Abstract ID 131403Poster Board 569 Chemokine receptors 2 and 5 (CCR2 and CCR5, respectively) are highly homologous G-protein coupled receptors on the surface of select immune cells with distinct functions and endogenous agonists but with select shared antagonists. These chemokine receptors are promising targets for small molecule therapeutics in the treatment of various cancers and autoimmune diseases, despite only one currently approved therapeutic for CCR5. Rational design of future therapeutics relies on a structural understanding of the determinants of antagonist selectivity as high affinity targeting of only one receptor is often desired. Further complicating therapeutic development, antagonists of human CCR2 (hCCR2) are often inactive to mouse CCR2 (mCCR2). Here, through rationally-guided gain-of-function mutagenesis, we characterized the molecular determinants of CCR2/CCR5 and human/mouse antagonist selectivity. Binding poses for a panel of selective and dual-specific antagonists were generated through molecular docking. Using these poses, we hypothesized that a single bulky residue in TM2 of hCCR5 (Y892.63) and mCCR2 (Y1142.63) dictates selectivity by obstructing binding of the minor pocket-targeting orthosteric antagonists with large substituents. By contrast, the corresponding S1012.63 of hCCR2 is permissive to the binding of such antagonists because it allows them to occupy space otherwise filled by tyrosine. To validate this hypothesis, we introduced a Y892.63S mutation into human CCR5 and tested the ability of a previously CCR2-selective antagonist cherney08a_22 to inhibit chemokine-induced suppression of forskolin-stimulated cAMP in HeLa cells expressing the mutant. Intracellular cAMP levels were measured with the BRET-based CAMYEL reporter. These experiments confirmed that indeed the CCR5(Y892.63S) mutant became sensitive to cherney08a_22. We also demonstrated how the same residue could impart selectivity against human CCR5 and mouse CCR2 to antagonists with a medium-size (acetamide) substituent. An alternative hypothesis was assessed for CCR2-selective antagonists predicted to bind towards TM5 and the major pocket. Selectivity for these hCCR2 antagonists was suspected to be conveyed by residues F1093.33, S18045.52, or I1985.42 of human CCR5, which are distinct from their CCR2 counterparts. However, the introduction of CCR2-mimicking F1093.33H and S18045.52P mutations in hCCR5 was not sufficient to produce sensitivity to major pocket-targeting CCR2-selective antagonist PF-4136309. We therefore concluded that its selectivity mechanism is due to the hCCR2 residue R2065.42 which is I1985.42 in hCCR5. Our findings provide a better framework for the rational development of selective and dual-affinity antagonists for CCR2 and CCR5 to overcome past deficiencies in design of potent and selective inhibitors.
Chemokines are involved in cell migration and activation during routine immune surveillance, inflammation and even cancer metastasis. The migration of chemokine receptor- bearing cells, including leukocytes and tumor cells, occurs in response to the secretion of chemokines, which accumulate on cell surfaces through interaction with glycosaminoglycans (GAGs) where they effectively serve as traffic signals to guide cell movement. Engagement of chemokines with their receptors subsequently causes the activation of signaling pathways that result in firm adhesion and extravasation of the cell into tissue, and in the case of leukocytes, activation of defense mechanisms. However, in cancer cells, the signaling pathways can be exploited or redirected, resulting in responses like survival, growth and proliferation. Herein, a structural and functional approach was used to address specific questions about the interactions of chemokines (i) with GAGs and (ii) with chemokine receptors in the context of cancer. Technically, the use of mass spectrometry has been a strong theme throughout these studies. In Chapter 2, a novel application of hydroxyl radical footprinting coupled with mass spectrometry was used to characterize the GAG binding specificity of the chemokine, MCP-3/CCL7. Potential GAG binding epitopes, identified by mass spectrometry, were then validated by mutagenesis and functional assays. In Chapter 3 and 4, a phosphoproteomic mass spectrometry strategy was used to elucidate CXCL12- mediated survival signaling through the receptor, CXCR4, in cells from patients with chronic lymphocytic leukemia (CLL). While signaling cascades involved in chemokine- mediated migration are well established, pathways involved in cell survival and proliferation in cancer, are not. Methods developed for phosphopeptide enrichment, and subsequent analysis via mass spectrometry are described in Chapter 3, and interesting/novel phosphoproteins, potentially involved in CXCL12-mediated CLL survival are described in Chapter 4. In Chapter 5, a functional approach was taken to elucidate the roles of receptors CXCR4 and CXCR7 in breast cancer growth and metastasis. The data show that CXCR7 affects the functional activity of CXCR4 in vitro, and decreases the extent of lung metastases in vivo, without inhibiting primary tumor growth. Overall, these studies serve to better understand some of the regulatory mechanisms that control chemokine function in normal physiology and in cancer
Heparan sulfates (HS) are linear sulfated polysaccharides that modulate a wide range of physiological and disease-processes. Variations in HS epimerization and sulfation provide enormous structural diversity, which is believed to underpin protein binding and regulatory properties. The ligand requirements of HS-binding proteins have, however, been defined in only a few cases. We describe here a synthetic methodology that can rapidly provide a library of well-defined HS oligosaccharides. It is based on the use of modular disaccharides to assemble several selectively protected tetrasaccharides that were subjected to selective chemical modifications such as regioselective O- and N-sulfation and selective de-sulfation. A number of the resulting compounds were subjected to enzymatic modifications by 3-O-sulfotransferases-1 (3-OST1) to provide 3-O-sulfated derivatives. The various approaches for diversification allowed one tetrasaccharide to be converted into 12 differently sulfated derivatives. By employing tetrasaccharides with different backbone compositions, a library of 47 HS-oligosaccharides was prepared and the resulting compounds were used to construct a HS microarray. The ligand requirements of a number of HS-binding proteins including fibroblast growth factor 2 (FGF-2), and the chemokines CCL2, CCL5, CCL7, CCL13, CXCL8, and CXCL10 were examined using the array. Although all proteins recognized multiple compounds, they exhibited clear differences in structure-binding characteristics. The HS microarray data guided the selection of compounds that could interfere in biological processes such as cell proliferation. Although the library does not cover the entire chemical space of HS-tetrasaccharides, the binding data support a notion that changes in cell surface HS composition can modulate protein function.
Both chemokine oligomerization and binding to glycosaminoglycans (GAGs) are required for their function in cell recruitment. Interactions with GAGs facilitate the formation of chemokine gradients, which provide directional cues for migrating cells. In contrast, chemokine oligomerization is thought to contribute to the affinity of GAG interactions by providing a more extensive binding surface than single subunits alone. However, the importance of chemokine oligomerization to GAG binding has not been extensively quantified. Additionally, the ability of chemokines to form different oligomers has been suggested to impart specificity to GAG interactions, but most studies have been limited to heparin. In this study, several differentially oligomerizing chemokines (CCL2, CCL3, CCL5, CCL7, CXCL4, CXCL8, CXCL11 and CXCL12) and select oligomerization-deficient mutants were systematically characterized by surface plasmon resonance to determine their relative affinities for heparin, heparan sulfate (HS) and chondroitin sulfate-A (CS-A). Wild-type chemokines demonstrated a hierarchy of binding affinities for heparin and HS that was markedly dependent on oligomerization. These results were corroborated by their relative propensity to accumulate on cells and the critical role of oligomerization in cell presentation. CS-A was found to exhibit greater chemokine selectivity than heparin or HS, as it only bound a subset of chemokines; moreover, binding to CS-A was ablated with oligomerization-deficient mutants. Overall, this study definitively demonstrates the importance of oligomerization for chemokine–GAG interactions, and demonstrates diversity in the affinity and specificity of different chemokines for GAGs. These data support the idea that GAG interactions provide a mechanism for fine-tuning chemokine function.
Abstract 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 S101 2.63 and V244 6.36 are implicated as determinants of CCR2/CCR5 and human/mouse orthosteric and allosteric antagonist selectivity, respectively, and the role of S101 2.63 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.
The control of cell migration by chemokines involves interactions with two types of receptors: seven transmembrane chemokine‐type G protein‐coupled receptors and cell surface or extracellular matrix‐associated glycosaminoglycans. Coordinated interaction of chemokines with both types of receptors is required for directional migration of cells in numerous physiological and pathological processes. Accumulated structural information, culminating most recently in the structure of a chemokine receptor in complex with a chemokine, has led to a view where chemokine oligomers bind to glycosaminoglycans through epitopes formed when chemokine subunits come together, while chemokine monomers bind to receptors in a pseudo two‐step mechanism of receptor activation. Exploitation of this structural knowledge has and will continue to provide important information for therapeutic strategies, as described in this review.
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