Homology modeling of human CCR5 and analysis of its binding properties through molecular docking and molecular dynamics simulation

In this study, homology modeling, molecular docking and molecular dynamics simulation were performed to explore structural features and binding mechanism of some inhibitors of chemokine receptor type 5 (CCR5), and to construct a model for designing new CCR5 inhibitors for preventing HIV attachment to the host cell. A homology modeling procedure was employed to construct a 3D model of CCR5. For this procedure, the X-ray crystal structure of bovine rhodopsin (1F88A) at 2.80? resolution was used as template. After inserting the constructed model into a hydrated lipid bilayer, a 20ns molecular dynamics (MD) simulation was performed on the whole system. After reaching the equilibrium, twenty-four CCR5 inhibitors were docked in the active site of the obtained model. The binding models of the investigated antagonists indicate the mechanism of binding of the studied compounds to the CCR5 obviously. Moreover, 3D pictures of inhibitor-protein complex provided precious data regarding the binding orientation of each antagonist into the active site of this protein. One additional 20 ns MD simulation was performed on the initial structure of the CCR5-ligand 21 complex, resulted from the previous docking calculations, embedded in a hydrated POPE bilayer to explore the effects of the presence of lipid bilayer in the vicinity of CCR5-ligand complex. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.     

Structure prediction of GPCRs using piecewise homologs and application to the human CCR5 chemokine receptor: validation through agonist and antagonist docking

This article describes the construction and validation of a three-dimensional model of the human CC chemokine receptor 5 (CCR5) receptor using multiple homology modeling. A new methodology is presented where we built each secondary structural model of the protein separately from distantly related homologs of known structure. The reliability of our approach for G-protein coupled receptors was assessed through the building of the human C-X-C chemokine receptor type 4 (CXCR4) receptor of known crystal structure. The models are refined using molecular dynamics simulations and energy minimizations using CHARMM, a classical force field for proteins. Finally, docking models of both the natural agonists and the antagonists of the receptors CCR5 and CXCR4 are proposed. This study explores the possible binding process of ligands to the receptor cavity of chemokine receptors at molecular and atomic levels. We proposed few crucial residues in receptors binding to agonist/antagonist for further validation through experimental analysis. In particular, our study provides better understanding of the blockage mechanism of the chemokine receptors CCR5 and CXCR4, and may help the identification of new lead compounds for drug development in HIV infection, inflammatory diseases, and cancer metastasis.     

Molecular docking and 3D QSAR studies on 1-amino-2-phenyl-4-(piperidin-1-yl)-butanes based on the structural modeling of human CCR5 receptor

In the present study, we have used an approach combining protein structure modeling, molecular dynamics (MD) simulation, automated docking, and 3D QSAR analyses to investigate the detailed interactions of CCR5 with their antagonists. Homology modeling and MD simulation were used to build the 3D model of CCR5 receptor based on the high-resolution X-ray structure of bovine rhodopsin. A series of 64 CCR5 antagonists, 1-amino-2-phenyl-4-(piperidin-1-yl)-butanes, were docked into the putative binding site of the 3D model of CCR5 using the docking method, and the probable interaction model between CCR5 and the antagonists were obtained. The predicted binding affinities of the antagonists to CCR5 correlate well with the antagonist activities, and the interaction model could be used to explain many mutagenesis results. All these indicate that the 3D model of antagonist-CCR5 interaction is reliable. Based on the binding conformations and their alignment inside the binding pocket of CCR5, three-dimensional structure-activity relationship (3D QSAR) analyses were performed on these antagonists using comparative molecular field analysis (CoMFA) and comparative molecular similarity analysis (CoMSIA) methods. Both CoMFA and CoMSIA provide statistically valid models with good correlation and predictive power. The q(2)(r(cross)(2)) values are 0.568 and 0.587 for CoMFA and CoMSIA, respectively. The predictive ability of these models was validated by six compounds that were not included in the training set. Mapping these models back to the topology of the active site of CCR5 leads to a better understanding of antagonist-CCR5 interaction. These results suggest that the 3D model of CCR5 can be used in structure-based drug design and the 3D QSAR models provide clear guidelines and accurate activity predictions for novel antagonist design.     

Ligand supported homology modeling and docking evaluation of CCR2: docked pose selection by consensus scoring

Chemokine receptor 2 (CCR2) is a G-protein coupled receptor (GPCR) and a crucial target for various inflammatory and autoimmune diseases. The structure based antagonists design for many GPCRs, including CCR2, is restricted by the lack of an experimental three dimensional structure. Homology modeling is widely used for the study of GPCR-ligand binding. Since there is substantial diversity for the ligand binding pocket and binding modes among GPCRs, the receptor-ligand binding mode predictions should be derived from homology modeling with supported ligand information. Thus, we modeled the binding of our proprietary CCR2 antagonist using ligand supported homology modeling followed by consensus scoring the docking evaluation based on all modeled binding sites. The protein-ligand model was then validated by visual inspection of receptor-ligand interaction for consistency of published site-directed mutagenesis data and virtual screening a decoy compound database. This model was able to successfully identify active compounds within the decoy database. Finally, additional hit compounds were identified through a docking-based virtual screening of a commercial database, followed by a biological assay to validate CCR2 inhibitory activity. Thus, this procedure can be employed to screen a large database of compounds to identify new CCR2 antagonists.     

Selection of CC chemokine receptor 5-binding peptide from a phage display peptide library

Human CC chemokine receptor (CCR) 5 is a G protein-coupled receptor involved in a broad range of human diseases that mediates HIV-1 viral entry into cells. Certain small molecule receptor antagonists to CCR5 have been useful in therapy for these diseases. In this study, CCR5-expressing CHO cells (CHO/CCR5 cells) were used to select CCR5-binding peptides from a phage-displayed 12-mers peptide library. All of the 30 clones selected from the library showed specific binding to CHO/CCR5 cells by enzyme linked immunosorbent assay (ELISA). Seventeen out of the 30 clones shared the amino acid motif AFDWTFVPSLIL. The motif-containing phages and synthetic peptide AFDWTFVPSLIL blocked the binding of mAb 2D7 to CHO/CCR5 cells and competitively inhibited the ability of chemokine regulated on activation normal T cell expressed and secreted (RANTES) binding to CHO/CCR5 cells. Furthermore, the peptide AFDWTFVPSLIL also inhibited RANTES induced increase in the intracellular Ca2+ level in CHO/CCR5 cells. These results suggest that the peptide AFDWTFVPSLIL was specific for CCR5 and that it might become a CCR5 antagonist.     

Allosteric model of maraviroc binding to CC chemokine receptor 5 (CCR5)

Maraviroc is a nonpeptidic small molecule human immunodeficiency virus type 1 (HIV-1) entry inhibitor that has just entered the therapeutic arsenal for the treatment of patients. We recently demonstrated that maraviroc binding to the HIV-1 coreceptor, CC chemokine receptor 5 (CCR5), prevents it from binding the chemokine CCL3 and the viral envelope glycoprotein gp120 by an allosteric mechanism. However, incomplete knowledge of ligand-binding sites and the lack of CCR5 crystal structures have hampered an in-depth molecular understanding of how the inhibitor works. Here, we addressed these issues by combining site-directed mutagenesis (SDM) with homology modeling and docking. Six crystal structures of G-protein-coupled receptors were compared for their suitability for CCR5 modeling. All CCR5 models had equally good geometry, but that built from the recently reported dimeric structure of the other HIV-1 coreceptor CXCR4 bound to the peptide CVX15 (Protein Data Bank code 3OE0) best agreed with the SDM data and discriminated CCR5 from non-CCR5 binders in a virtual screening approach. SDM and automated docking predicted that maraviroc inserts deeply in CCR5 transmembrane cavity where it can occupy three different binding sites, whereas CCL3 and gp120 lie on distinct yet overlapped regions of the CCR5 extracellular loop 2. Data suggesting that the transmembrane cavity remains accessible for maraviroc in CCL3-bound and gp120-bound CCR5 help explain our previous observation that the inhibitor enhances dissociation of preformed ligand-CCR5 complexes. Finally, we identified residues in the predicted CCR5 dimer interface that are mandatory for gp120 binding, suggesting that receptor dimerization might represent a target for new CCR5 entry inhibitors.     

Identification of the binding site for a novel class of CCR2b chemokine receptor antagonists: binding to a common chemokine receptor motif within the helical bundle

Monocyte chemoattracant-1 (MCP-1) stimulates leukocyte chemotaxis to inflammatory sites, such as rheumatoid arthritis, atherosclerosis, and asthma, by use of the MCP-1 receptor, CCR2, a member of the G-protein-coupled seven-transmembrane receptor superfamily. These studies identified a family of antagonists, spiropiperidines. One of the more potent compounds blocks MCP-1 binding to CCR2 with a K(d) of 60 nm, but it is unable to block binding to CXCR1, CCR1, or CCR3. These compounds were effective inhibitors of chemotaxis toward MCP-1 but were very poor inhibitors of CCR1-mediated chemotaxis. The compounds are effective blockers of MCP-1-driven inhibition of adenylate cyclase and MCP-1- and MCP-3-driven cytosolic calcium influx; the compounds are not agonists for these pathways. We showed that glutamate 291 (Glu(291)) of CCR2 is a critical residue for high affinity binding and that this residue contributes little to MCP-1 binding to CCR2. The basic nitrogen present in the spiropiperidine compounds may be the interaction partner for Glu(291), because the basicity of this nitrogen was essential for affinity; furthermore, a different class of antagonists, a class that does not have a basic nitrogen (2-carboxypyrroles), were not affected by mutations of Glu(291). In addition to the CCR2 receptor, spiropiperidine compounds have affinity for several biogenic amine receptors. Receptor models indicate that the acidic residue, Glu(291), from transmembrane-7 of CCR2 is in a position similar to the acidic residue contributed from transmembrane-3 of biogenic amine receptors, which may account for the shared affinity of spiropiperidines for these two receptor classes. The models suggest that the acid-base pair, Glu(291) to piperidine nitrogen, anchors the spiropiperidine compound within the transmembrane ovoid bundle. This binding site may overlap with the space required by MCP-1 during binding and signaling; thus the small molecule ligands act as antagonists. An acidic residue in transmembrane region 7 is found in most chemokine receptors and is rare in other serpentine receptors. The model of the binding site may suggest ways to make new small molecule chemokine receptor antagonists, and it may rationalize the design of more potent and selective antagonists.     

The CC chemokine eotaxin (CCL11) is a partial agonist of CC chemokine receptor 2b

Despite sharing considerable homology with the members of the monocyte chemoattractant protein (MCP) family, the CC chemokine eotaxin (CCL11) has previously been reported to signal exclusively via the receptor CC chemokine receptor 3 (CCR3). Using the monocyte cell line THP-1, we investigated the relative abilities of eotaxin and MCPs 1-4 to induce CCR2 signaling, employing assays of directed cell migration and intracellular calcium flux. Surprisingly, 1 microm concentrations of eotaxin were able to recruit THP-1 cells in chemotaxis assays, and this migration was sensitive to antagonism of CCR2 but not CCR3. Radiolabeled eotaxin binding assays performed on transfectants bearing CCR2b or CCR3 confirmed eotaxin binding to CCR2 with a K(d) of 7.50 +/- 3.30 nm, compared with a K(d) of 1.68 +/- 0.91 nm at CCR3. In addition, whereas 1 microm concentrations of eotaxin were able to recruit CCR2b transfectants, substimulatory concentrations of eotaxin inhibited MCP-1-induced chemotaxis of CCR2b transfectants and also inhibited MCP-1-induced intracellular calcium flux of THP-1 cells. Collectively, these findings suggest that eotaxin is a partial agonist of the CCR2b receptor. A greater understanding of the interaction of CCR2 with all of its ligands, both full and partial agonists, may aid the rational design of specific antagonists that hold great promise as future therapeutic treatments for a variety of inflammatory disorders.     

Synthesis and evaluation of cis-3,4-disubstituted piperidines as potent CC chemokine receptor 2 (CCR2) antagonists

A series of cis-3,4-disubstituted piperidines was synthesized and evaluated as CC chemokine receptor 2 (CCR2) antagonists. Compound 24 emerged with an attractive profile, possessing excellent binding (CCR2 IC(50)=3.4 nM) and functional antagonism (calcium flux IC(50)=2.0 nM and chemotaxis IC(50)=5.4 nM). Studies to explore the binding of these piperidine analogs utilized a key CCR2 receptor mutant (E291A) with compound 14 and revealed a significant reliance on Glu291 for binding.     

CC and CX3C chemokines differentially interact with the N terminus of the human cytomegalovirus-encoded US28 receptor

Human cytomegalovirus (HCMV) is the causative agent of life-threatening systemic diseases in immunocompromised patients as well as a risk factor for vascular pathologies, like atherosclerosis, in immunocompetent individuals. HCMV encodes a G-protein-coupled receptor (GPCR), referred to as US28, that displays homology to the human chemokine receptor CCR1 and binds several chemokines of the CC family as well as the CX3C chemokine fractalkine with high affinity. Most importantly, following HCMV infection, US28 activates several intracellular pathways, either constitutively or in a chemokine-dependent manner. In this study, our goal was to understand the molecular interactions between chemokines and the HCMV-encoded US28 receptor. To achieve this goal, a double approach has been used, consisting in the analysis of both receptor and ligand mutants. This approach has led us to identify several amino acids located in the N terminus of US28 that differentially contribute to the high affinity binding of CC versus CX3C chemokines. Additionally, our results highlight the importance of secondary modifications occurring at US28, such as sulfation, for ligand recognition. Finally, the effects of chemokine dimerization and interaction with glycosaminoglycans (GAGs) on chemokine binding and activation of US28 were investigated as well using CCL4 as model ligand. In line with the two-state model describing chemokine/receptor interaction, we show that an aromatic residue in the N-loop region of CCL4 promotes tight binding to US28, whereas receptor activation depends on the presence of the N terminus of CCL4, as shown previously for CCR5.     

Site-directed mutagenesis of CC chemokine receptor 1 reveals the mechanism of action of UCB 35625, a small molecule chemokine receptor antagonist

The chemokine receptor CCR1 and its principal ligand, CCL3/MIP-1alpha, have been implicated in the pathology of several inflammatory diseases including rheumatoid arthritis, multiple sclerosis, and asthma. As such, these molecules are the focus of much research with the ultimate aim of developing novel therapies. We have described previously a non-competitive small molecule antagonist of CCR1 (UCB 35625), which we hypothesized interacted with amino acids located within the receptor transmembrane (TM) helices (Sabroe, I., Peck, M. J., Jan Van Keulen, B., Jorritsma, A., Simmons, G., Clapham, P. R., Williams, T. J., and Pease, J. E. (2000) J. Biol. Chem. 275, 25985-25992). Here we describe an approach to identifying the mechanism by which the molecule antagonizes CCR1. Thirty-three point mutants of CCR1 were expressed transiently in L1.2 cells, and the cells were assessed for their capacity to migrate in response to CCL3 in the presence or absence of UCB 35625. Cells expressing the mutant constructs Y41A (TM helix 1, or TM1), Y113A (TM3), and E287A (TM7) were responsive to CCL3 but resistant to the antagonist, consistent with a role for the TM helices in CCR1 interactions with UCB 35625. Subsequent molecular modeling successfully docked the compound with CCR1 and suggests that the antagonist ligates TM1, 2, and 7 of CCR1 and severely impedes access to TM2 and TM3, a region thought to be perturbed by the chemokine amino terminus during the process of receptor activation. Insights into the mechanism of action of these compounds may facilitate the development of more potent antagonists that show promise as future therapeutic agents in the treatment of inflammatory disease.     

Small molecule receptor agonists and antagonists of CCR3 provide insight into mechanisms of chemokine receptor activation

Chemokine receptor CCR3 is highly expressed by eosinophils and signals in response to binding of the eotaxin family of chemokines, which are up-regulated in allergic disorders. Consequently, CCR3 blockade is of interest as a possible therapeutic approach for the treatment of allergic disease. We have described previously a bispecific antagonist of CCR1 and CCR3 named UCB35625 that was proposed to interact with the transmembrane residues Tyr-41, Tyr-113, and Glu-287 of CCR1, all of which are conserved in CCR3. Here, we show that cells expressing the CCR3 constructs Y113A and E287Q are insensitive to antagonism by UCB35625 and also exhibit impaired chemotaxis in response to CCL11/eotaxin, suggesting that these residues are important for antagonist binding and also receptor activation. Furthermore, mutation of the residue Tyr-113 to alanine was found to turn the antagonist UCB35625 into a CCR3 agonist. Screens of small molecule libraries identified a novel specific agonist of CCR3 named CH0076989. This was able to activate eosinophils and transfectants expressing both wild-type CCR3 and a CCR1-CCR3 chimeric receptor lacking the CCR3 amino terminus, indicating that this region of CCR3 is not required for CH0076989 binding. A direct interaction with the transmembrane helices of CCR3 was supported by mutation of the residues Tyr-41, Tyr-113, and Glu-287 that resulted in complete loss of CH0076989 activity, suggesting that the compound mimics activation by CCL11. We conclude that both agonists and antagonists of CCR3 appear to occupy overlapping sites within the transmembrane helical bundle, suggesting a fine line between agonism and antagonism of chemokine receptors.     

HHV8-encoded vMIP-I selectively engages chemokine receptor CCR8. Agonist and antagonist profiles of viral chemokines.

Uncertainty regarding viral chemokine function is mirrored by an incomplete knowledge of host chemokine receptor usage by the virally encoded proteins. One such molecule is vMIP-I, a C-C type chemokine of undefined function and binding specificity, encoded by the Kaposi's sarcoma herpesvirus HHV-8. We report here that vMIP-I binds to and induces cytosolic [Ca(2+)] signals in human T cells selectively through CCR8, a CC chemokine receptor associated with Th2 lymphocytes. Furthermore, using a panel of 65 different human, viral, and rodent chemokines, we have established a comprehensive ligand binding "fingerprint" for CCR8. The receptor exhibits marked "high" affinity (K(d) < 15 nM) only for four chemokines, three of them of viral origin: vMIP-I, vMIP-II, vMCC-I, and human I-309. A previously unreported second class of lower affinity ligands includes MCP-3 and possibly two other viral chemokines. vMIP-I and I-309 appear to act as CCR8 agonists: binding to and inducing cytosolic [Ca(2+)] elevation through the receptor. By contrast, vMIP-II and vMCC-I act as potent antagonists: binding without inducing signaling, and blocking the effects of I-309 and vMIP-I. These results suggest a ligand hierarchy for CCR8, identifying vMIP-I as a selective viral chemokine agonist. CCR8 may thus engage a specific subset of chemokines with the potential to regulate each other during viral infection and immune regulation.     

Predictions of CCR1 chemokine receptor structure and BX 471 antagonist binding followed by experimental validation

A major challenge in the application of structure-based drug design methods to proteins belonging to the superfamily of G protein-coupled receptors (GPCRs) is the paucity of structural information (1). The 19 chemokine receptors, belonging to the Class A family of GPCRs, are important drug targets not only for autoimmune diseases like multiple sclerosis but also for the blockade of human immunodeficiency virus type 1 entry (2). Using the MembStruk computational method (3), we predicted the three-dimensional structure of the human CCR1 receptor. In addition, we predicted the binding site of the small molecule CCR1 antagonist BX 471, which is currently in Phase II clinical trials (4). Based on the predicted antagonist binding site we designed 17 point mutants of CCR1 to validate the predictions. Subsequent competitive ligand binding and chemotaxis experiments with these mutants gave an excellent correlation to these predictions. In particular, we find that Tyr-113 and Tyr-114 on transmembrane domain 3 and Ile-259 on transmembrane 6 contribute significantly to the binding of BX 471. Finally, we used the predicted and validated structure of CCR1 in a virtual screening validation of the Maybridge data base, seeded with selective CCR1 antagonists. The screen identified 63% of CCR1 antagonists in the top 5% of the hits. Our results indicate that rational drug design for GPCR targets is a feasible approach.     

Toward the Discovery of Vaccine Adjuvants: Coupling In Silico Screening and In Vitro Analysis of Antagonist Binding to Human and Mouse CCR4 Receptors

Background

Adjuvants enhance or modify an immune response that is made to an antigen. An antagonist of the chemokine CCR4 receptor can display adjuvant-like properties by diminishing the ability of CD4+CD25+ regulatory T cells (Tregs) to down-regulate immune responses.

Methodology

Here, we have used protein modelling to create a plausible chemokine receptor model with the aim of using virtual screening to identify potential small molecule chemokine antagonists. A combination of homology modelling and molecular docking was used to create a model of the CCR4 receptor in order to investigate potential lead compounds that display antagonistic properties. Three-dimensional structure-based virtual screening of the CCR4 receptor identified 116 small molecules that were calculated to have a high affinity for the receptor; these were tested experimentally for CCR4 antagonism. Fifteen of these small molecules were shown to inhibit specifically CCR4-mediated cell migration, including that of CCR4⁺ Tregs.

Significance

Our CCR4 antagonists act as adjuvants augmenting human T cell proliferation in an in vitro immune response model and compound SP50 increases T cell and antibody responses in vivo when combined with vaccine antigens of Mycobacterium tuberculosis and Plasmodium yoelii in mice.     

3-Amino-1-alkyl-cyclopentane carboxamides as small molecule antagonists of the human and murine CC chemokine receptor 2

A series of low molecular weight antagonists of both the human and murine CC chemokine receptor 2, containing a 1-alkyl-3-(3-methyl-4-spiroindenylpiperidine)-substituted cyclopentanecarboxamide, is described. A SAR study of the C(1) substituent revealed that short, branched alkyl groups such as isopropyl, isobutyl, or cyclopropyl are optimal for both human and murine CCR2 binding activity.     

C-C chemokine receptor 3 antagonism by the beta-chemokine macrophage inflammatory protein 4, a property strongly enhanced by an amino-terminal alanine-methionine swap

Allergic reactions are characterized by the infiltration of tissues by activated eosinophils, Th2 lymphocytes, and basophils. The beta-chemokine receptor CCR3, which recognizes the ligands eotaxin, eotaxin-2, monocyte chemotactic protein (MCP) 3, MCP4, and RANTES, plays a central role in this process, and antagonists to this receptor could have potential therapeutic use in the treatment of allergy. We describe here a potent and specific CCR3 antagonist, called Met-chemokine beta 7 (Ckbeta7), that prevents signaling through this receptor and, at concentrations as low as 1 nM, can block eosinophil chemotaxis induced by the most potent CCR3 ligands. Met-Ckbeta7 is a more potent CCR3 antagonist than Met- and aminooxypentane (AOP)-RANTES and, unlike these proteins, exhibits no partial agonist activity and is highly specific for CCR3. Thus, this antagonist may be of use in ameliorating leukocyte infiltration associated with allergic inflammation. Met-Ckbeta7 is a modified form of the beta-chemokine macrophage inflammatory protein (MIP) 4 (alternatively called pulmonary and activation-regulated chemokine (PARC), alternative macrophage activation-associated C-C chemokine (AMAC) 1, or dendritic cell-derived C-C chemokine (DCCK) 1). Surprisingly, the unmodified MIP4 protein, which is known to act as a T cell chemoattractant, also exhibits this CCR3 antagonistic activity, although to a lesser extent than Met-Ckbeta7, but to a level that may be of physiological relevance. MIP4 may therefore use chemokine receptor agonism and antagonism to control leukocyte movement in vivo. The enhanced activity of Met-Ckbeta7 is due to the alteration of the extreme N-terminal residue from an alanine to a methionine.     

An explorative study on Staphylococcus aureus MurE inhibitor: induced fit docking,binding free energy calculation,and molecular dynamics

Staphylococcus aureus MurE enzyme catalyzes the addition of l-lysine as third residue of the peptidoglycan peptide moiety. Due to the high substrate specificity and its ubiquitous nature among bacteria, MurE enzyme is considered as one of the potential target for the development of new therapeutic agents. In the present work, induced fit docking (IFD), binding free energy calculation, and molecular dynamics (MD) simulation were carried out to elucidate the inhibition potential of 2-thioxothiazolidin-4-one based inhibitor 1 against S. aureus MurE enzyme. The inhibitor 1 formed majority of hydrogen bonds with the central domain residues Asn151, Thr152, Ser180, Arg187, and Lys219. Binding free-energy calculation by MM-GBSA approach showed that van der Waals (ΔG_vdW, ?57.30?kcal/mol) and electrostatic solvation (ΔG_solv, ?36.86?kcal/mol) energy terms are major contributors for the inhibitor binding. Further, 30-ns MD simulation was performed to validate the stability of ligand–protein complex and also to get structural insight into mode of binding. Based on the IFD and MD simulation results, we designed four new compounds D1–D4 with promising binding affinity for the S. aureus MurE enzyme. The designed compounds were subjected to the extra-precision docking and binding free energy was calculated for complexes. Further, a 30-ns MD simulation was performed for D1/4C13 complex.     

Identification of chemokine receptor CCR4 antagonist

The present study reports the identification and hits to leads optimization of chemokine receptor CCR4 antagonists. Compound 12 is a high affinity, non-cytotoxic antagonist of CCR4 that blocks the functional activity mediated by the receptor.     

Allosteric and orthosteric sites in CC chemokine receptor (CCR5), a chimeric receptor approach

Chemokine receptors play a major role in immune system regulation and have consequently been targets for drug development leading to the discovery of several small molecule antagonists. Given the large size and predominantly extracellular receptor interaction of endogenous chemokines, small molecules often act more deeply in an allosteric mode. However, opposed to the well described molecular interaction of allosteric modulators in class C 7-transmembrane helix (7TM) receptors, the interaction in class A, to which the chemokine receptors belong, is more sparsely described. Using the CCR5 chemokine receptor as a model system, we studied the molecular interaction and conformational interchange required for proper action of various orthosteric chemokines and allosteric small molecules, including the well known CCR5 antagonists TAK-779, SCH-C, and aplaviroc, and four novel CCR5 ago-allosteric molecules. A chimera was successfully constructed between CCR5 and the closely related CCR2 by transferring all extracellular regions of CCR2 to CCR5, i.e. a Trojan horse that resembles CCR2 extracellularly but signals through a CCR5 transmembrane unit. The chimera bound CCR2 (CCL2 and CCL7), but not CCR5 chemokines (CCL3 and CCL5), with CCR2-like high affinities and potencies throughout the CCR5 signaling unit. Concomitantly, high affinity binding of small molecule CCR5 agonists and antagonists was retained in the transmembrane region. Importantly, whereas the agonistic and antagonistic properties were preserved, the allosteric enhancement of chemokine binding was disrupted. In summary, the Trojan horse chimera revealed that orthosteric and allosteric sites could be structurally separated and still act together with transmission of agonism and antagonism across the different receptor units.