Physiologically relevant aspirin concentrations trigger immunostimulatory cytokine production by human leukocytes

Acetylsalicylic acid is a globally used non-steroidal anti-inflammatory drug (NSAID) with diverse pharmacological properties, although its mechanism of immune regulation during inflammation (especially at in vivo relevant doses) remains largely speculative. Given the increase in clinical perspective of Acetylsalicylic acid in various diseases and cancer prevention, this study aimed to investigate the immunomodulatory role of physiological Acetylsalicylic acid concentrations (0.005, 0.02 and 0.2 mg/ml) in a human whole blood of infection-induced inflammation. We describe a simple, highly reliable whole blood assay using an array of toll-like receptor (TLR) ligands 1–9 in order to systematically explore the immunomodulatory activity of Acetylsalicylic acid plasma concentrations in physiologically relevant conditions. Release of inflammatory cytokines and production of prostaglandin E2 (PGE2) were determined directly in plasma supernatant. Experiments demonstrate for the first time that plasma concentrations of Acetylsalicylic acid significantly increased TLR ligand-triggered IL-1β, IL-10, and IL-6 production in a dose-dependent manner. In contrast, indomethacin did not exhibit this capacity, whereas cyclooxygenase (COX)-2 selective NSAID, celecoxib, induced a similar pattern like Acetylsalicylic acid, suggesting a possible relevance of COX-2. Accordingly, we found that exogenous addition of COX downstream product, PGE2, attenuates the TLR ligand-mediated cytokine secretion by augmenting production of anti-inflammatory cytokines and inhibiting release of pro-inflammatory cytokines. Low PGE2 levels were at least involved in the enhanced IL-1β production by Acetylsalicylic acid

The Chemokine Receptor CXCR3 Isoform B Drives Breast Cancer Stem Cells

We are seeking to identify molecular targets that are relevant to breast cancer cells with stem-like properties. There is growing evidence that cancer stem cells (CSCs) are supported by inflammatory mediators expressed in the tumor microenvironment. The chemokine receptor CXCR3 binds the interferon-γ-inducible, ELR-negative CXC chemokines CXCL9, CXCL10, and CXCL11 and malignant cells have co-opted this receptor to promote tumor cell migration and invasion. There are 2 major isoforms of CXCR3: CXCR3A and CXCR3B. The latter is generated from alternative splicing and results in a protein with a longer N-terminal domain. CXCR3 isoform A is generally considered to play a major role in tumor metastasis. When the entire tumor cell population is examined, CXCR3 isoform B is usually detected at much lower levels than CXCR3A and for this, and other reasons, was not considered to drive tumor progression. We have shown that CXCR3B is significantly upregulated in the subpopulation of breast CSCs in comparison with the bulk tumor cell population in 3 independent breast cancer cell lines (MDA-MB-231, SUM159, and T47D). Modulation of CXCR3B levels by knock in strategies increases CSC populations identified by aldehyde dehydrogenase activity or CD44+CD24− phenotype as well as tumorsphere-forming capacity. The reverse is seen when CXCR3B is gene-silenced. CXCL11 and CXCL10 directly induce CSC. We also report that novel CXCR3 allosteric modulators BD064 and BD103 prevent the induction of CSCs. BD103 inhibited experimental metastasis. This protective effect is associated with the reversal of CXCR3 ligand-mediated activation of STAT3, ERK1/2, CREB, and NOTCH1 pathways. We propose that CXCR3B, expressed on CSC, should be explored further as a novel therapeutic target

Ligand biased and probe-dependent modulation of the chemokine receptor CXCR3 signaling by negative allosteric modulators

Over the last decade functional selectivity (or bias) has evolved from being a peculiar phenomenon to being recognized as an essential feature of synthetic ligands targeting G protein-coupled receptors (GPCRs). The chemokine receptor CXCR3 is an outstanding platform to study various aspects of biased signaling, because nature itself uses functional selectivity to manipulate the receptor signaling. At the same time CXCR3 is an attractive therapeutic target in autoimmune diseases and cancer. Here we report the discovery of a small molecule (1b) that can selectively inhibit CXCL11-dependent G protein activation over β-arrestin recruitment (with a 190-fold selectivity). The compound also demonstrates probe-dependent activity, i.e. it inhibits CXCL11- over CXCL10-mediated G protein activation with a 12-fold selectivity. Together with previously reported biased negative allosteric modulator from our group, the present study provides additional support to our hypothesis of multiple binding orientations for synthetic ligands of CXCR3

Thrombocyte apheresis cassettes as a novel source of viable peripheral blood mononuclear cells

BACKGROUND Traditionally, white blood cells (WBCs) are collected from buffy coats or freshly drawn blood. However, the increasing demand for peripheral blood mononuclear cells (PBMCs) in the research phases of immunological therapy development makes it necessary to identify alternative sources of these cells. STUDY DESIGN AND METHODS Leukapheresis products are cost intensive and not offered by all blood banks. Therefore, thrombocyte apheresis cassettes (TACs), plateletpheresis waste products, were investigated as a possible low‐cost and easily accessible blood source for research laboratories. The recovery rate, phenotype, and functionality of WBC subsets from TAC are unknown and were investigated in comparison to frequently used blood resources via flow cytometry. RESULTS On average, TACs provide 30.3 × 106/mL PBMCs, situating themselves between peripheral whole blood (WB; 5.35 × 106/mL) and leukoreduction system chamber (LRSC; 163.9 × 106/mL) yields. Frequencies of CD14, CD3, CD4, CD8, CD56, CD19, and CD11c positive cells in TACs correlate with normal proportions of WBC populations. Stimulation of TAC‐derived PBMCs by lipopolysaccharide (LPS) and resiquimod (R848) showed no significant differences in expression levels of human leukocyte antigen (HLA)‐DR, DQ, DP, and CD86 or cytokine secretion compared to other blood source derived PBMC. Following stimulation with LPS or R848, comparable levels of tumor necrosis factor‐α, interleukin‐10, and interleukin‐1β could be measured between TAC, LRSC, and WB. Additionally, TAC‐derived T cells retained their proliferation capability and were able to produce interferon‐γ following T‐cell receptor stimulation. CONCLUSION TACs provide a cost‐effective source of viable and functional human blood cells that can readily be used for clinical and laboratory investigations after plateletpheresis preparation

SERPINC1 c.1247dupC: a novel SERPINC1 gene mutation associated with familial thrombosis results in a secretion defect and quantitative antithrombin deficiency

Abstract Background Antithrombin (AT) is an important anticoagulant in hemostasis. We describe here the characterization of a novel AT mutation associated with clinically relevant thrombosis. A pair of sisters with confirmed type I AT protein deficiency was genetically analyzed on suspicion of an inherited SERPINC1 mutation. A frameshift mutation, c.1247dupC, was identified and the effect of this mutation was examined on the cellular and molecular level. Methods Plasmids for the expression of wild-type (WT) and mutated SERPINC1 coding sequence (CDS) fused to green fluorescent protein (GFP) or hemagglutinin (HA) tag were transfected into HEK293T cells. Subcellular localization and secretion of the respective fusion proteins were analyzed by confocal laser scanning microscopy and Western blot. Results The c.1247dupC mutation results in a frameshift in the CDS of the SERPINC1 gene and a subsequently altered amino acid sequence (p.Ser417LysfsTer48). This alteration affects the C-terminus of the AT antigen and results in impaired secretion as confirmed by GFP- and HA-tagged mutant AT analyzed in HEK293T cells. Conclusion The p.Ser417LysfsTer48 mutation leads to impaired secretion, thus resulting in a quantitative AT deficiency. This is in line with the type I AT deficiency observed in the patients

Identifying Modulators of CXC Receptors 3 and 4 with Tailored Selectivity Using Multi-Target Docking

The G protein-coupled receptors of the C–X–C subfamily form a group among the chemokine receptors whose endogenous ligands are peptides with a common Cys–X–Cys motif. The CXC chemokine receptors 3 and 4 (CXCR3, CXCR4), which are investigated in this study, are linked to severe diseases such as cancer, multiple sclerosis, and HIV infections. Of particular interest, this receptor pair potentially forms a target for a polypharmacological drug treatment. Considering known ligands from public databases, such dual binders have not been identified yet. We therefore applied large-scale docking to the structure of CXCR4 and a homology model of CXCR3 with the goal to predict such dual binders, as well as compounds selective for either one of the receptors. Using signaling and biochemical assays, we showed that more than 50% of these predictions were correct in each category, yielding ligands with excellent binding efficiencies. These results highlight that docking is a suitable tool for the identification of ligands with tailored binding profiles to GPCRs, even when using homology models. More importantly, we present novel CXCR3–CXCR4 dual modulators that might pave the road to understanding the mechanisms of polypharmacological inhibition of these receptors

Discovery of 2-aminopyrimidines as potent agonists for the bitter taste receptor TAS2R14

The bitter taste receptor TAS2R14 is a G protein-coupled receptor that is found on the tongue as well as in the human airway smooth muscle and other extraoral tissues. Because its activation causes bronchodilatation, TAS2R14 is a potential target for the treatment of asthma or chronic obstructive pulmonary disease. Structural variations of flufenamic acid, a nonsteroidal anti-inflammatory drug, led us to 2-aminopyridines showing considerable efficacy and potency in an IP-One accumulation assay. In combination with a bioisosteric exchange of the carboxylic moiety by a tetrazole unit, a set of promising new TAS2R14 agonists was developed. The most potent ligand 28.1 (EC50 = 72 nM) revealed a six-fold higher potency than flufenamic acid and a maximum efficacy of 129%. Besides its unprecedented TAS2R14 activation, 28.1 revealed marked selectivity over a panel of 24 non-bitter taste human GPCRs

Molecular Mechanisms of Biased and Probe-Dependent Signaling at CXC-Motif Chemokine Receptor CXCR3 Induced by Negative Allosteric Modulators

Our recent explorations of allosteric modulators with improved properties resulted in the identification of two biased negative allosteric modulators, BD103 (N-1-{[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrinni-din2yl]ethyl}-4-(4-fluorobutoxy)-N-[(1 -methylpiperidin-4-yl)methyl}]butanannide) and BD064 (5-[(N-{1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrinnidin-2-yl]ethyl-2-[4-fluoro-3-(trifluoromethyl)phenyl]acetannido)methyl]-2-fluorophenyl}boronic acid), that exhibited probe-dependent inhibition of CXC-motif chemokine receptor CXCR3 signaling. With the intention to elucidate the structural mechanisms underlying their selectivity and probe dependence, we used site-directed mutagenesis combined with homology modeling and docking to identify amino acids of CXCR3 that contribute to modulator binding, signaling, and transmission of cooperativity. With the use of allosteric radioligand RAMX3 ([H-3]N-{1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrinnidin-2-yl]ethyl}-2-[4-fluoro-3-(trifluoronnethyl)phenyl]-N-[(1-methylpiperidin-4-yl)methyl] acetamide), we identified that F131(3.32) and Y308(7.43) contribute specifically to the binding pocket of BD064, whereas D186(4.60) solely participates in the stabilization of binding conformation of BD103. The influence of mutations on the ability of negative allosteric modulators to inhibit chemokine-mediated activation (CXCL11 and CXCL10) was assessed with the bioluminescence resonance energy transfer-based cAMP and beta-arrestin recruitment assay. Obtained data revealed complex molecular mechanisms governing biased and probe-dependent signaling at CXCR3. In particular, F131(3.32), S304(7.39), and Y308(7.43) emerged as key residues for the compounds to modulate the chemokine response. Notably, D186(4.60), W268(6.48), and S304(7.39) turned out to play a role in signal pathway selectivity of CXCL10, as mutations of these residues led to a G protein-active but beta-arrestin-inactive conformation. These diverse effects of mutations suggest the existence of ligand- and pathway-specific receptor conformations and give new insights in the sophisticated signaling machinery between allosteric ligands, chemokines, and their receptors, which can provide a powerful platform for the development of new allosteric drugs with improved pharmacological properties

Boronic Acids as Probes for Investigation of Allosteric Modulation of the Chemokine Receptor CXCR3

The chemokine receptor CXCR3 is a G protein-coupled receptor, which conveys extracellular signals into cells by changing its conformation upon agonist binding. To facilitate the mechanistic understanding of allosteric modulation of CXCR3, we combined computational modeling with the synthesis of novel chemical tools containing boronic acid moiety, site-directed mutagenesis, and detailed functional characterization. The design of boronic acid derivatives was based on the predictions from homology modeling and docking. The choice of the boronic acid moiety was dictated by its unique ability to interact with proteins in a reversible covalent way, thereby influencing conformational dynamics of target biomolecules. During the synthesis of the library we have developed a novel approach for the purification of drug-like boronic acids. To validate the predicted binding mode and to identify amino acid residues responsible for the transduction of signal through CXCR3, we conducted a site-directed mutagenesis study. With the use of allosteric radioligand RAMX3 we were able to establish the existence of a second allosteric binding pocket in CXCR3, which enables different binding modes of structurally closely related allosteric modulators of CXCR3. We have also identified residues Trp109^2.60 and Lys300^7.35 inside the transmembrane bundle of the receptor as crucial for the regulation of the G protein activation. Furthermore, we report the boronic acid <b>14</b> as the first biased negative allosteric modulator of the receptor. Overall, our data demonstrate that boronic acid derivatives represent an outstanding tool for determination of key receptor–ligand interactions and induction of ligand-biased signaling