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1.
FASEB J ; 34(6): 7825-7846, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32297676

RESUMO

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.


Assuntos
Células Epiteliais Alveolares/patologia , Fibrose Pulmonar Idiopática/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Alvéolos Pulmonares/patologia , Células Epiteliais Alveolares/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Citocinas/metabolismo , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Pulmão/metabolismo , Pulmão/patologia , Alvéolos Pulmonares/metabolismo , Células-Tronco/metabolismo , Células-Tronco/patologia
2.
J Pharmacol Exp Ther ; 361(3): 355-365, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28351853

RESUMO

Brain-derived neurotrophic factor (BDNF) is a central modulator of neuronal development and synaptic plasticity in the central nervous system. This renders the BDNF-modulated tropomyosin receptor kinase B (TrkB) a promising drug target to treat synaptic dysfunctions. Using GRowth factor-driven expansion and INhibition of NotCH (GRINCH) during maturation, the so-called GRINCH neurons were derived from human-induced pluripotent stem cells. These GRINCH neurons were used as model cells for pharmacologic profiling of two TrkB-agonistic antibodies, hereafter referred to as AB2 and AB20 In next-generation sequencing studies, AB2 and AB20 stimulated transcriptional changes, which extensively overlapped with BDNF-driven transcriptional modulation. In regard to TrkB phosphorylation, both AB2 and AB20 were only about half as efficacious as BDNF; however, with respect to the TrkB downstream signaling, AB2 and AB20 displayed increased efficacy values, providing a stimulation at least comparable to BDNF in respect to VGF transcription, as well as of AKT and cAMP response element-binding protein phosphorylation. In a complex structure of the TrkB-d5 domain with AB20, determined by X-ray crystallography, the AB20 binding site was found to be allosteric in regard to the BDNF binding site, whereas AB2 was known to act orthosterically with BDNF. In agreement with this finding, AB2 and AB20 acted synergistically at greater concentrations to drive TrkB phosphorylation. Although TrkB downstream signaling declined faster after pulse stimulation with AB20 than with AB2, AB20 restimulated TrkB phosphorylation more efficiently than AB2. In conclusion, both antibodies displayed some limitations and some benefits in regard to future applications as therapeutic agents.


Assuntos
Anticorpos Monoclonais/farmacologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Receptor trkB/agonistas , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Fator Neurotrófico Derivado do Encéfalo/química , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Células CHO , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Humanos , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Imunoglobulina G/farmacologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios/metabolismo , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptor trkB/química , Receptor trkB/metabolismo
3.
SLAS Discov ; : 100185, 2024 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-39341280

RESUMO

Human induced pluripotent stem cell (iPSC)-derived macrophages (IDMs) present a valuable substitute for monocyte-derived macrophages (MDMs) in order to study inflammation pathways in vitro. Through optimization of an IDM differentiation protocol, a six-fold increase in the production yield of myeloid progenitors was achieved. The derived IDMs were further characterized with respect to nucleotide-binding oligomerization domain (NOD) and receptor-interacting serine/threonine-protein kinase 2 (RIPK2) signaling, a key regulatory pathway for autoimmune diseases. The IDM cells recapitulated MDM biology with respect to the proinflammatory chemokine and inflammatory cytokine fingerprint more closely than THP-1 cells. When assessing RIPK2 modulation effect on tumor necrosis factor α (TNF-α), a cardinal mediator of inflammation, a similar pharmacological effect of RIPK2 inhibitors was observed in IDMs and MDMs. Additionally, IDMs and MDMs displayed a similar transcription and pathway profile in response to NOD1/2 stimulation and pharmacological inhibition of RIPK2. In summary, the enhanced myeloid production yield in the improved IDM differentiation protocol offers new opportunities for utilizing physiologically relevant macrophage models in the context of inflammatory diseases.

4.
SLAS Discov ; 28(4): 149-162, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37072070

RESUMO

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.


Assuntos
Células-Tronco Pluripotentes Induzidas , Humanos , Macrófagos , Diferenciação Celular , Descoberta de Drogas
5.
Exp Cell Res ; 317(1): 42-50, 2011 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-20858487

RESUMO

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.


Assuntos
Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Biópsia , Técnicas de Cultura de Células , Células Cultivadas , Método Duplo-Cego , Regulação para Baixo/efeitos dos fármacos , Feminino , Humanos , Masculino , Fosforilação/efeitos dos fármacos , Placebos , Inibidores de Proteínas Quinases/uso terapêutico , Transporte Proteico/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologia , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia
6.
J Chem Inf Model ; 51(8): 1897-905, 2011 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-21761911

RESUMO

Computer-based chemogenomics approaches compare macromolecular drug targets based on their amino acid sequences or derived properties, by similarity of their ligands, or according to ligand-target interaction models. Here we present ARTS (Assay Related Target Similarity) as a quantitative index that estimates target similarity directly from measured affinities of a set of probe compounds. This approach reduces the risk of deducing artificial target relationships from mutually inactive compounds. ARTS implements a scoring scheme that matches intertarget similarity based on dose-response measurements. While all experimentally derived target similarities have a tendency to be data set-dependent, we demonstrate that ARTS depends less on the used data set than the commonly used Pearson correlation or Tanimoto index.


Assuntos
Algoritmos , Química Farmacêutica/métodos , Descoberta de Drogas/métodos , Genômica/métodos , Preparações Farmacêuticas/análise , Receptores Acoplados a Proteínas G/análise , Sequência de Aminoácidos , Sítios de Ligação , Simulação por Computador , Bases de Dados de Proteínas , Relação Dose-Resposta a Droga , Desenho de Fármacos , Humanos , Ligantes , Modelos Químicos , Modelos Moleculares , Preparações Farmacêuticas/química , Ligação Proteica , Ensaio Radioligante , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Relação Estrutura-Atividade
7.
Am J Respir Crit Care Med ; 181(5): 438-45, 2010 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-20007923

RESUMO

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).


Assuntos
Receptores ErbB/antagonistas & inibidores , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Administração por Inalação , Adulto , Idoso , Broncoscopia , Método Duplo-Cego , Fator de Crescimento Epidérmico/metabolismo , Feminino , Volume Expiratório Forçado/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Mucinas/metabolismo , Resultado do Tratamento
8.
Sci Rep ; 11(1): 17028, 2021 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-34426605

RESUMO

In order to circumvent the limited access and donor variability of human primary alveolar cells, directed differentiation of human pluripotent stem cells (hiPSCs) into alveolar-like cells, provides a promising tool for respiratory disease modeling and drug discovery assays. In this work, a unique, miniaturized 96-Transwell microplate system is described where hiPSC-derived alveolar-like cells were cultured at an air-liquid interface (ALI). To this end, hiPSCs were differentiated into lung epithelial progenitor cells (LPCs) and subsequently matured into a functional alveolar type 2 (AT2)-like epithelium with monolayer-like morphology. AT2-like cells cultured at the physiological ALI conditions displayed characteristics of AT2 cells with classical alveolar surfactant protein expressions and lamellar-body like structures. The integrity of the epithelial barriers between the AT2-like cells was confirmed by applying a custom-made device for 96-parallelized transepithelial electric resistance (TEER) measurements. In order to generate an IPF disease-like phenotype in vitro, the functional AT2-like cells were stimulated with cytokines and growth factors present in the alveolar tissue of IPF patients. The cytokines stimulated the secretion of pro-fibrotic biomarker proteins both on the mRNA (messenger ribonucleic acid) and protein level. Thus, the hiPSC-derived and cellular model system enables the recapitulation of certain IPF hallmarks, while paving the route towards a miniaturized medium throughput approach of pharmaceutical drug discovery.


Assuntos
Ar , Técnicas de Cultura de Células , Células-Tronco Pluripotentes Induzidas/citologia , Miniaturização , Modelos Biológicos , Alvéolos Pulmonares/citologia , Biomarcadores/metabolismo , Diferenciação Celular , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Fenótipo , Alvéolos Pulmonares/ultraestrutura , Fibrose Pulmonar/patologia , Transcrição Gênica
10.
Commun Chem ; 3(1): 75, 2020 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-36703453

RESUMO

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.

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