Small molecule-based fluorescent probes as tool compounds for chemokine receptors CXCR4 and ACKR3

Chemokine receptors CXCR4 and ACKR3 form, together with their shared ligand CXCL12, a highly intricate signalling axis that recently has emerged as a promising target for the treatment of cancer. To further the understanding of how these receptors function, and how they are involved in the various processes of tumour development and metastasis, efforts were made to develop fluorescent tool compounds. In this thesis is described the design, synthesis, and biological characterisation of small molecule-based fluorescent probes for CXCR4 and ACKR3. It examines the use of these fluorescent probes as toolkit compounds for the study of ligand-receptor interactions and the characterisation of novel ligands. It furthermore shows their application in live cell microscopy. A thorough evaluation of SAR of reported small molecule CXCR4 receptor inhibitors, in combination with in silico design, informed the synthetic strategy for linker and fluorophore conjugation. This project explored three chemical classes of CXCR4 receptor inhibitors, leading to the development of multiple high affinity probes that show specific binding in a novel fluorescence-based NanoBRETTM binding assay (pKd values ranging 6.6-7.1). Using the same NanoBRETTM approach their utility in competition binding experiments was assessed, where they showed to be fully displaceable by established small molecule inhibitors. Additionally, the ability of these fluorescent probes to affect CXCL12-mediated signalling of CXCR4 was assessed in a functional GloSensor™ cAMP assay. Here, the antagonist nature of the small molecules these probes were based on was found to be retained in the final probes. Finally, live cell confocal microscopy using selected compounds (6,6-dimethyl-5,6-dihydroimidazo[2,1-b]thiazol-3-yl)methyl(E)-N-cyclohexyl-N'-((1r,4r)-4-(6-(2-(4-((E)-2-(5,5-difluoro-7-(thiophen-2-yl)-5H-4λ4,5λ4-dipyrrolo[1,2-c:2',1'f][1,3,2]diazaborinin-3-yl)vinyl)phenoxy)acetamido)hexanamido)cyclo-hexyl)carbamimidothioate (9), (S,E)-6-(2-(4-(2-(5,5-difluoro-7-(thiophen-2-yl)-5H-4λ4,5λ4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-3-yl)vinyl)phenoxy)acetamido)-N-(3-(3-((methyl(5,6,7,8-tetrahydroquinolin-8-yl)amino)methyl)isoquinolin-4-yl)propyl)hexanamide (22), and (S,E)-N-(4-(((1H-benzo[d]imidazol-2-yl)methyl)(5,6,7,8-tetrahydroquinolin-8-yl)amino)butyl)-6-(2-(4-(2-(5,5-difluoro-7-(thiophen-2-yl)-5H-4λ4,5λ4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-3-yl)vinyl)phenoxy)acetamido)hexanamide (30) showed clear localisation of the fluorescent ligands on the cell membrane, localising neatly at sites of high receptor expression. Utilizing a similar approach high affinity fluorescent probes were developed for the atypical chemokine receptor ACKR3. An homology model of ACKR3 bound to small molecule agonist GD301 provided rational for the structural modification of this ligand and the incorporation of BODIPY dyes. A series of fluorescent derivatives were synthesised and assessed in a NanoBRETTM binding assay on whole cells. Nine fluorescent ligands were developed, of which six showed good affinity towards the receptor (pKd values ranging 6.1-7.1). Competition binding experiments using the same NanoBRETTM approach revealed these fluorescent conjugates interact with the receptor in a competitive manner, and suggests these probes are viable toolkit compounds in the characterisation of small molecule ligands. Finally, live cell confocal imaging with selected probe (R,E)-3-(2-(6-(3-(5,5-difluoro-7,9-dimethyl-5H-4λ4,5λ4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-2-yl)propanamido)hexanamido)ethoxy)-N-(3-(2-fluorophenyl)-2-methylallyl)-4,5-dimethoxy-N-(2-(1-methylpyrrolidin-2-yl)ethyl)benzamide (40a) showed good localisation of the probe on the cell membrane. Upon binding to the receptor, the probe was furthermore able to induce internalisation over time, suggesting an agonist-like response. Treatment with a high concentration of competitive ligand resulted in complete dissipation of the fluorescent signal of the probe, revealing very low levels of nonspecific binding. Overall, this project has led to the successful development of fluorescent probes for the chemokine receptors CXCR4 and ACKR3. With their high affinity for the receptor, alongside their excellent fluorescent properties, these compounds provide valuable means to study these receptors in vitro, and facilitate live imaging techniques such as confocal microscopy and flow cytometry

Modulators of CXCR4 and CXCR7/ACKR3 Function

Copyright © 2019 by The Author(s). The two G protein-coupled receptors (GPCRs) C-X-C chemokine receptor type 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3) are part of the class A chemokine GPCR family and represent important drug targets for human immunodeficiency virus (HIV) infection, cancer, and inflammation diseases. CXCR4 is one of only three chemokine receptors with a US Food and Drug Administration approved therapeutic agent, the small-molecule modulator AMD3100. In this review, known modulators of the two receptors are discussed in detail. Initially, the structural relationship between receptors and ligands is reviewed on the basis of common structural motifs and available crystal structures. To date, no atypical chemokine receptor has been crystallized, which makes ligand design and predictions for these receptors more difficult. Next, the selectivity, receptor activation, and the resulting ligand-induced signaling output of chemokines and other peptide ligands are reviewed. Binding of pepducins, a class of lipid-peptides whose basis is the internal loop of a GPCR, to CXCR4 is also discussed. Finally, small-molecule modulators of CXCR4 and ACKR3 are reviewed. These modulators have led to the development of radio- and fluorescently labeled tool compounds, enabling the visualization of ligand binding and receptor characterization both in vitro and in vivo. SIGNIFICANCE STATEMENT: To investigate the pharmacological modulation of CXCR4 and ACKR3, significant effort has been focused on the discovery and development of a range of ligands, including small-molecule modulators, pepducins, and synthetic peptides. Imaging tools, such as fluorescent probes, also play a pivotal role in the field of drug discovery. This review aims to provide an overview of the aforementioned modulators that facilitate the study of CXCR4 and ACKR3 receptors

Frequency and evolution of sleep-wake disturbances after ischemic stroke: A 2-year prospective study of 437 patients.

OBJECTIVE In the absence of systematic and longitudinal data, this study prospectively assessed both frequency and evolution of sleep-wake disturbances (SWD) after stroke. METHODS In 437 consecutively recruited patients with ischemic stroke or transient ischemic attack (TIA), stroke characteristics and outcome were assessed within the 1st week and 3.2 ± 0.3 years (M±SD) after the acute event. SWD were assessed by interview and questionnaires at 1 and 3 months as well as 1 and 2 years after the acute event. Sleep disordered breathing (SDB) was assessed by respirography in the acute phase and repeated in one fifth of the participants 3 months and 1 year later. RESULTS Patients (63.8% male, 87% ischemic stroke and mean age 65.1 ± 13.0 years) presented with mean NIHSS-score of 3.5 ± 4.5 at admission. In the acute phase, respiratory event index was >15/h in 34% and >30/h in 15% of patients. Over the entire observation period, the frequencies of excessive daytime sleepiness (EDS), fatigue and insomnia varied between 10-14%, 22-28% and 20-28%, respectively. Mean insomnia and EDS scores decreased from acute to chronic stroke, whereas restless legs syndrome (RLS) percentages (6-9%) and mean fatigue scores remained similar. Mean self-reported sleep duration was enhanced at acute stroke (month 1: 07:54 ± 01:27h) and decreased at chronic stage (year 2: 07:43 ± 01:20h). CONCLUSIONS This study documents a high frequency of SDB, insomnia, fatigue and a prolonged sleep duration after stroke/TIA, which can persist for years. Considering the negative effects of SWD on physical, brain and mental health these data suggest the need for a systematic assessment and management of post-stroke SWD

CXCL17 is an allosteric inhibitor of CXCR4 through a mechanism of action involving glycosaminoglycans

CXCL17 is a chemokine principally expressed by mucosal tissues, where it facilitates chemotaxis of monocytes, dendritic cells, and macrophages and has antimicrobial properties. CXCL17 is also implicated in the pathology of inflammatory disorders and progression of several cancers, and its expression is increased during viral infections of the lung. However, the exact role of CXCL17 in health and disease requires further investigation, and there is a need for confirmed molecular targets mediating CXCL17 functional responses. Using a range of bioluminescence resonance energy transfer (BRET)–based assays, here we demonstrated that CXCL17 inhibited CXCR4-mediated signaling and ligand binding. Moreover, CXCL17 interacted with neuropillin-1, a VEGFR2 coreceptor. In addition, we found that CXCL17 only inhibited CXCR4 ligand binding in intact cells and demonstrated that this effect was mimicked by known glycosaminoglycan binders, surfen and protamine sulfate. Disruption of putative GAG binding domains in CXCL17 prevented CXCR4 binding. This indicated that CXCL17 inhibited CXCR4 by a mechanism of action that potentially required the presence of a glycosaminoglycan-containing accessory protein. Together, our results revealed that CXCL17 is an endogenous inhibitor of CXCR4 and represents the next step in our understanding of the function of CXCL17 and regulation of CXCR4 signaling

Small Molecule Fluorescent Ligands for the Atypical Chemokine Receptor 3 (ACKR3)

The atypical chemokine receptor 3 (ACKR3) is a receptor that induces cancer progression and metastasis in multiple cell types. Therefore, new chemical tools are required to study the role of ACKR3 in cancer and other diseases. In this study, fluorescent probes, based on a series of small molecule ACKR3 agonists, were synthesized. Three fluorescent probes, which showed specific binding to ACKR3 through a luminescence-based NanoBRET binding assay (pKd ranging from 6.8 to 7.8) are disclosed. Due to their high affinity at the ACKR3, we have shown their application in both competition binding experiments and confocal microscopy studies showing the cellular distribution of this receptor

Conversion of daily pegvisomant to weekly pegvisomant combined with long-acting somatostatin analogs, in controlled acromegaly patients

The efficacy of combined treatment in active acromegaly with both long-acting somatostatin analogs (SRIF) and pegvisomant (PEG-V) has been well established. The aim was to describe the PEG-V dose reductions after the conversion from daily PEG-V to combination treatment. To clarify the individual beneficial and adverse effects, in two acromegaly patients, who only normalized their insulin like growth factor (IGF-I) levels with high-dose pegvisomant therapy. We present two cases of a 31 and 44 years old male with gigantism and acromegaly that were controlled subsequently by surgery, radiotherapy, SRIF analogs and daily PEG-V treatment. They were converted to combined treatment of monthly SSA and (twice) weekly PEG-V. High dose SSA treatment was added while the PEG-V dose was decreased during carful monitoring of the IGF-I. After switching from PEG-V monotherapy to SRIF analogs plus pegvisomant combination therapy IGF-I remained normal. However, the necessary PEG-V dose, to normalize IGF-I differed significantly between these two patients. One patient needed twice weekly 100 mg, the second needed 60 mg once weekly on top of their monthly lanreotide Autosolution injections of 120 mg. The weekly dose reduction was 80 and 150 mg. After the introducing of lanreotide, fasting glucose and glycosylated haemoglobin concentrations increased. Diabetic medication had to be introduced or increased. No changes in liver tests or in pituitary adenoma size were observed. In these two patients, PEG-V in combination with long-acting SRIF analogs was as effective as PEG-V monotherapy in normalizing IGF-I levels, although significant dose-reductions in PEG-V could be achieved. However, there seems to be a wide variation in the reduction of PEG-V dose, which can be obtained after conversion to combined treatment

Plasma lipid profiles discriminate bacterial from viral infection in febrile children

Fever is the most common reason that children present to Emergency Departments. Clinical signs and symptoms suggestive of bacterial infection are often non-specific, and there is no definitive test for the accurate diagnosis of infection. The 'omics' approaches to identifying biomarkers from the host-response to bacterial infection are promising. In this study, lipidomic analysis was carried out with plasma samples obtained from febrile children with confirmed bacterial infection (n = 20) and confirmed viral infection (n = 20). We show for the first time that bacterial and viral infection produces distinct profile in the host lipidome. Some species of glycerophosphoinositol, sphingomyelin, lysophosphatidylcholine and cholesterol sulfate were higher in the confirmed virus infected group, while some species of fatty acids, glycerophosphocholine, glycerophosphoserine, lactosylceramide and bilirubin were lower in the confirmed virus infected group when compared with confirmed bacterial infected group. A combination of three lipids achieved an area under the receiver operating characteristic (ROC) curve of 0.911 (95% CI 0.81 to 0.98). This pilot study demonstrates the potential of metabolic biomarkers to assist clinicians in distinguishing bacterial from viral infection in febrile children, to facilitate effective clinical management and to the limit inappropriate use of antibiotics

Improved functionalization of oleic acid-coated iron oxide nanoparticles for biomedical applications

Superparamagnetic iron oxide nanoparticles can providemultiple benefits for biomedical applications in aqueous environments such asmagnetic separation or magnetic resonance imaging. To increase the colloidal stability and allow subsequent reactions, the introduction of hydrophilic functional groups onto the particles’ surface is essential. During this process, the original coating is exchanged by preferably covalently bonded ligands such as trialkoxysilanes. The duration of the silane exchange reaction, which commonly takes more than 24 h, is an important drawback for this approach. In this paper, we present a novel method, which introduces ultrasonication as an energy source to dramatically accelerate this process, resulting in high-quality waterdispersible nanoparticles around 10 nmin size. To prove the generic character, different functional groups were introduced on the surface including polyethylene glycol chains, carboxylic acid, amine, and thiol groups. Their colloidal stability in various aqueous buffer solutions as well as human plasma and serum was investigated to allow implementation in biomedical and sensing applications.status: publishe