Application of human iPSC-derived macrophages in a miniaturized high-content-imaging-based efferocytosis assay.

Macrophages play a pivotal role in drug discovery due to their key regulatory functions in health and disease. Overcoming the limited availability and donor variability of human monocyte-derived macrophages (MDMs), human induced pluripotent stem cell (iPSC)-derived macrophages (IDMs) could provide a promising tool for both disease modeling and drug discovery. To access large numbers of model cells for medium- to high-throughput application purposes, an upscaled protocol was established for differentiation of iPSCs into progenitor cells and subsequent maturation into functional macrophages. These IDM cells resembled MDMs both with respect to surface marker expression and phago- as well as efferocytotic function. A statistically robust high-content-imaging assay was developed to quantify the efferocytosis rate of IDMs and MDMs allowing for measurements both in the 384- and 1536-well microplate format. Validating the applicability of the assay, inhibitors of spleen tyrosine kinase (Syk) were shown to modulate efferocytosis in IDMs and MDMs with comparable pharmacology. The miniaturized cellular assay with the upscaled provision of macrophages opens new routes to pharmaceutical drug discovery in the context of efferocytosis-modulating substances.

Development of a miniaturized 96-Transwell air-liquid interface human small airway epithelial model.

In order to overcome the challenges associated with a limited number of airway epithelial cells that can be obtained from clinical sampling and their restrained capacity to divide ex vivo, miniaturization of respiratory drug discovery assays is of pivotal importance. Thus, a 96-well microplate system was developed where primary human small airway epithelial (hSAE) cells were cultured at an air-liquid interface (ALI). After four weeks of ALI culture, a pseudostratified epithelium containing basal, club, goblet and ciliated cells was produced. The 96-well ALI cultures displayed a cellular composition, ciliary beating frequency, and intercellular tight junctions similar to 24-well conditions. A novel custom-made device for 96-parallelized transepithelial electric resistance (TEER) measurements, together with dextran permeability measurements, confirmed that the 96-well culture developed a tight barrier function during ALI differentiation. 96-well hSAE cultures were responsive to transforming growth factor ß1 (TGF-ß1) and tumor necrosis factor α (TNF-α) in a concentration dependent manner. Thus, the miniaturized cellular model system enables the recapitulation of a physiologically responsive, differentiated small airway epithelium, and a robotic integration provides a medium throughput approach towards pharmaceutical drug discovery, for instance, in respect of fibrotic distal airway/lung diseases.

Recapitulating idiopathic pulmonary fibrosis related alveolar epithelial dysfunction in a human iPSC-derived air-liquid interface model.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease of unknown cause that is characterized by progressive fibrotic lung remodeling. An abnormal emergence of airway epithelial-like cells within the alveolar compartments of the lung, herein termed bronchiolization, is often observed in IPF. However, the origin of this dysfunctional distal lung epithelium remains unknown due to a lack of suitable human model systems. In this study, we established a human induced pluripotent stem cell (iPSC)-derived air-liquid interface (ALI) model of alveolar epithelial type II (ATII)-like cell differentiation that allows us to investigate alveolar epithelial progenitor cell differentiation in vitro. We treated this system with an IPF-relevant cocktail (IPF-RC) to mimic the pro-fibrotic cytokine milieu present in IPF lungs. Stimulation with IPF-RC during differentiation increases secretion of IPF biomarkers and RNA sequencing (RNA-seq) of these cultures reveals significant overlap with human IPF patient data. IPF-RC treatment further impairs ATII differentiation by driving a shift toward an airway epithelial-like expression signature, providing evidence that a pro-fibrotic cytokine environment can influence the proximo-distal differentiation pattern of human lung epithelial cells. In conclusion, we show for the first time, the establishment of a human model system that recapitulates aspects of IPF-associated bronchiolization of the lung epithelium in vitro.

A small-molecule inhibitor of lectin-like oxidized LDL receptor-1 acts by stabilizing an inactive receptor tetramer state.

The C-type lectin family member lectin-like oxidized LDL receptor-1 (LOX-1) has been object of intensive research. Its modulation may offer a broad spectrum of therapeutic interventions ranging from cardiovascular diseases to cancer. LOX-1 mediates uptake of oxLDL by vascular cells and plays an important role in the initiation of endothelial dysfunction and its progression to atherosclerosis. So far only a few compounds targeting oxLDL-LOX-1 interaction are reported with a limited level of characterization. Here we describe the identification and characterization of BI-0115, a selective small molecule inhibitor of LOX-1 that blocks cellular uptake of oxLDL. Identified by a high throughput screening campaign, biophysical analysis shows that BI-0115 binding triggers receptor inhibition by formation of dimers of the homodimeric ligand binding domain. The structure of LOX-1 bound to BI-0115 shows that inter-ligand interactions at the receptor interfaces are key to the formation of the receptor tetramer thereby blocking oxLDL binding.

Phenotypic analysis of chemokine-driven actin reorganization in primary human neutrophils.

The chemokine-driven activation of CXC-type chemokine receptors 1/2 (CXCR1/2) and the subsequent reorganization of the neutrophilic actin are early key events in the induction of neutrophil migration toward centers of inflammation. In this study, an image analysis algorithm was developed to detect subtle chemokine-induced changes in the actin cytoskeleton of primary human neutrophils. By this means, a discrete early step of neutrophil activation was dissected that could be initiated by concentrations of growth-related oncogen α (Gro-α) or interleukin-8 (IL-8) just above their resting-state plasma levels. The associated half-maximal effective concentration (EC50) values for Gro-α and IL-8 of 8 and 22 pM, respectively, are between two and three orders of magnitude below the so-far reported EC50 values of these chemokines for the induction of neutrophilic calcium release, integrin expression, degranulation, and receptor internalization. Sch527123, a known inhibitor of CXCR2 (KD=49 pM) and with a lower potency/affinity also of CXCR1 (KD=3.9 nM), antagonized actin remodeling with half-maximal inhibitory concentration (IC50) values of 400 pM for the CXCR2-specific agonist Gro-α and of 36 nM for the CXCR1/2-promiscuous agonist IL-8. This observation indicates that the here-described early step of chemokine-driven actin reorganization is modulated by both CXCR1 and CXCR2. Thus, the imaging-based assay format, as developed in this work, may be employed in a phenotypic screening campaign to identify inhibitors of an early step in CXCR1/2-induced neutrophilic chemotaxis.

High-content analysis of CCR2 antagonists on human primary monocytes.

The monocyte chemoattractant protein 1 (MCP-1)-driven activation of CC-type chemokine receptor 2 (CCR2) is one of the early key events to induce monocyte migration toward centers of inflammation. In this work, the authors analyzed MCP-1 internalization into primary human monocytes using partially automated liquid handling, automated fluorescence microscopic imaging, and a specific image analysis algorithm. A fluorophore-conjugated form of MCP-1 was rapidly endocytosed and retained by the monocytes. The CCR2 dependency of the MCP-1 internalization was demonstrated by the use of BMS CCR2 22, a CCR2-specific antagonist. The apparent inhibitory potencies of a series of small-molecule CCR2 antagonists were determined and compared in five assay formats, including the high-content analysis assay described in this work. Interestingly, some but not all antagonists showed markedly different inhibitory behaviors in the five readout systems, with an up to more than 100-fold difference between the highest and the lowest apparent inhibitory potencies. These findings raise the distinct possibility that some CCR2 antagonists are capable of discriminating between different functional states of the CCR2 receptor(s) and suggest strategies for the identification of functionally selective CCR2 antagonists with increased therapeutic advantage over nonselective antagonists.

In vivo inhibition of epidermal growth factor receptor autophosphorylation prevents receptor internalization.

The question whether epidermal growth factor (EGF)-induced receptor endocytosis requires the prior autophosphorylation via the EGF receptor (EGFR) kinase domain has been a matter of long-standing debate. In the airway epithelial cell line NCI-H292, the EGFR kinase domain inhibitor BIBW 2948 BS was found to inhibit both autophosphorylation and subsequent internalization of the endogenous EGFR with similar IC50 values. Applying an ex vivo EGFR internalization assay in a clinical study, the in vivo effect of inhalatively administered BIBW 2948 BS was determined directly at the targeted receptor in airway tissues from COPD patients. In these experiments, the in vivo inhibition of the EGFR kinase domain prevented the EGF-induced internalization of EGFR.

High content screening of CXCR2-dependent signalling pathways.

Stimulation of CXC-type chemokine receptor 2 (CXCR2)-transfected cells by Gro-alpha or IL-8 induced (i) CXCR2 internalization, (ii) phosphorylation of ERK1/2 (pERK) and (iii) translocation of nuclear factor of activated T cells (NFAT) into the nucleus. Employing high content screening (HCS; i.e. fluorimetric imaging combined with image analysis) these three ligand-induced events were quantified by using a CXCR2-specific antibody, an antibody recognizing phosphorylated ERK1/2 (pERK) and a red fluorescent protein (RFP) in fusion to transiently overexpressed NFAT, respectively. As an RFP, we applied a recently developed mutant of an Entacmaea quadricolor fluorescent protein with favorable properties for HCS, such as high fluorescence brightness, photostability, large Stokes shift, and stability with regard to formaldehyde. Receptor internalization was closely coupled with ERK signalling both when analyzed in regard of stimulation by physiological CXCR2 ligands and when observed in the presence of antagonistic test compounds. A means of increasing the throughput or of broadening the pharmacological characterization of test compounds is the use of multiplexed imaging. Indeed, CXCR2 internalization could be multiplexed with the NFAT nuclear translocation by fixation at approximately 45 min after Gro-alpha stimulation. This multiplexing demonstrated that Gro-alpha-induced CXCR2 internalization was tightly correlated with Gro-alpha-induced NFAT translocation, also on the single cell level. The analysis of ERK phosphorylation, NFAT translocation and receptor internalization enabled the profiling of antagonistic test compounds with respect to G-protein signalling and possible receptor desensitization liabilities.

Safety and efficacy of an inhaled epidermal growth factor receptor inhibitor (BIBW 2948 BS) in chronic obstructive pulmonary disease.

RATIONALE: Epidermal growth factor receptor (EGFR) activation is implicated in mucin hypersecretion in chronic obstructive pulmonary disease (COPD). OBJECTIVES: To investigate the safety and efficacy of an inhaled EGFR antagonist (BIBW 2948) in COPD. METHODS: Multicenter, double-blind, placebo-controlled trial of 4 weeks of treatment with two doses of BIBW 2948 (15 and 30 mg twice a day) on safety and mucin-related outcomes in 48 patients with COPD. The effect of BIBW 2948 on EGFR activation in airway epithelial cells was assessed using an ex vivo assay. Efficacy measures included the volume of mucin in the airway epithelium (Vs mu,bala) in bronchial biopsies and the expression of mucin genes in bronchial brushings. MEASUREMENTS AND MAIN RESULTS: Inhaled BIBW 2948 induced a dose-related inhibition of EGFR internalization (reflecting decreased EGFR activation) in epithelial cells from treated subjects. However, BIBW 2948 was associated with a dose-related increase in adverse events, including reversible liver enzyme elevation (n = 2), and reduction in FEV(1). The changes in mucin stores and mucin gene expression were not significantly different in the pooled BIBW 2948 group versus placebo (volume of mucin per surface area of basal lamina = 0.22 +/- 7.11 vs. 0.47 +/- 8.06 microm(3)/microm(2); P = 0.93). However, in the 30 mg twice a day group, the reduction in epithelial mucin stores was greatest in subjects with the greatest degree of EGFR inhibition (Pearson r = 0.98; 95% confidence interval, 0.71-0.99). CONCLUSIONS: Four-week treatment with BIBW 2948 did not significantly decrease epithelial mucin stores and was poorly tolerated in patients with COPD. Ex vivo analyses suggest that higher doses may be more effective at both EGFR inhibition and decreases in mucin stores but that adverse events should be expected. Clinical trial registered with www.clinicaltrials.gov (NCT00423137).

CXCR2 inverse agonism detected by arrestin redistribution.

To study CXCR2 modulated arrestin redistribution, the authors employed arrestin as a fusion protein containing either the Aequorea victoria-derived enhanced green fluorescent protein (EGFP) or a recently developed mutant of eqFP611, a red fluorescent protein derived from Entacmaea quadricolor. This mutant, referred to as RFP611, had earlier been found to assume a dimeric quarternary structure. It was therefore employed in this work as a "tandem" (td) construct for pseudo-monomeric fusion protein labeling. Both arrestin fusion proteins, containing either td-RFP611 (Arr-td-RFP611) or enhanced green fluorescent protein (EGFP; Arr-EGFP), were found to colocalize with internalized fluorescently labeled Gro-alpha a few minutes after Gro-alpha addition. Intriguingly, however, Arr-td-RFP611 and Arr-EGFP displayed distinct cellular distribution patterns in the absence of any CXCR2-activating ligand. Under these conditions, Arr-td-RFP611 showed a largely homogeneous cytosolic distribution, whereas Arr-EGFP segregated, to a large degree, into granular spots. These observations indicate a higher sensitivity of Arr EGFP to the constitutive activity of CXCR2 and, accordingly, an increased arrestin redistribution to coated pits and endocytic vesicles. In support of this interpretation, the authors found the known CXCR2 antagonist Sch527123 to act as an inverse agonist with respect to Arr-EGFP redistribution. The inverse agonistic properties of Sch527123 were confirmed in vitro in a guanine nucleotide binding assay, revealing an IC(50) value similar to that observed for Arr-EGFP redistribution. Thus, the redistribution assay, when based on Arr-EGFP, enables the profiling of antagonistic test compounds with respect to inverse agonism. When based on Arr-td-RFP611, the assay may be employed to study CXCR2 agonism or neutral antagonism.

Automated high content screening for phosphoinositide 3 kinase inhibition using an AKT 1 redistribution assay.

High Content Screening (HCS), a combination of fluorescence microscopic imaging and automated image analysis, has become a frequently applied tool to study test compound effects in cellular disease-modelling systems. In this work, we established a medium to high throughput HCS assay in the 384-well format to measure cellular type I phosphoinositide 3 kinase (PI3K) activity. Type I PI3K is involved in several intracellular pathways such as cell survival, growth and differentiation as well as immunological responses. As a cellular model system we used Chinese Hamster Ovary (CHO) cells that had been stably transfected with human insulin receptor (hIR) and an AKT1-enhanced green fluorescent protein (EGFP) fusion construct. Upon stimulation of the hIR with insulin-like growth factor-1 (IGF-1), PI3K was activated to phosphorylate phosphatidylinositol (PtdIns)-4,5-bisphosphate at the 3-position, resulting in the recruitment of AKT1-EGFP to the plasma membrane. The AKT1-EGFP redistribution assay was robust and displayed little day-to-day variability, the quantification of the fluorescence intensity associated with plasma membrane spots delivered good Z' statistics. A novel format of compound dose-response testing was employed using serial dilutions of test compounds across consecutive microtiter plates (MTPs). The dose response testing of a PI3K inhibitor series provided reproducible IC50 values. The profiling of the redistribution assay with isoform-selective inhibitors indicates that PI3Kalpha is the main isoform activated in the CHO host cells after IGF-1 stimulation. Toxic compound side effects could be determined using automated image analysis. We conclude that the AKT1-EGFP redistribution assay represents a solid medium/high throughput screening (MTS/HTS) format to determine the cellular activity of PI3K inhibitors under conditions of growth factor stimulation.

Peptide protein binding assay using ImageFlashPlates or Imaging Beads.

Imaging devices used for the measurement of radioligand-receptor binding assays are typically based on charge-coupled device (CCD) cameras, which are more sensitive for red-shifted scintillation. In the past, red-shifted scintillants had only been integrated into microspheres, referred to as scintillation proximity assay (SPA) Imaging Beads. More recently, ImageFlashPlates have been developed that emit light at 615 nm when exposed to beta-radiation. In this article, we report the establishment of peptide-protein binding assays using either streptavidin-coated ImageFlashPlates or Imaging Beads in a low volume 384-well format. In these assays, we employed a biotinylated peptide X and a [33P]-phosphorylated protein Y as the binding partner. The FlashPlates required a washing step, the bead-filled microtiter plates (MTPs) needed a centrifugation step for optimal performance in the scintillation measurements. Both the peptide X-loaded FlashPlates and the beads displayed saturable binding of [33P]-phosphorylated protein Y with a similar scintillation efficiency. A KD value of about 30 nmol/l was measured using the bead-based assay. Due to the washing step in the FlashPlate experiment, approximately two-thirds of the [33P]-phosphorylated protein Y were withdrawn from equilibrium binding. This resulted in correspondingly lower scintillation signals for the FlashPlate experiment. For this reason, the FlashPlate produced a Z' value of 0.64 that was lower than the Z' value of 0.87 for the beads. Using a reference inhibitor in a competition assay produced similar IC50 values for the bead-based assay as for the FlashPlate. Depending on the local automation environment either the centrifugation step for the beads or the washing step for the FlashPlates may be considered more or less of a challenge. Low volume 384-well high-throughput screening (HTS) applicable assay formats are achievable using either the ImageFlashPlates or the Imaging Beads.

Automated high throughput screening for serine kinase inhibitors using a LEADseeker scintillation proximity assay in the 1536-well format.

High-throughput screening in the 1536-well format has been largely restricted to solution-based and cell-based screens. In this article, we show the feasibility of a completely automated, robust scintillation proximity assay in the 1536-well format that is suitable to identify inhibitors for a serine/threonine kinase from a compound library. The introduction of [(33)P]phosphate into a biotinylated peptide substrate mirrors the activity of the kinase. The peptide is immobilized on streptavidin-coated LEADseeker imaging beads and [(33)P]phosphate incorporation is detected with the LEADseeker imaging system of Amersham Pharmacia Biotech. To improve the liquid handling procedures for imaging bead suspensions in the low microliter range, we developed a novel trough with an integrated stirring function. A comparison of the 1536-well assay to a 384-well assay revealed a comparable assay quality with Z' factors of about 0.7 for the 384-well format and 0.6 for the 1536-well format. In an automated screen of a random compound collection, 94.4% of the inhibitory compounds could be identified with both assay formats. Dose-response curves were performed for a selection of identified kinase inhibitors and revealed similar IC(50) values for both assay formats.