Molecular determinants of antagonist interactions with chemokine receptors CCR2 and CCR5.

By driving monocyte chemotaxis, the chemokine receptor CCR2 shapes inflammatory responses and the formation of tumor microenvironments. This makes it a promising target in inflammation and immuno-oncology; however, despite extensive efforts, there are no FDA-approved CCR2-targeting therapeutics. Cited challenges include the redundancy of the chemokine system, suboptimal properties of compound candidates, and species differences that confound the translation of results from animals to humans. Structure-based drug design can rationalize and accelerate the discovery and optimization of CCR2 antagonists to address these challenges. The prerequisites for such efforts include an atomic-level understanding of the molecular determinants of action of existing antagonists. In this study, using molecular docking and artificial-intelligence-powered compound library screening, we uncover the structural principles of small molecule antagonism and selectivity towards CCR2 and its sister receptor CCR5. CCR2 orthosteric inhibitors are shown to universally occupy an inactive-state-specific tunnel between receptor helices 1 and 7; we also discover an unexpected role for an extra-helical groove accessible through this tunnel, suggesting its potential as a new targetable interface for CCR2 and CCR5 modulation. By contrast, only shape complementarity and limited helix 8 hydrogen bonding govern the binding of various chemotypes of allosteric antagonists. CCR2 residues S1012.63 and V2446.36 are implicated as determinants of CCR2/CCR5 and human/mouse orthosteric and allosteric antagonist selectivity, respectively, and the role of S1012.63 is corroborated through experimental gain-of-function mutagenesis. We establish a critical role of induced fit in antagonist recognition, reveal strong chemotype selectivity of existing structures, and demonstrate the high predictive potential of a new deep-learning-based compound scoring function. Finally, this study expands the available CCR2 structural landscape with computationally generated chemotype-specific models well-suited for structure-based antagonist design.

Ligand-Dependent Mechanisms of C-C Chemokine Receptor 5 (CCR5) Trafficking Revealed by APEX2 Proximity Labeling Proteomics.

CC chemokine receptor 5 (CCR5) contributes to inflammatory responses by driving cell migration and scavenging chemokine to shape directional chemokine gradients. A drug against CCR5 has been approved for blocking HIV entry into cells. However, targeting CCR5 for the treatment of inflammatory diseases and cancer has had limited success because of the complex biology and pharmacology of this receptor. CCR5 is activated by many natural and engineered chemokines that elicit distinct receptor signaling and trafficking responses, including some that sequester the receptor inside the cell. The sequestration phenomenon may be therapeutically exploitable, but the mechanisms by which different ligands traffic CCR5 to different cellular locations are poorly understood. Here we employed live cell ascorbic acid peroxidase proximity labeling and quantitative mass spectrometry proteomics for unbiased discovery of temporally resolved protein neighborhoods of CCR5 following stimulation with its endogenous agonist, CCL5, and two CCL5 variants that promote intracellular retention of the receptor. Along with targeted pharmacological assays, the data reveals distinct ligand-dependent CCR5 trafficking patterns with temporal resolution. All three chemokines internalize CCR5 via ß-arrestin- dependent, clathrin-mediated endocytosis but to different extents, with different kinetics and with varying dependencies on GPCR kinase subtypes. The agonists differ in their ability to target the receptor to lysosomes for degradation, as well as to the Golgi compartment and the trans-Golgi network, and these trafficking patterns translate into distinct levels of ligand scavenging. The results provide insight into the molecular mechanisms behind CCR5 intracellular sequestration and suggest actionable patterns for the development of chemokine-based CCR5 targeting molecules. Significance Statement: CCR5 plays a crucial role in the immune system and is important in numerous physiological and pathological processes such as inflammation, cancer and HIV transmission. Along with its functional diversity, different CCR5 ligands can induce distinct receptor signaling responses and trafficking behaviors; the latter includes intracellular receptor sequestration which offers a potential therapeutic strategy for inhibiting CCR5 function. Using time-resolved proximity labeling proteomics and targeted pharmacological experiments, this study reveals the molecular basis for receptor sequestration including information that can be exploited for the development of CCR5 targeting molecules that promote retention of the receptor inside the cell.

Divergent regulation of α-arrestin ARRDC3 function by ubiquitination.

The α-arrestin ARRDC3 is a recently discovered tumor suppressor in invasive breast cancer that functions as a multifaceted adaptor protein to control protein trafficking and cellular signaling. However, the molecular mechanisms that control ARRDC3 function are unknown. Other arrestins are known to be regulated by posttranslational modifications, suggesting that ARRDC3 may be subject to similar regulatory mechanisms. Here we report that ubiquitination is a key regulator of ARRDC3 function and is mediated primarily by two proline-rich PPXY motifs in the ARRDC3 C-tail domain. Ubiquitination and the PPXY motifs are essential for ARRDC3 function in regulating GPCR trafficking and signaling. Additionally, ubiquitination and the PPXY motifs mediate ARRDC3 protein degradation, dictate ARRDC3 subcellular localization, and are required for interaction with the NEDD4-family E3 ubiquitin ligase WWP2. These studies demonstrate a role for ubiquitination in regulating ARRDC3 function and reveal a mechanism by which ARRDC3 divergent functions are controlled.

A long isoform of GIV/Girdin contains a PDZ-binding module that regulates localization and G-protein binding.

PDZ domains are one of the most abundant protein domains in eukaryotes and are frequently found on junction-localized scaffold proteins. Various signaling molecules bind to PDZ proteins via PDZ-binding motifs (PBM) and fine-tune cellular signaling. However, how such interaction affects protein function is difficult to predict and must be solved empirically. Here we describe a long isoform of the guanine nucleotide exchange factor GIV/Girdin (CCDC88A) that we named GIV-L, which is conserved throughout evolution, from invertebrates to vertebrates, and contains a PBM. Unlike GIV, which lacks PBM and is cytosolic, GIV-L localizes onto cell junctions and has a PDZ interactome (as shown through annotating Human Cell Map and BioID-proximity labeling studies), which impacts GIV-L's ability to bind and activate trimeric G-protein, Gαi, through its guanine-nucleotide exchange modulator (GEM) module. This GEM module is found exclusively in vertebrates. We propose that the two functional modules in GIV may have evolved sequentially: the ability to bind PDZ proteins via the PBM evolved earlier in invertebrates, whereas G-protein binding and activation may have evolved later only among vertebrates. Phenotypic studies in Caco-2 cells revealed that GIV and GIV-L may have antagonistic effects on cell growth, proliferation (cell cycle), and survival. Immunohistochemical analysis in human colon tissues showed that GIV expression increases with a concomitant decrease in GIV-L during cancer initiation. Taken together, these findings reveal how regulation in GIV/CCDC88A transcript helps to achieve protein modularity, which allows the protein to play opposing roles either as a tumor suppressor (GIV-L) or as an oncogene (GIV).

Design and Characterization of an Intracellular Covalent Ligand for CC Chemokine Receptor 2.

Covalently acting inhibitors constitute a large and growing fraction of approved small-molecule therapeutics as well as useful tools for a variety of in vitro and in vivo applications. Here, we aimed to develop a covalent antagonist of CC chemokine receptor 2 (CCR2), a class A GPCR that has been pursued as a therapeutic target in inflammation and immuno-oncology. Based on a known intracellularly binding CCR2 antagonist, several covalent derivatives were synthesized and characterized by radioligand binding and functional assays. These studies revealed compound 14 as an intracellular covalent ligand for CCR2. In silico modeling followed by site-directed mutagenesis confirmed that 14 forms a covalent bond with one of three proximal cysteine residues, which can be engaged interchangeably. To our knowledge, compound 14 represents the first covalent ligand reported for CCR2. Due to its unique properties, it may represent a promising tool for ongoing and future studies of CCR2 pharmacology.

Receptor tyrosine kinases activate heterotrimeric G proteins via phosphorylation within the interdomain cleft of Gαi.

The molecular mechanisms by which receptor tyrosine kinases (RTKs) and heterotrimeric G proteins, two major signaling hubs in eukaryotes, independently relay signals across the plasma membrane have been extensively characterized. How these hubs cross-talk has been a long-standing question, but answers remain elusive. Using linear ion-trap mass spectrometry in combination with biochemical, cellular, and computational approaches, we unravel a mechanism of activation of heterotrimeric G proteins by RTKs and chart the key steps that mediate such activation. Upon growth factor stimulation, the guanine-nucleotide exchange modulator dissociates Gαi•ßγ trimers, scaffolds monomeric Gαi with RTKs, and facilitates the phosphorylation on two tyrosines located within the interdomain cleft of Gαi. Phosphorylation triggers the activation of Gαi and inhibits second messengers (cAMP). Tumor-associated mutants reveal how constitutive activation of this pathway impacts cell's decision to "go" vs. "grow." These insights define a tyrosine-based G protein signaling paradigm and reveal its importance in eukaryotes.

TLR4 signaling and macrophage inflammatory responses are dampened by GIV/Girdin.

Sensing of pathogens by Toll-like receptor 4 (TLR4) induces an inflammatory response; controlled responses confer immunity but uncontrolled responses cause harm. Here we define how a multimodular scaffold, GIV (a.k.a. Girdin), titrates such inflammatory response in macrophages. Upon challenge with either live microbes or microbe-derived lipopolysaccharides (a ligand for TLR4), macrophages with GIV mount a more tolerant (hypo-reactive) transcriptional response and suppress proinflammatory cytokines and signaling pathways (i.e., NFkB and CREB) downstream of TLR4 compared to their GIV-depleted counterparts. Myeloid-specific gene-depletion studies confirmed that the presence of GIV ameliorates dextran sodium sulfate-induced colitis and sepsis-induced death. The antiinflammatory actions of GIV are mediated via its C-terminally located TIR-like BB-loop (TILL) motif which binds the cytoplasmic TIR modules of TLR4 in a manner that precludes receptor dimerization; such dimerization is a prerequisite for proinflammatory signaling. Binding of GIV's TILL motif to TIR modules inhibits proinflammatory signaling via other TLRs, suggesting a convergent paradigm for fine-tuning macrophage inflammatory responses.

Functional anatomy of the full-length CXCR4-CXCL12 complex systematically dissected by quantitative model-guided mutagenesis.

Because of their prominent roles in development, cancer, and HIV, the chemokine receptor CXCR4 and its ligand CXCL12 have been the subject of numerous structural and functional studies, but the determinants of ligand binding, selectivity, and signaling are still poorly understood. Here, building on our latest structural model, we used a systematic mutagenesis strategy to dissect the functional anatomy of the CXCR4-CXCL12 complex. Key charge swap mutagenesis experiments provided evidence for pairwise interactions between oppositely charged residues in the receptor and chemokine, confirming the accuracy of the predicted orientation of the chemokine relative to the receptor and providing insight into ligand selectivity. Progressive deletion of N-terminal residues revealed an unexpected contribution of the receptor N terminus to chemokine signaling. This finding challenges a longstanding "two-site" hypothesis about the essential features of the receptor-chemokine interaction in which the N terminus contributes only to binding affinity. Our results suggest that although the interaction of the chemokine N terminus with the receptor-binding pocket is the key driver of signaling, the signaling amplitude depends on the extent to which the receptor N terminus binds the chemokine. Together with systematic characterization of other epitopes, these data enable us to propose an experimentally consistent structural model for how CXCL12 binds CXCR4 and initiates signal transmission through the receptor transmembrane domain.

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.

Crosslinking-guided geometry of a complete CXC receptor-chemokine complex and the basis of chemokine subfamily selectivity.

Chemokines and their receptors are orchestrators of cell migration in humans. Because dysregulation of the receptor-chemokine system leads to inflammation and cancer, both chemokines and receptors are highly sought therapeutic targets. Yet one of the barriers for their therapeutic targeting is the limited understanding of the structural principles behind receptor-chemokine recognition and selectivity. The existing structures do not include CXC subfamily complexes and lack information about the receptor distal N-termini, despite the importance of the latter in signaling, regulation, and bias. Here, we report the discovery of the geometry of the complex between full-length CXCR4, a prototypical CXC receptor and driver of cancer metastasis, and its endogenous ligand CXCL12. By comprehensive disulfide cross-linking, we establish the existence and the structure of a novel interface between the CXCR4 distal N-terminus and CXCL12 ß1-strand, while also recapitulating earlier findings from nuclear magnetic resonance, modeling and crystallography of homologous receptors. A cross-linking-informed high-resolution model of the CXCR4-CXCL12 complex pinpoints the interaction determinants and reveals the occupancy of the receptor major subpocket by the CXCL12 proximal N terminus. This newly found positioning of the chemokine proximal N-terminus provides a structural explanation of CXC receptor-chemokine selectivity against other subfamilies. Our findings challenge the traditional two-site understanding of receptor-chemokine recognition, suggest the possibility of new affinity and signaling determinants, and fill a critical void on the structural map of an important class of therapeutic targets. These results will aid the rational design of selective chemokine-receptor targeting small molecules and biologics with novel pharmacology.

Druggable exosites of the human kino-pocketome.

Small molecules binding at any of the multiple regulatory sites on the molecular surface of a protein kinase may stabilize or disrupt the corresponding interaction, leading to consequent modulation of the kinase cellular activity. As such, each of these sites represents a potential drug target. Even targeting sites outside the immediate ATP site, the so-called exosites, may cause desirable biological effects through an allosteric mechanism. Targeting exosites can alleviate adverse effects and toxicity that is common when ATP-site compounds bind promiscuously to many other types of kinases. In this study we have identified, catalogued, and annotated all potentially druggable exosites on the protein kinase domains within the existing structural human kinome. We then priority-ranked these exosites by those most amenable to drug design. In order to identify pockets that are either consistent across the kinome, or unique and specific to a particular structure, we have also implemented a normalized representation of all pockets, and displayed these graphically. Finally, we have built a database and designed a web-based interface for users interested in accessing the 3-dimensional representations of these pockets. We envision this information will assist drug discovery efforts searching for untargeted binding pockets in the human kinome.

Discovery of holoenzyme-disrupting chemicals as substrate-selective CK2 inhibitors.

CK2 is a constitutively active protein kinase overexpressed in numerous malignancies. Interaction between CK2α and CK2ß subunits is essential for substrate selectivity. The CK2α/CK2ß interface has been previously targeted by peptides to achieve functional effects; however, no small molecules modulators were identified due to pocket flexibility and open shape. Here we generated numerous plausible conformations of the interface using the fumigation modeling protocol, and virtually screened a compound library to discover compound 1 that suppressed CK2α/CK2ß interaction in vitro and inhibited CK2 in a substrate-selective manner. Orthogonal SPR, crystallography, and NMR experiments demonstrated that 4 and 6, improved analogs of 1, bind to CK2α as predicted. Both inhibitors alter CK2 activity in cells through inhibition of CK2 holoenzyme formation. Treatment with 6 suppressed MDA-MB231 triple negative breast cancer cell growth and induced apoptosis. Altogether, our findings exemplify an innovative computational-experimental approach and identify novel non-peptidic inhibitors of CK2 subunit interface disclosing substrate-selective functional effects.

Nilotinib, an approved leukemia drug, inhibits smoothened signaling in Hedgehog-dependent medulloblastoma.

Dysregulation of the seven-transmembrane (7TM) receptor Smoothened (SMO) and other components of the Hedgehog (Hh) signaling pathway contributes to the development of cancers including basal cell carcinoma (BCC) and medulloblastoma (MB). However, SMO-specific antagonists produced mixed results in clinical trials, marked by limited efficacy and high rate of acquired resistance in tumors. Here we discovered that Nilotinib, an approved inhibitor of several kinases, possesses an anti-Hh activity, at clinically achievable concentrations, due to direct binding to SMO and inhibition of SMO signaling. Nilotinib was more efficacious than the SMO-specific antagonist Vismodegib in inhibiting growth of two Hh-dependent MB cell lines. It also reduced tumor growth in subcutaneous MB mouse xenograft model. These results indicate that in addition to its known activity against several tyrosine-kinase-mediated proliferative pathways, Nilotinib is a direct inhibitor of the Hh pathway. The newly discovered extension of Nilotinib's target profile holds promise for the treatment of Hh-dependent cancers.

The Angiotensin Receptor Blocker Losartan Suppresses Growth of Pulmonary Metastases via AT1R-Independent Inhibition of CCR2 Signaling and Monocyte Recruitment.

Inflammatory monocytes have been shown to play key roles in cancer metastasis through promotion of tumor cell extravasation, growth, and angiogenesis. Monocyte recruitment to metastases is mediated primarily via the CCL2-CCR2 chemotactic axis. Thus, disruption of this axis represents an attractive therapeutic target for the treatment of metastatic disease. Losartan, a type I angiotensin II receptor (AT1R) antagonist, has been previously shown to have immunomodulatory actions involving monocyte and macrophage activity. However, the exact mechanisms accounting for these effects have not been fully elucidated. Therefore, we investigated the effects of losartan and its primary metabolite on CCL2-mediated monocyte recruitment and CCR2 receptor function using mouse tumor models and in vitro human monocyte cultures. We show, in this study, that losartan and its metabolite potently inhibit monocyte recruitment through the noncompetitive inhibition of CCL2-induced ERK1/2 activation, independent of AT1R activity. Studies in experimental metastasis models demonstrated that losartan treatment significantly reduced the metastatic burden in mice, an effect associated with a significant decrease in CD11b+/Ly6C+-recruited monocytes in the lungs. Collectively, these results indicate that losartan can exert antimetastatic activity by inhibiting CCR2 signaling and suppressing monocyte recruitment and therefore suggest that losartan (and potentially other AT1R blocker drugs) could be repurposed for use in cancer immunotherapy.

2-Aminothiazole Derivatives as Selective Allosteric Modulators of the Protein Kinase CK2. 2. Structure-Based Optimization and Investigation of Effects Specific to the Allosteric Mode of Action.

Protein CK2 has gained much interest as an anticancer drug target in the past decade. We had previously described the identification of a new allosteric site on the catalytic α-subunit, along with first small molecule ligands based on the 4-(4-phenylthiazol-2-ylamino)benzoic acid scaffold. In the present work, structure optimizations guided by a binding model led to the identification of the lead compound 2-hydroxy-4-((4-(naphthalen-2-yl)thiazol-2-yl)amino)benzoic acid (27), showing a submicromolar potency against purified CK2α (IC50 = 0.6 µM). Furthermore, 27 induced apoptosis and cell death in 786-O renal cell carcinoma cells (EC50 = 5 µM) and inhibited STAT3 activation even more potently than the ATP-competitive drug candidate CX-4945 (EC50 of 1.6 µM vs 5.3 µM). Notably, the potencies of our allosteric ligands to inhibit CK2 varied depending on the individual substrate. Altogether, the novel allosteric pocket was proved a druggable site, offering an excellent perspective to develop efficient and selective allosteric CK2 inhibitors.

2-Aminothiazole Derivatives as Selective Allosteric Modulators of the Protein Kinase CK2. 1. Identification of an Allosteric Binding Site.

CK2 is a ubiquitous Ser/Thr protein kinase involved in the control of various signaling pathways and is known to be constitutively active. In the present study, we identified aryl 2-aminothiazoles as a novel class of CK2 inhibitors, which displayed a non-ATP-competitive mode of action and stabilized an inactive conformation of CK2 in solution. Enzyme kinetics studies, STD NMR, circular dichroism spectroscopy, and native mass spectrometry experiments demonstrated that the compounds bind in an allosteric pocket outside the ATP-binding site. Our data, combined with molecular docking studies, strongly suggested that this new binding site was located at the interface between the αC helix and the flexible glycine-rich loop. A first hit optimization led to compound 7, exhibiting an IC50 of 3.4 µM against purified CK2α in combination with a favorable selectivity profile. Thus, we identified a novel class of CK2 inhibitors targeting an allosteric pocket, offering great potential for further optimization into anticancer drugs.

Convergence of Wnt, growth factor, and heterotrimeric G protein signals on the guanine nucleotide exchange factor Daple.

Cellular proliferation, differentiation, and morphogenesis are shaped by multiple signaling cascades, and their dysregulation plays an integral role in cancer progression. Three cascades that contribute to oncogenic potential are those mediated by Wnt proteins and the receptor Frizzled (FZD), growth factor receptor tyrosine kinases (RTKs), and heterotrimeric G proteins and associated GPCRs. Daple is a guanine nucleotide exchange factor (GEF) for the G protein Gαi Daple also binds to FZD and the Wnt/FZD mediator Dishevelled (Dvl), and it enhances ß-catenin-independent Wnt signaling in response to Wnt5a-FZD7 signaling. We identified Daple as a substrate of multiple RTKs and non-RTKs and, hence, as a point of convergence for the three cascades. We found that phosphorylation near the Dvl-binding motif in Daple by both RTKs and non-RTKs caused Daple/Dvl complex dissociation and augmented the ability of Daple to bind to and activate Gαi, which potentiated ß-catenin-independent Wnt signals and stimulated epithelial-mesenchymal transition (EMT) similarly to Wnt5a/FZD7 signaling. Although Daple acts as a tumor suppressor in the healthy colon, the concurrent increased abundance of Daple and epidermal growth factor receptor (EGFR) in colorectal tumors was associated with poor patient prognosis. Thus, the Daple-dependent activation of Gαi and the Daple-dependent enhancement of ß-catenin-independent Wnt signals are not only stimulated by Wnt5a/FZD7 to suppress tumorigenesis but also hijacked by growth factor-activated RTKs to enhance tumor progression. These findings identify a cross-talk paradigm among growth factor RTKs, heterotrimeric G proteins, and the Wnt/FZD pathway in cancer.

What Do Structures Tell Us About Chemokine Receptor Function and Antagonism?

Chemokines and their cell surface G protein-coupled receptors are critical for cell migration, not only in many fundamental biological processes but also in inflammatory diseases and cancer. Recent X-ray structures of two chemokines complexed with full-length receptors provided unprecedented insight into the atomic details of chemokine recognition and receptor activation, and computational modeling informed by new experiments leverages these insights to gain understanding of many more receptor:chemokine pairs. In parallel, chemokine receptor structures with small molecules reveal the complicated and diverse structural foundations of small molecule antagonism and allostery, highlight the inherent physicochemical challenges of receptor:chemokine interfaces, and suggest novel epitopes that can be exploited to overcome these challenges. The structures and models promote unique understanding of chemokine receptor biology, including the interpretation of two decades of experimental studies, and will undoubtedly assist future drug discovery endeavors.

Structural basis of ligand interaction with atypical chemokine receptor 3.

Chemokines drive cell migration through their interactions with seven-transmembrane (7TM) chemokine receptors on cell surfaces. The atypical chemokine receptor 3 (ACKR3) binds chemokines CXCL11 and CXCL12 and signals exclusively through ß-arrestin-mediated pathways, without activating canonical G-protein signalling. This receptor is upregulated in numerous cancers making it a potential drug target. Here we collected over 100 distinct structural probes from radiolytic footprinting, disulfide trapping, and mutagenesis to map the structures of ACKR3:CXCL12 and ACKR3:small-molecule complexes, including dynamic regions that proved unresolvable by X-ray crystallography in homologous receptors. The data are integrated with molecular modelling to produce complete and cohesive experimentally driven models that confirm and expand on the existing knowledge of the architecture of receptor:chemokine and receptor:small-molecule complexes. Additionally, we detected and characterized ligand-induced conformational changes in the transmembrane and intracellular regions of ACKR3 that elucidate fundamental structural elements of agonism in this atypical receptor.

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.