokine superfamily, which binds to its receptor, C-C motif chemokine receptor-2 (CCR2), for the induction of chemotactic activity and an increase of calcium influx. It exerts multiple effects on a variety of cells, including monocytes, macrophages, osteoclasts, basophils, and endothelial cells, and is involved in a diverse range of diseases. This review discusses the molecular structure and role of CCL2 and CCR2 in skeletal biology and disease. Molecular structure analyses reveal that CCL2 shares a conserved C-C motif; however, it has only limited sequence homology with other CCL family members. Likewise, CCR2, as a member of the G-protein-coupled seven-transmembrane receptor superfamily, shares conserved cysteine residues, but exhibits very limited sequence homology with other CCR family members. In the skeletal system, the expression of CCL2 is regulated by a variety of factors, such as parathyroid hormone/parathyroid hormone-related peptide, interleukin 1b, tumor necrosis factor-α and transforming growth factor-beta, RANKL, and mechanical forces. The interaction of CCL2 and CCR2 activates several signaling cascades, including PI3K/Akt/ERK/NF-κB, PI3K/MAPKs, and JAK/STAT-1/STAT-3. Understanding the role of CCL2 and CCR2 will facilitate the development of novel therapies for skeletal disorders, including rheumatoid arthritis, osteolysis and other inflammatory diseases related to abnormal chemotaxis.

Chemokine Receptor 2-targeted Molecular Imaging in Pulmonary Fibrosis. A Clinical Trial.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a progressive inflammatory lung disease without effective molecular markers of disease activity or treatment responses. Monocyte and interstitial macrophages that express the C-C motif CCR2 (chemokine receptor 2) are active in IPF and central to fibrosis.Objectives: To phenotype patients with IPF for potential targeted therapy, we developed 64Cu-DOTA-ECL1i, a radiotracer to noninvasively track CCR2+ monocytes and macrophages using positron emission tomography (PET).Methods: CCR2+ cells were investigated in mice with bleomycin- or radiation-induced fibrosis and in human subjects with IPF. The CCR2+ cell populations were localized relative to fibrotic regions in lung tissue and characterized using immunolocalization, single-cell mass cytometry, and Ccr2 RNA in situ hybridization and then correlated with parallel quantitation of lung uptake by 64Cu-DOTA-ECL1i PET.Measurements and Main Results: Mouse models established that increased 64Cu-DOTA-ECL1i PET uptake in the lung correlates with CCR2+ cell infiltration associated with fibrosis (n = 72). As therapeutic models, the inhibition of fibrosis by IL-1ß blockade (n = 19) or antifibrotic pirfenidone (n = 18) reduced CCR2+ macrophage accumulation and uptake of the radiotracer in mouse lungs. In lung tissues from patients with IPF, CCR2+ cells concentrated in perifibrotic regions and correlated with radiotracer localization (n = 21). Human imaging revealed little lung uptake in healthy volunteers (n = 7), whereas subjects with IPF (n = 4) exhibited intensive signals in fibrotic zones.Conclusions: These findings support a role for imaging CCR2+ cells within the fibrogenic niche in IPF to provide a molecular target for personalized therapy and monitoring.Clinical trial registered with www.clinicaltrials.gov (NCT03492762).

Rational Design of Monodispersed Mutants of Proteins by Identifying Aggregation Contact Sites Using Solubilizing Agents.

Suppression of protein aggregation is a subject of growing importance in the treatment of protein aggregation diseases, an urgent worldwide human health problem, and the production of therapeutic proteins, such as antibody drugs. We previously reported a method to identify compounds that suppress aggregation, based on screening using multiple terminal deletion mutants. We now present a method to determine the aggregation contact sites of proteins, using such solubilizing compounds, to design monodispersed mutants. We applied this strategy to the chemokine receptor-binding domain (CRBD) of FROUNT, which binds to the membrane-proximal C-terminal intracellular region of CCR2. Initially, the backbone NMR signals were assigned to a certain extent by available methods, and the putative locations of five α-helices were identified. Based on NMR chemical shift perturbations upon varying the protein concentrations, the first and third helices were found to contain the aggregation contact sites. The two helices are amphiphilic, and based on an NMR titration with 1,6-hexanediol, a CRBD solubilizing compound, the contact sites were identified as the hydrophobic patches located on the hydrophilic sides of the two helices. Subsequently, we designed multiple mutants targeting amino acid residues on the contact sites. Based on their NMR spectra, a doubly mutated CRBD (L538E/P612S) was selected from the designed mutants, and its monodispersed nature was confirmed by other biophysical methods. We then assessed the CCR2-binding activities of the mutants. Our method is useful for the protein structural analyses, the treatment of protein aggregation diseases, and the improvement of therapeutic proteins.

Structural Basis for Allosteric Ligand Recognition in the Human CC Chemokine Receptor 7.

The CC chemokine receptor 7 (CCR7) balances immunity and tolerance by homeostatic trafficking of immune cells. In cancer, CCR7-mediated trafficking leads to lymph node metastasis, suggesting the receptor as a promising therapeutic target. Here, we present the crystal structure of human CCR7 fused to the protein Sialidase NanA by using data up to 2.1 Å resolution. The structure shows the ligand Cmp2105 bound to an intracellular allosteric binding pocket. A sulfonamide group, characteristic for various chemokine receptor ligands, binds to a patch of conserved residues in the Gi protein binding region between transmembrane helix 7 and helix 8. We demonstrate how structural data can be used in combination with a compound repository and automated thermal stability screening to identify and modulate allosteric chemokine receptor antagonists. We detect both novel (CS-1 and CS-2) and clinically relevant (CXCR1-CXCR2 phase-II antagonist Navarixin) CCR7 modulators with implications for multi-target strategies against cancer.

Structural basis for ligand modulation of the CCR2 conformational landscape.

CC chemokine receptor 2 (CCR2) is a part of the chemokine receptor family, an important class of therapeutic targets. These class A G-protein coupled receptors (GPCRs) are involved in mammalian signaling pathways and control cell migration toward endogenous CC chemokine ligands, named for the adjacent cysteine motif on their N terminus. Chemokine receptors and their associated ligands are involved in a wide range of diseases and thus have become important drug targets. CCR2, in particular, promotes the metastasis of cancer cells and is also implicated in autoimmunity-driven type-1 diabetes, diabetic nephropathy, multiple sclerosis, asthma, atherosclerosis, neuropathic pain, and rheumatoid arthritis. Although promising, CCR2 antagonists have been largely unsuccessful to date. Here, we investigate the effect of an orthosteric and an allosteric antagonist on CCR2 dynamics by coupling long-timescale molecular dynamics simulations with Markov-state model theory. We find that the antagonists shift CCR2 into several stable inactive conformations that are distinct from the crystal structure conformation and disrupt a continuous internal water and sodium ion pathway, preventing transitions to an active-like state. Several metastable conformations present a cryptic drug-binding pocket near the allosteric site that may be amenable to targeting with small molecules. Without antagonists, the apo dynamics reveal intermediate conformations along the activation pathway that provide insight into the basal dynamics of CCR2 and may also be useful for future drug design.

Structural refinement and prediction of potential CCR2 antagonists through validated multi-QSAR modeling studies.

Chemokines trigger numerous inflammatory responses and modulate the immune system. The interaction between monocyte chemoattractant protein-1 and chemokine receptor 2 (CCR2) may be the cause of atherosclerosis, obesity, and insulin resistance. However, CCR2 is also implicated in other inflammatory diseases such as rheumatoid arthritis, multiple sclerosis, asthma, and neuropathic pain. Therefore, there is a paramount importance of designing potent and selective CCR2 antagonists despite a number of drug candidates failed in clinical trials. In this article, 83 CCR2 antagonists by Jhonson and Jhonson Pharmaceuticals have been considered for robust validated multi-QSAR modeling studies to get an idea about the structural and pharmacophoric requirements for designing more potent CCR2 antagonists. All these QSAR models were validated and statistically reliable. Observations resulted from different modeling studies correlated and validated results of other ones. Finally, depending on these QSAR observations, some new molecules were proposed that may exhibit higher activity against CCR2.

Arginine 313 of the putative 8th helix mediates Gαq/14 coupling of human CC chemokine receptors CCR2a and CCR2b.

In man, two CC chemokine receptor isoforms, CCR2a and CCR2b, are present that belong to the rhodopsin-like G protein-coupled receptor family, and couple to Gi and Gq family members. The CCR2 receptors are known to regulate canonical functions of chemokines such as directed migration of leukocytes, and to potentially control non-canonical functions such as differentiation, proliferation, and gene transcription of immune and non-immune cells. We recently reported on the activation of phospholipase C isoenzymes and RhoA GTPases by coupling of the two CCR2 receptors to members of the Gq family, in particular Gαq and Gα14. So far little is known about the structural requirements for the CCR2/Gq/14 interaction. Interestingly, the CCR2 receptor isoforms are identical up to arginine 313 (R313) that is part of the putative 8th helix in CCR2 receptors, and the 8th helix has been implicated in the interaction of rhodopsin-like G protein-coupled receptors with Gαq. In the present work we describe that the 8th helix of both CCR2a and CCR2b is critically involved in selectively activating Gαq/14-regulated signaling. Refined analysis using various CCR2a and CCR2b mutants and analyzing their cellular signaling, e.g. ligand-dependent (i) activation of phospholipase C isoenzymes, (ii) stimulation of serum response factor-mediated gene transcription, (iii) activation of mitogen-activated protein kinases, (iv) internalization, and (v) changes in intracellular calcium concentrations, identified arginine 313 within the amino terminal portion of helix 8 to play a role for the agonist-mediated conformational changes and the formation of a Gαq/14 binding surface. We show that R313 determines Gαq/14 protein-dependent but not Gi protein-dependent cellular signaling, and plays no role in Gq/Gi-independent receptor internalization, indicating a role of R313 in biased signaling of CCR2 receptors.

Crystal Structure of CC Chemokine Receptor 2A in Complex with an Orthosteric Antagonist Provides Insights for the Design of Selective Antagonists.

We determined two crystal structures of the chemokine receptor CCR2A in complex with the orthosteric antagonist MK-0812. Full-length CCR2A, stabilized by rubredoxin and a series of five mutations were resolved at 3.3 Å. An N- and C-terminally truncated CCR2A construct was crystallized in an alternate crystal form, which yielded a 2.7 Å resolution structure using serial synchrotron crystallography. Our structures provide a clear structural explanation for the observed key role of residue E2917.39 in high-affinity binding of several orthosteric CCR2 antagonists. By combining all the structural information collected, we generated models of co-structures for the structurally diverse pyrimidine amide class of CCR2 antagonists. Even though the representative Ex15 overlays well with MK-0812, it also interacts with the non-conserved H1213.33, resulting in a significant selectivity over CCR5. Insights derived from this work will facilitate drug discovery efforts directed toward highly selective CCR2 antagonists with potentially superior efficacy.

Pyrrolone Derivatives as Intracellular Allosteric Modulators for Chemokine Receptors: Selective and Dual-Targeting Inhibitors of CC Chemokine Receptors 1 and 2.

The recent crystal structures of CC chemokine receptors 2 and 9 (CCR2 and CCR9) have provided structural evidence for an allosteric, intracellular binding site. The high conservation of residues involved in this site suggests its presence in most chemokine receptors, including the close homologue CCR1. By using [3H]CCR2-RA-[ R], a high-affinity, CCR2 intracellular ligand, we report an intracellular binding site in CCR1, where this radioligand also binds with high affinity. In addition, we report the synthesis and biological characterization of a series of pyrrolone derivatives for CCR1 and CCR2, which allowed us to identify several high-affinity intracellular ligands, including selective and potential multitarget antagonists. Evaluation of selected compounds in a functional [35S]GTPγS assay revealed that they act as inverse agonists in CCR1, providing a new manner of pharmacological modulation. Thus, this intracellular binding site enables the design of selective and multitarget inhibitors as a novel therapeutic approach.

Closely related, yet unique: Distinct homo- and heterodimerization patterns of G protein coupled chemokine receptors and their fine-tuning by cholesterol.

Chemokine receptors, a subclass of G protein coupled receptors (GPCRs), play essential roles in the human immune system, they are involved in cancer metastasis as well as in HIV-infection. A plethora of studies show that homo- and heterodimers or even higher order oligomers of the chemokine receptors CXCR4, CCR5, and CCR2 modulate receptor function. In addition, membrane cholesterol affects chemokine receptor activity. However, structural information about homo- and heterodimers formed by chemokine receptors and their interplay with cholesterol is limited. Here, we report homo- and heterodimer configurations of the chemokine receptors CXCR4, CCR5, and CCR2 at atomistic detail, as obtained from thousands of molecular dynamics simulations. The observed homodimerization patterns were similar for the closely related CC chemokine receptors, yet they differed significantly between the CC receptors and CXCR4. Despite their high sequence identity, cholesterol modulated the CC homodimer interfaces in a subtype-specific manner. Chemokine receptor heterodimers display distinct dimerization patterns for CXCR4/CCR5 and CXCR4/CCR2. Furthermore, associations between CXCR4 and CCR5 reveal an increased cholesterol-sensitivity as compared to CXCR4/CCR2 heterodimerization patterns. This work provides a first comprehensive structural overview over the complex interaction network between chemokine receptors and indicates how heterodimerization and the interaction with the membrane environment diversifies the function of closely related GPCRs.

A Natural CCR2 Antagonist Relieves Tumor-associated Macrophage-mediated Immunosuppression to Produce a Therapeutic Effect for Liver Cancer.

Hepatocellular carcinoma (HCC) is a common malignant tumor in the digestive tract with limited therapeutic choices. Although sorafenib, an orally administered multikinase inhibitor, has produced survival benefits for patients with advanced HCC, favorable clinical outcomes are limited due to individual differences and resistance. The application of immunotherapy, a promising approach for HCC is urgently needed. Macrophage infiltration, mediated by the CCL2/CCR2 axis, is a potential immunotherapeutic target. Here, we report that a natural product from Abies georgei, named 747 and related in structure to kaempferol, exhibits sensitivity and selectivity as a CCR2 antagonist. The specificity of 747 on CCR2 was demonstrated via calcium flux, the binding domain of CCR2 was identified in an extracellular loop by chimera binding assay, and in vivo antagonistic activity of 747 was confirmed through a thioglycollate-induced peritonitis model. In animals, 747 elevated the number of CD8+ T cells in tumors via blocking tumor-infiltrating macrophage-mediated immunosuppression and inhibited orthotopic and subcutaneous tumor growth in a CD8+ T cell-dependent manner. Further, 747 enhanced the therapeutic efficacy of low-dose sorafenib without obvious toxicity, through elevating the numbers of intra-tumoral CD8+ T cells and increasing death of tumor cells. Thus, we have discovered a natural CCR2 antagonist and have provided a new perspective on development of this antagonist for treatment of HCC. In mouse models of HCC, 747 enhanced the tumor immunosuppressive microenvironment and potentiated the therapeutic effect of sorafenib, indicating that the combination of an immunomodulator with a chemotherapeutic drug could be a new approach for treating HCC.

Insights into CC Chemokine Ligand 2/Chemokine Receptor 2 Molecular Recognition: A Step Forward toward Antichemotactic Agents.

Chemokine ligand 2 (CCL2), also known as monocyte chemoattractant protein 1 (MCP-1), is a chemokine that recruits immune cells to inflammatory sites by interacting with G protein-coupled receptor CCR2. The CCL2/CCR2 axis is also involved in pathological processes such as tumor growth and metastasis and hence is currently considered as an important drug target. CCL2 exists in a dynamic monomer-dimer equilibrium that is modulated by CCR2 binding. We used solution nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics simulations to study the interactions between CCL2 and a sulfopeptide corresponding to the N-terminal sequence of CCR2 (CCR218-31). Peptide binding induced the dissociation of CCL2 into monomers, forming stable CCL2/CCR218-31 complexes. NMR relaxation measurements indicated that residues around the CCR218-31 binding site, which are located at the dimer interface, undergo a complex regime of motions. NMR data were used to construct a three-dimensional structural model of the CCL2/CCR218-31 complex, revealing that CCR218-31 occupies a binding site juxtaposed to the dimer interface, partially replacing monomer-monomer contacts, explaining why CCR218-31 binding weakens the dimer interface and induces dissociation. We found that the main interactions governing receptor binding are highly stable salt bridges with conserved chemokine residues as well as hydrophobic interactions. These data provide new insights into the structure-function relationship of the CCL2-CCR2 interaction and may be helpful for the design of novel antichemotactic agents.

Key determinants of selective binding and activation by the monocyte chemoattractant proteins at the chemokine receptor CCR2.

Chemokines and their receptors collectively orchestrate the trafficking of leukocytes in normal immune function and inflammatory diseases. Different chemokines can induce distinct responses at the same receptor. In comparison to monocyte chemoattractant protein-1 (MCP-1; also known as CCL2), the chemokines MCP-2 (CCL8) and MCP-3 (CCL7) are partial agonists of their shared receptor CCR2, a key regulator of the trafficking of monocytes and macrophages that contribute to the pathology of atherosclerosis, obesity, and type 2 diabetes. Through experiments with chimeras of MCP-1 and MCP-3, we identified the chemokine amino-terminal region as being the primary determinant of both the binding and signaling selectivity of these two chemokines at CCR2. Analysis of CCR2 mutants showed that the chemokine amino terminus interacts with the major subpocket in the transmembrane helical bundle of CCR2, which is distinct from the interactions of some other chemokines with the minor subpockets of their receptors. These results suggest the major subpocket as a target for the development of small-molecule inhibitors of CCR2.

Biochemical and biophysical characterization of cytokine-like protein 1 (CYTL1).

Cytokine-like protein 1 (CYTL1) is a small widely expressed secreted protein lacking significant primary sequence homology to any other known protein. CYTL1 expression appears to be highest in the hematopoietic system and in chondrocytes; however, maintenance of cartilage in mouse models of arthritis is its only reported function in vivo. Despite lacking sequence homology to chemokines, CYTL1 is predicted by computational methods to fold like a chemokine, and has been reported to function as a chemotactic agonist at the chemokine receptor CCR2 in mouse monocyte/macrophages. Nevertheless, since chemokines are defined by structure and chemokine receptors are able to bind many non-chemokine ligands, direct determination of the CYTL1 tertiary structure will ultimately be required to know whether it actually folds as a chemokine and therefore is a chemokine. Towards this goal, we have developed a method for producing functional recombinant human CYTL1 in bacteria, and we provide new evidence about the biophysical and biochemical properties of recombinant CYTL1. Circular dichroism analysis showed that, like chemokines, CYTL1has a higher content of beta-sheet than alpha-helix secondary structure. Furthermore, recombinant CYTL1 promoted calcium flux in chondrocytes. Nevertheless, unlike chemokines, CYTL1 had limited affinity to proteoglycans. Together, these properties further support cytokine-like properties for CYTL1 with some overlap with the chemokines.

Structural Analysis of Chemokine Receptor-Ligand Interactions.

This review focuses on the construction and application of structural chemokine receptor models for the elucidation of molecular determinants of chemokine receptor modulation and the structure-based discovery and design of chemokine receptor ligands. A comparative analysis of ligand binding pockets in chemokine receptors is presented, including a detailed description of the CXCR4, CCR2, CCR5, CCR9, and US28 X-ray structures, and their implication for modeling molecular interactions of chemokine receptors with small-molecule ligands, peptide ligands, and large antibodies and chemokines. These studies demonstrate how the integration of new structural information on chemokine receptors with extensive structure-activity relationship and site-directed mutagenesis data facilitates the prediction of the structure of chemokine receptor-ligand complexes that have not been crystallized. Finally, a review of structure-based ligand discovery and design studies based on chemokine receptor crystal structures and homology models illustrates the possibilities and challenges to find novel ligands for chemokine receptors.

Partial Agonist and Biased Signaling Properties of the Synthetic Enantiomers J113863/UCB35625 at Chemokine Receptors CCR2 and CCR5.

Biased agonism at G protein-coupled receptors constitutes a promising area of research for the identification of new therapeutic molecules. In this study we identified two novel biased ligands for the chemokine receptors CCR2 and CCR5 and characterized their functional properties. We showed that J113863 and its enantiomer UCB35625, initially identified as high affinity antagonists for CCR1 and CCR3, also bind with low affinity to the closely related receptors CCR2 and CCR5. Binding of J113863 and UCB35625 to CCR2 or CCR5 resulted in the full or partial activation of the three Gi proteins and the two Go isoforms. Unlike chemokines, the compounds did not activate G12 Binding of J113863 to CCR2 or CCR5 also induced the recruitment of ß-arrestin 2, whereas UCB35625 did not. UCB35625 induced the chemotaxis of L1.2 cells expressing CCR2 or CCR5. In contrast, J113863 induced the migration of L1.2-CCR2 cells but antagonized the chemokine-induced migration of L1.2-CCR5 cells. We also showed that replacing the phenylalanine 3.33 in CCR5 TM3 by the corresponding histidine of CCR2 converts J113863 from an antagonist for cell migration and a partial agonist in other assays to a full agonist in all assays. Further analyses indicated that F3.33H substitution strongly increased the activation of G proteins and ß-arrestin 2 by J113863. These results highlight the biased nature of the J113863 and UCB35625 that act either as antagonist, partial agonist, or full agonist according to the receptor, the enantiomer, and the signaling pathway investigated.

Structure of CC chemokine receptor 2 with orthosteric and allosteric antagonists.

CC chemokine receptor 2 (CCR2) is one of 19 members of the chemokine receptor subfamily of human class A G-protein-coupled receptors. CCR2 is expressed on monocytes, immature dendritic cells, and T-cell subpopulations, and mediates their migration towards endogenous CC chemokine ligands such as CCL2 (ref. 1). CCR2 and its ligands are implicated in numerous inflammatory and neurodegenerative diseases including atherosclerosis, multiple sclerosis, asthma, neuropathic pain, and diabetic nephropathy, as well as cancer. These disease associations have motivated numerous preclinical studies and clinical trials (see http://www.clinicaltrials.gov) in search of therapies that target the CCR2-chemokine axis. To aid drug discovery efforts, here we solve a structure of CCR2 in a ternary complex with an orthosteric (BMS-681 (ref. 6)) and allosteric (CCR2-RA-[R]) antagonist. BMS-681 inhibits chemokine binding by occupying the orthosteric pocket of the receptor in a previously unseen binding mode. CCR2-RA-[R] binds in a novel, highly druggable pocket that is the most intracellular allosteric site observed in class A G-protein-coupled receptors so far; this site spatially overlaps the G-protein-binding site in homologous receptors. CCR2-RA-[R] inhibits CCR2 non-competitively by blocking activation-associated conformational changes and formation of the G-protein-binding interface. The conformational signature of the conserved microswitch residues observed in double-antagonist-bound CCR2 resembles the most inactive G-protein-coupled receptor structures solved so far. Like other protein-protein interactions, receptor-chemokine complexes are considered challenging therapeutic targets for small molecules, and the present structure suggests diverse pocket epitopes that can be exploited to overcome obstacles in drug design.

Photoluminescent Mesoporous Silicon Nanoparticles with siCCR2 Improve the Effects of Mesenchymal Stromal Cell Transplantation after Acute Myocardial Infarction.

BACKGROUND: Despite the benefits of mesenchymal stromal cell (MSC) transplantation in cardiac tissue, detailed in vivo observations have shown that MSCs only survive for a brief period after transplantation due to harsh microenvironmental conditions, including ischemia, inflammation and anoikis, in the infarcted myocardium. Thus, new strategies are needed to enhance MSC survival and inhibit cardiac remodeling. Studies have now demonstrated that chemokine [C-C motif] ligand 2 (CCL2) and its cognate receptor C-C chemokine receptor 2 (CCR2) promote excessive Ly6C(high) inflammatory monocyte infiltration at the infarct in response to ischemic myocardial injury. Therefore, decreasing the activities of these monocytes immediately after acute myocardial infarction (AMI) could be beneficial for AMI patients. OBJECTIVES: This study tested the hypothesis that therapeutic siRNA-loaded photoluminescent mesoporous silicon nanoparticles (PMSNs) targeting CCR2 expression in Ly6C(high) inflammatory monocytes decrease the accumulation of these cells in the infarct, improve the efficacy of MSC transplantation and attenuate myocardial remodeling. METHODS: PMSNs carrying therapeutic siCCR2 were first synthesized without the inclusion of fluorescent materials or dyes. After AMI BALB/c mice were established, 10(5) 5-ethynyl-2'- deoxyuridine (EdU)-labeled MSCs suspended in 100 µl of phosphate buffered saline (PBS) were injected into the border zone of the infarct of each mouse. PMSNs-siCCR2 (25 µg/g) were also intravenously injected via the tail vein immediately following AMI induction. Control mice were injected with an equal amount of PMSNs without siCCR2. PMSNs-siCCR2 were examined in vivo using near-infrared imaging technology. The therapeutic effects of PMSNs-siCCR2 for MSC transplantation were determined at the mRNA, protein and functional levels. RESULTS: PMSNs-siCCR2 circulated freely in vivo and were cleared in a relatively short period of time (t(½)=37 min) with no evidence of toxicity. The therapeutic PMSNs-siCCR2 showed higher levels of cellular accumulation in Ly6C(high) monocytes in the spleen and more efficient degradation of CCR2 compared with the control (8.04%±2.17% vs. 20.02%±4.55%, p<0.001). Subsequently, the PMSNs-siCCR2 decreased the accumulation of CD11b-positive monocytes at the infarct (49.3%±17.34% vs. 61.32%±22.43%, p<0.001) on day 1. Increased survival of transplanted MSCs (13±3/mm(2) vs. 4±1/mm(2), p<0.001) and significantly decreased TdT-mediated dUTP nick end labeling (TUNEL)(+) cardiac myocytes (17.44%±6.26% vs. 39.49%±13.28%, p<0.001) were then identified in the infarct zone three days after AMI induction in the PMSNs-siCCR2 group. Three weeks after MSC injection, significant increases were observed in the vascular density (235.5±39.6/mm(2) vs. 147.4±20.3/mm(2), p<0.001) and the cardiac myosin-positive area (21.7%±8.4% vs. 13.2%±4.4%, p<0.001) of the infarct border zone. In addition, significant amelioration of left ventricular (LV) remodeling (thickness of the LV posterior walls) (0.84±0.11 mm vs. 0.61±0.08 mm, p<0.001) was also observed at the same time compared with the control group. CONCLUSIONS: PMSNs-siCCR2-mediated CCR2 gene silencing in Ly6C(high) monocytes improved the effectiveness of MSC transplantation and selectively ameliorated myocardial remodeling after AMI. These results suggest that PMSNs-siCCR2 could potentially be used to develop an anti-inflammatory therapy for post-AMI MSC transplantation.