ABSTRACT
CC chemokine receptor 2 and CCL2 are highly involved in cancer growth and metastasis, and immune escape. Raised sodium ion concentrations in solid tumours have also been correlated to metastasis and immune modulation. Sodium ions can modulate class A G protein-coupled receptors through the sodium ion binding site characterized by a highly conserved aspartic acid residue (D2.50), also present in CCR2. Hence, we further explored this binding site in CCR2 by radioligand binding studies and mutagenesis. Modulation of three distinctly binding radioligands by sodium ions and amiloride derivates was investigated. Sodium ions were observed to be relatively weak modulators of antagonist binding, but substantially increased 125I-CCL2 dissociation from CCR2. 6-Substituted Hexamethylene Amiloride (HMA) modulated all tested radioligands. Induced-fit docking of HMA in the presumed sodium ion binding site of CCR2 confirmed its binding site. Finally, investigation of (cancer-associated) mutations in the sodium ion binding site showed a markedly decreased expression compared to wild type. Only two mutants, G123A3.35 and G127K3.39, were able to be bound by [3H]INCB3344 and [3H]CCR2-RA-[R]. Thus, mutagenesis showed that the sodium ion binding site residues, which are distinct from other class A GPCRs and related to chemokine receptor evolution, are crucial for receptor integrity. Moreover, the tested mutations appeared to have no effect on modulation observed by HMA or a minor effect on sodium chloride modulation on the tested radioligands. All in all, these results invite further exploration of the CCR2 sodium ion binding site in (cancer) biology, and potentially as a third druggable binding site.
ABSTRACT
Tumor tissues often contain high extracellular adenosine, promoting an immunosuppressed environment linked to mesenchymal transition and immune evasion. Here, we show that loss of the epithelial transcription factor, GRHL2, triggers NT5E/CD73 ecto-enzyme expression, augmenting the conversion of AMP to adenosine. GRHL2 binds an intronic NT5E sequence and is negatively correlated with NT5E/CD73 in breast cancer cell lines and patients. Remarkably, the increased adenosine levels triggered by GRHL2 depletion in MCF-7 breast cancer cells do not suppress but mildly increase CD8 T cell recruitment, a response mimicked by a stable adenosine analog but prevented by CD73 inhibition. Indeed, NT5E expression shows a positive rather than negative association with CD8 T cell infiltration in breast cancer patients. These findings reveal a GRHL2-regulated immune modulation mechanism in breast cancers and show that extracellular adenosine, besides its established role as a suppressor of T cell-mediated cytotoxicity, is associated with enhanced T cell recruitment.
ABSTRACT
BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest types of cancer and the chemotherapies such as gemcitabine/nab-paclitaxel are confronted with intrinsic or acquired resistance. The aim of this study was to investigate mechanisms underlying paclitaxel resistance in PDAC and explore strategies to overcome it. METHODS: Three paclitaxel (PR) and gemcitabine resistant (GR) PDAC models were established. Transcriptomics and proteomics were used to identify conserved mechanisms of drug resistance. Genetic and pharmacological approaches were used to overcome paclitaxel resistance. RESULTS: Upregulation of ABCB1 through locus amplification was identified as a conserved feature unique to PR cells. ABCB1 was not affected in any of the GR models and no cross resistance was observed. The ABCB1 inhibitor verapamil or siRNA-mediated ABCB1 depletion sensitized PR cells to paclitaxel and prevented efflux of ABCB1 substrates in all models. ABCB1 expression was associated with a trend towards shorter survival in patients who had received gemcitabine/nab-paclitaxel treatment. A pharmacological screen identified known and novel kinase inhibitors that attenuate efflux of ABCB1 substrates and sensitize PR PDAC cells to paclitaxel. CONCLUSION: Upregulation of ABCB1 through locus amplification represents a novel, conserved mechanism of PDAC paclitaxel resistance. Kinase inhibitors identified in this study can be further (pre) clinically explored as therapeutic strategies to overcome paclitaxel resistance in PDAC.
Subject(s)
Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Humans , Paclitaxel/pharmacology , Paclitaxel/therapeutic use , Gemcitabine , Deoxycytidine/pharmacology , Deoxycytidine/therapeutic use , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Carcinoma, Pancreatic Ductal/drug therapy , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/pathology , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , ATP Binding Cassette Transporter, Subfamily B/geneticsABSTRACT
Integrins mediate adhesive interactions between cells and their environment, including neighboring cells and extracellular matrix (ECM). These heterodimeric transmembrane receptors bind extracellular ligands with their globular head domains and connect to the cytoskeleton through multi-protein interactions at their cytoplasmic tails. Integrin containing cell-matrix adhesions are dynamic force-responsive protein complexes that allow bidirectional mechanical coupling of cells with their environment. This allows cells to sense and modulate tissue mechanics and regulates intracellular signaling impacting on cell faith, survival, proliferation, and differentiation programs. Dysregulation of these functions has been extensively reported in cancer and associated with tumor growth, invasion, angiogenesis, metastasis, and therapy resistance. This central role in multiple hallmarks of cancer and their localization on the cell surface makes integrins attractive targets for cancer therapy. However, despite a wealth of highly encouraging preclinical data, targeting integrin adhesion complexes in clinical trials has thus far failed to meet expectations. Contributing factors to therapeutic failure are 1) variable integrin expression, 2) redundancy in integrin function, 3) distinct roles of integrins at various disease stages, and 4) sequestering of therapeutics by integrin-containing tumor-derived extracellular vesicles. Despite disappointing clinical results, new promising approaches are being investigated that highlight the potential of integrins as targets or prognostic biomarkers. Improvement of therapeutic delivery at the tumor site via integrin binding ligands is emerging as another successful approach that may enhance both efficacy and safety of conventional therapeutics. In this review we provide an overview of recent encouraging preclinical findings, we discuss the apparent disagreement between preclinical and clinical results, and we consider new opportunities to exploit the potential of integrin adhesion complexes as targets for cancer therapy.
ABSTRACT
Despite the fact that different genetic programmes drive metastasis of solid tumours, the ultimate outcome is the same: tumour cells are empowered to pass a series of physical hurdles to escape the primary tumour and disseminate to other organs. Epithelial-to-mesenchymal transition (EMT) has been proposed to drive the detachment of individual cells from primary tumour masses and facilitate the subsequent establishment of metastases in distant organs. However, this concept has been challenged by observations from pathologists and from studies in animal models, in which partial and transient acquisition of mesenchymal traits is seen but tumour cells travel collectively rather than as individuals. In this review, we discuss how crosstalk between a hybrid E/M state and variations in the mechanical aspects of the tumour microenvironment can provide tumour cells with the plasticity required for strategies to navigate surrounding tissues en route to dissemination. Targeting such plasticity provides therapeutic opportunities to combat metastasis.
Subject(s)
Cell Plasticity/physiology , Epithelial-Mesenchymal Transition/physiology , Neoplasm Invasiveness/pathology , Neoplasms/pathology , Tumor Microenvironment/physiology , Animals , Humans , Neoplasms/metabolism , Receptor Cross-Talk/physiologyABSTRACT
Herein we report the design and synthesis of a series of highly selective CCR2 antagonists as 18 F-labeled PET tracers. The derivatives were evaluated extensively for their off-target profile at 48 different targets. The most potent and selective candidate was applied inâ vivo in a biodistribution study, demonstrating a promising profile for further preclinical development. This compound represents the first potential nonpeptidic PET tracer for the imaging of CCR2 receptors.
Subject(s)
Drug Development , Radiopharmaceuticals/pharmacology , Receptors, CCR2/antagonists & inhibitors , Dose-Response Relationship, Drug , Humans , Molecular Structure , Positron-Emission Tomography , Radiopharmaceuticals/chemistry , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Intracellular Space/drug effects , Intracellular Space/metabolism , Pyrroles/chemistry , Pyrroles/pharmacology , Receptors, CCR1/antagonists & inhibitors , Receptors, CCR2/antagonists & inhibitors , Allosteric Regulation/drug effects , Humans , Ligands , Molecular Docking Simulation , Protein Conformation , Pyrroles/chemical synthesis , Pyrroles/metabolism , Receptors, CCR1/chemistry , Receptors, CCR1/metabolism , Receptors, CCR2/chemistry , Receptors, CCR2/metabolism , Structure-Activity RelationshipABSTRACT
Metastases are a major cause of cancer mortality. AXL, a receptor tyrosine kinase aberrantly expressed in many tumors, is a potent oncogenic driver of metastatic cell motility and has been identified as broadly relevant in cancer drug resistance. Despite its frequent association with changes in cancer phenotypes, the precise mechanism leading to AXL activation is incompletely understood. In addition to its ligand growth arrest specific-6 (Gas6), activation of AXL requires the lipid moiety phosphatidylserine (PS). Phosphatidylserine is only available to mediate AXL activation when it is externalized on cell membranes, an event that occurs during certain physiologic processes such as apoptosis. Here, it is reported that exposure of cancer cells to phosphatidylserine-containing vesicles, including synthetic liposomes and apoptotic bodies, contributes to enhanced migration of tumor cells via a PS-Gas6-AXL signaling axis. These findings suggest that anticancer treatments that induce fractional cell killing enhance the motility of surviving cells in AXL-expressing tumors, which may explain the widespread role of AXL in limiting therapeutic efficacy.Implications: This study demonstrates that motility behavior of AXL-expressing tumor cells can be elicited by Gas6-bearing apoptotic bodies generated from tumor treatment with therapeutics that produce killing of a portion of the tumor cells present but not all, hence generating potentially problematic invasive and metastatic behavior of the surviving tumor cells. Mol Cancer Res; 15(12); 1656-66. ©2017 AACR.
Subject(s)
Drug Resistance, Neoplasm/genetics , Intercellular Signaling Peptides and Proteins/genetics , Neoplasms/genetics , Proto-Oncogene Proteins/genetics , Receptor Protein-Tyrosine Kinases/genetics , Apoptosis/genetics , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Extracellular Vesicles/genetics , Gene Expression Regulation, Neoplastic , Humans , Liposomes/metabolism , Neoplasms/drug therapy , Neoplasms/metabolism , Neoplasms/pathology , Phosphatidylserines/genetics , Phosphatidylserines/metabolism , Signal Transduction/genetics , Axl Receptor Tyrosine KinaseABSTRACT
The AXL receptor is a TAM (Tyro3, AXL, MerTK) receptor tyrosine kinase (RTK) important in physiological inflammatory processes such as blood clotting, viral infection, and innate immune-mediated cell clearance. Overexpression of the receptor in a number of solid tumors is increasingly appreciated as a key drug resistance and tumor dissemination mechanism. Although the ligand-receptor (Gas6-AXL) complex structure is known, literature reports on ligand-mediated signaling have provided conflicting conclusions regarding the influence of other factors such as phosphatidylserine binding, and a detailed, mechanistic picture of AXL activation has not emerged. Integrating quantitative experiments with mathematical modeling, we show here that AXL operates to sense local spatial heterogeneity in ligand concentration, a feature consistent with its physiological role in inflammatory cell responses. This effect arises as a result of an intricate reaction-diffusion interaction. Our results demonstrate that AXL functions distinctly from other RTK families, a vital insight for envisioned design of AXL-targeted therapeutic intervention.
ABSTRACT
Animal models suggest that the chemokine ligand 2/CC-chemokine receptor 2 (CCL2/CCR2) axis plays an important role in the development of inflammatory diseases. However, CCR2 antagonists have failed in clinical trials because of a lack of efficacy. We previously described a new approach for the design of CCR2 antagonists by the use of structure-kinetics relationships (SKRs). Herein we report new findings on the structure-affinity relationships (SARs) and SKRs of the reference compound MK-0483, its diastereomers, and its structural analogues as CCR2 antagonists. The SARs of the 4-arylpiperidine group suggest that lipophilic hydrogen-bond-accepting substituents at the 3-position are favorable. However, the SKRs suggest that a lipophilic group with a certain size is desired [e.g., 3-Br: Ki =2.8 nM, residence time (t(res))=243 min; 3-iPr: Ki =3.6 nM, t(res) =266 min]. Alternatively, additional substituents and further optimization of the molecule, while keeping a carboxylic acid at the 3-position, can also prolong t(res); this was most prominently observed in MK-0483 (Ki =1.2 nM, t(res) =724 min) and a close analogue (Ki =7.8 nM) with a short residence time.
Subject(s)
Cyclopentanes/pharmacology , Piperidines/pharmacology , Receptors, CCR2/antagonists & inhibitors , Animals , Cyclopentanes/chemical synthesis , Cyclopentanes/chemistry , Dose-Response Relationship, Drug , Kinetics , Molecular Conformation , Piperidines/chemical synthesis , Piperidines/chemistry , Structure-Activity Relationship , Time FactorsABSTRACT
Activation of chemokine CC receptors subtype 2 (CCR2) plays an important role in chronic inflammatory processes such as atherosclerosis, multiple sclerosis and rheumatoid arthritis. A diverse set of spirocyclic butanamides 4 (N-benzyl-4-(3,4-dihydrospiro[[2]benzopyran-1,4'-piperidin]-1'-yl)butanamides) was prepared by different combination of spirocyclic piperidines 8 (3,4-dihydrospiro[[2]benzopyran-1,4'-piperidines]) and γ-halobutanamides 11. A key step in the synthesis of spirocyclic piperidines 8 was an Oxa-Pictet-Spengler reaction of ß-phenylethanols 5 with piperidone acetal 6. The substituted γ-hydroxybutanamides 11c-e were prepared by hydroxyethylation of methyl acetates 13 with ethylene sulfate giving the γ-lactones 14c and 14e. Aminolysis of the γ-lactones 14c and 14e with benzylamines provided the γ-hydroxybutanamides 15c-e, which were converted into the bromides 11c-e by an Appel reaction using polymer-bound PPh3. In radioligand binding assays the spirocyclic butanamides 4 did not displace the iodinated radioligand (125)I-CCL2 from the human CCR2. However, in the Ca(2+)-flux assay using human CCR2 strong antagonistic activity of butanamides 4 was detected. Analysis of the IC50-values led to clear relationships between the structure and the inhibition of the Ca(2+)-flux. 4g (4-(3,4-dihydrospiro[[2]benzopyran-1,4'-piperidin]-1'-yl)-N-[3,5-bis(trifluoromethylbenzyl)]-2-(4-fluorophenyl)butanamide) and 4o (N-[3,5-bis(trifluoromethyl)benzyl]-2-cyclopropyl-4-(3,4-dihydrospiro[[2]benzopyran-1,4'-piperidin]-1'-yl)butanamide) represent the most potent CCR2 antagonists with IC50-values of 89 and 17nM, respectively. Micromolar activities were found in the ß-arrestin recruitment assay with murine CCR2, but the structure-activity-relationships detected in the Ca(2+)-flux assay were confirmed.
Subject(s)
Receptors, CCR2/antagonists & inhibitors , Spiro Compounds/chemistry , Spiro Compounds/pharmacology , Structure-Activity Relationship , Arrestins/metabolism , Calcium/metabolism , Cell Line/drug effects , Chemistry Techniques, Synthetic , Drug Evaluation, Preclinical/methods , Humans , Inhibitory Concentration 50 , Iodine Radioisotopes , Radioligand Assay , Receptors, CCR2/metabolism , Spiro Compounds/chemical synthesis , Spiro Compounds/metabolism , beta-ArrestinsABSTRACT
Chemokine ligand 2 (CCL2) mediates chemotaxis of monocytes to inflammatory sites via interaction with its G protein-coupled receptor CCR2. Preclinical animal models suggest that the CCL2-CCR2 axis has a critical role in the development and maintenance of inflammatory disease states (e.g., multiple sclerosis, atherosclerosis, insulin resistance, restenosis, and neuropathic pain), which can be treated through inhibition of the CCR2 receptor. However, in clinical trials high-affinity inhibitors of CCR2 have often demonstrated a lack of efficacy. We have previously described a new approach for the design of high-affinity CCR2 antagonists, by taking their residence time (RT) on the receptor into account. Here, we report our findings on both structure-affinity relationship (SAR) and structure-kinetic relationship (SKR) studies for a series of 3-((inden-1-yl)amino)-1-isopropyl-cyclopentane-1-carboxamides as CCR2 antagonists. SAR studies showed that this class of compounds tolerates a vast diversity of substituents on the indenyl ring with only small changes in affinity. However, the SKR is affected greatly by minor modifications of the structure. The combination of SAR and SKR in the hit-to-lead process resulted in the discovery of a new high-affinity and long-residence-time CCR2 antagonist (compound 15a, Ki = 2.4 nM; RT = 714 min).
Subject(s)
Chemokine CCL2/antagonists & inhibitors , Cyclopentanes/chemical synthesis , Animals , Cell Line, Tumor , Chemokine CCL2/genetics , Cyclopentanes/chemistry , Cyclopentanes/pharmacology , Humans , Kinetics , Molecular Structure , Protein Binding , Stereoisomerism , Structure-Activity Relationship , Time Factors , TransfectionABSTRACT
CCR2 and CCR5 receptors play a key role in the development and progression of several inflammatory, cardiovascular and autoimmune diseases. Therefore, dual targeting of both receptors appeals as a promising strategy for the treatment of such complex, multifactorial disorders. Herein we report on the design, synthesis and biological evaluation of benzo[7]annulene- and [7]annulenothiophene-based selective and dual CCR2 and CCR5 receptor antagonists. Intermediates were designed in such a way that diversification could be introduced at the end of the synthesis. Starting from the lead compound TAK-779 (1), the quaternary ammonium moiety was exchanged by different non-charged moieties, the 4-methylphenyl moiety was extensively modified and the benzo[7]annulene core was replaced bioisosterically by the [7]annulenothiophene system. The naphthyl derivative 9h represents the most promising dual antagonist (Ki (CCR2) = 25 nM, IC50 (CCR5) = 17 nM), whereas the 6-isopropoxy-3-pyridyl and 4-methoxycarbonylphenyl derivatives 9k and 9r show more than 20-fold selectivity for the CCR2 (Ki = 19 nM) over the CCR5 receptor.
Subject(s)
CCR5 Receptor Antagonists/chemical synthesis , CCR5 Receptor Antagonists/pharmacology , Polycyclic Aromatic Hydrocarbons/chemistry , Polycyclic Aromatic Hydrocarbons/pharmacology , Receptors, CCR2/antagonists & inhibitors , Thiophenes/chemistry , Thiophenes/pharmacology , Animals , Binding Sites/drug effects , CCR5 Receptor Antagonists/chemistry , Dose-Response Relationship, Drug , Humans , Mice , Molecular Structure , Polycyclic Aromatic Hydrocarbons/chemical synthesis , Receptors, CCR2/metabolism , Receptors, CCR5/metabolism , Structure-Activity Relationship , Thiophenes/chemical synthesisABSTRACT
The chemokine receptor CCR2 is a G protein-coupled receptor that is involved in many diseases characterized by chronic inflammation, and therefore a large variety of CCR2 small molecule antagonists has been developed. On the basis of their chemical structures these antagonists can roughly be divided into two groups with most likely two topographically distinct binding sites. The aim of the current study was to identify the binding site of one such group of ligands, exemplified by three allosteric antagonists, CCR2-RA-[R], JNJ-27141491, and SD-24. We first used a chimeric CCR2/CCR5 receptor approach to obtain insight into the binding site of the allosteric antagonists and additionally introduced eight single point mutations in CCR2 to further characterize the putative binding pocket. All constructs were studied in radioligand binding and/or functional IP turnover assays, providing evidence for an intracellular binding site for CCR2-RA-[R], JNJ-27141491, and SD-24. For CCR2-RA-[R] the most important residues for binding were found to be the highly conserved tyrosine Y(7.53) and phenylalanine F(8.50) of the NPxxYx(5,6)F motif, as well as V(6.36) at the bottom of TM-VI and K(8.49) in helix-VIII. These findings demonstrate for the first time the presence of an allosteric intracellular binding site for CCR2 antagonists. This contributes to an increased understanding of the interactions of diverse ligands at CCR2 and may allow for a more rational design of future allosteric antagonists.
Subject(s)
Allosteric Site , Receptors, CCR2/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , CHO Cells , COS Cells , Cell Line, Tumor , Chlorocebus aethiops , Cricetinae , Cricetulus , Humans , Imidazoles/pharmacology , Ligands , Molecular Sequence Data , Point Mutation , Protein Binding , Pyrrolidines/pharmacology , Receptors, CCR2/antagonists & inhibitors , Receptors, CCR2/chemistry , Receptors, CCR2/genetics , Sulfonamides/pharmacologyABSTRACT
Chemokine receptors are widely expressed on a variety of immune cells and play a crucial role in normal physiology as well as in inflammatory and infectious diseases. The existence of 23 chemokine receptors and 48 chemokine ligands guarantees a tight control and fine-tuning of the immune system. Here, we discuss the multiple regulatory mechanisms of chemokine signalling at a systemic, cellular, and molecular level. In particular, we focus on the impact of biased signalling at the receptor level; an emerging concept in molecular pharmacology. An improved understanding of these mechanisms may provide a framework for more effective drug discovery and development at a target class that is so relevant for immune function.
Subject(s)
Receptors, Chemokine/metabolism , Signal Transduction , Animals , Chemokines/genetics , Chemokines/metabolism , Gene Expression Regulation , Humans , Immune System/cytology , Immune System/immunology , Immune System/metabolism , Receptors, G-Protein-Coupled/metabolismABSTRACT
Preclinical models of inflammatory diseases (e.g., neuropathic pain, rheumatoid arthritis, and multiple sclerosis) have pointed to a critical role of the chemokine receptor 2 (CCR2) and chemokine ligand 2 (CCL2). However, one of the biggest problems of high-affinity inhibitors of CCR2 is their lack of efficacy in clinical trials. We report a new approach for the design of high-affinity and long-residence-time CCR2 antagonists. We developed a new competition association assay for CCR2, which allows us to investigate the relation of the structure of the ligand and its receptor residence time [i.e., structure-kinetic relationship (SKR)] next to a traditional structure-affinity relationship (SAR). By applying combined knowledge of SAR and SKR, we were able to re-evaluate the hit-to-lead process of cyclopentylamines as CCR2 antagonists. Affinity-based optimization yielded compound 1 with good binding (Ki = 6.8 nM) but very short residence time (2.4 min). However, when the optimization was also based on residence time, the hit-to-lead process yielded compound 22a, a new high-affinity CCR2 antagonist (3.6 nM), with a residence time of 135 min.
Subject(s)
Cyclopentanes/chemical synthesis , Indans/chemical synthesis , Indenes/chemical synthesis , Receptors, CCR2/antagonists & inhibitors , Binding, Competitive , Cell Line, Tumor , Cyclopentanes/chemistry , Cyclopentanes/pharmacology , Humans , Indans/chemistry , Indans/pharmacology , Indenes/chemistry , Indenes/pharmacology , Kinetics , Ligands , Stereoisomerism , Structure-Activity Relationship , Tetrahydronaphthalenes/chemical synthesis , Tetrahydronaphthalenes/chemistry , Tetrahydronaphthalenes/pharmacologyABSTRACT
The chemokine receptor CCR2 is a G protein-coupled receptor that is activated primarily by the endogenous CC chemokine ligand 2 (CCL2). Many different small-molecule antagonists have been developed to inhibit this receptor, as it is involved in a variety of diseases characterized by chronic inflammation. Unfortunately, all these antagonists lack clinical efficacy, and therefore a better understanding of their mechanism of action is warranted. In this study, we examined the pharmacological properties of small-molecule CCR2 antagonists in radioligand binding and functional assays. Six structurally different antagonists were selected for this study, all of which displaced the endogenous agonist (125)I-CCL2 from CCR2 with nanomolar affinity. Two of these antagonists, INCB3344 [N-(2-(((3S,4S)-1-((1r,4S)-4-(benzo[d][1,3]dioxol-5-yl)-4-hydroxycyclohexyl)-4-ethoxypyrrolidin-3-yl)amino)-2-oxoethyl)-3-(trifluoromethyl)benzamide] and CCR2-RA, were radiolabeled to study the binding site in greater detail. We discovered that [(3)H]INCB3344 and [(3)H]CCR2-RA bind to distinct binding sites at CCR2, the latter being the first allosteric radioligand for CCR2. Besides the binding properties of the antagonists, we examined CCR2 inhibition in multiple functional assays, including a novel label-free whole-cell assay. INCB3344 competitively inhibited CCL2-induced G protein activation, whereas CCR2-RA showed a noncompetitive or allosteric mode of inhibition. These findings demonstrated that the CCR2 antagonists examined in this study can be classified into two groups with different binding sites and thereby different modes of inhibition. We have provided further insights in CCR2 antagonism, and these insights are important for the development of novel CCR2 inhibitors.
Subject(s)
Pyrrolidines/metabolism , Receptors, CCR2/antagonists & inhibitors , Receptors, CCR2/metabolism , Binding Sites/physiology , Cell Line , Chemokine CCL2/metabolism , Chemokine CCL2/pharmacology , Chemokines/metabolism , Chemokines/pharmacology , Humans , Protein Binding/physiology , Pyrrolidines/pharmacology , Receptors, CCR2/agonistsABSTRACT
PURPOSE: The aim of this study was to investigate the antitumor effects of HER2-directed combination therapy in ovarian cancer xenograft models to evaluate their potential. The combinations of trastuzumab and pertuzumab, and trastuzumab and aromatase inhibitor therapy were investigated. EXPERIMENTAL DESIGN: The effects of trastuzumab, pertuzumab, and letrozole on growth response, apoptosis, morphology, and gene and protein expression were evaluated in the SKOV3 ovarian cancer cell line xenograft and a panel of five human ovarian xenografts derived directly from clinical specimens. RESULTS: The combination of HER2-directed antibodies showed enhanced antitumor activity compared with single antibody therapy in the SKOV3 xenograft model. Apoptosis, morphology, and estrogen-regulated gene expression were modulated by these antibodies in both spatial and temporal manners. A panel of ovarian cancer xenografts showed differential growth responses to the combination of trastuzumab and pertuzumab. High HER2 expression and increasing HER3 protein expression on treatment were associated with growth response. In trastuzumab-treated SKOV3 tumors, there was a change in tumor morphology, with a reduction in frequency of estrogen receptor alpha (ERα)-negative clear cell areas. Trastuzumab, but not pertuzumab, increased expression of ERα in SKOV3 xenografts when analyzed by quantitative immunofluorescence. ERα and downstream signaling targets were modulated by trastuzumab alone and in combination. Trastuzumab enhanced the responsiveness of SKOV3 xenografts to letrozole when given in combination. CONCLUSIONS: These data suggest that trastuzumab in combination with pertuzumab could be an effective approach in high HER2-expressing ovarian cancers and could also enhance sensitivity to endocrine therapy in ERα-positive ovarian cancer.