ly-2157299 and Disease-Models--Animal

Uncontrolled and excessive fibrosis after myocardial infarction (MI) in the peri-infarct zone leads to left ventricular remodeling and deterioration of cardiac function. Inhibiting fibroblast activation during the mature phase of cardiac repair improves cardiac remodeling and function after MI. Here, we engineered a biocompatible microneedle (MN) patch using gelatin methacryloyl and loaded it with galunisertib, a transforming growth factor-beta (TGF-β)-specific inhibitor, to treat excessive cardiac fibrosis after MI. The MN patch could sustainably release galunisertib for more than 2 weeks and provide mechanical support for the fragile ventricular wall. After being applied to a rat model of MI, the galunisertib-loaded MN patch improved long-term cardiac function and reduced cardiac fibrosis by effectively inhibiting TGF-β depending on fibroblast activation. This strategy shows the potential of the MN patch as an advanced platform to locally deliver direct antifibrotic drugs to prevent myocardial fibrosis for the treatment of MI and the promotion of cardiac repair.

Topics: Animals; Disease Models, Animal; Fibrosis; Gelatin; Hydrogels; Methacrylates; Myocardial Infarction; Myocardium; Pyrazoles; Quinolines; Rats; Transforming Growth Factor beta

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

Topics: Animals; Antiviral Agents; Artificial Intelligence; Chlorocebus aethiops; Disease Models, Animal; Drug Evaluation, Preclinical; High-Throughput Screening Assays; Immunocompetence; Inhibitory Concentration 50; Methacycline; Mice, Inbred C57BL; Protease Inhibitors; Quantitative Structure-Activity Relationship; Small Molecule Libraries; Vero Cells; Zika Virus; Zika Virus Infection

Diamond Blackfan Anemia (DBA) is a congenital bone marrow failure syndrome associated with ribosomal gene mutations that lead to ribosomal insufficiency. DBA is characterized by anemia, congenital anomalies, and cancer predisposition. Treatment for DBA is associated with significant morbidity. Here, we report the identification of Nemo-like kinase (NLK) as a potential target for DBA therapy. To identify new DBA targets, we screen for small molecules that increase erythroid expansion in mouse models of DBA. This screen identified a compound that inhibits NLK. Chemical and genetic inhibition of NLK increases erythroid expansion in mouse and human progenitors, including bone marrow cells from DBA patients. In DBA models and patient samples, aberrant NLK activation is initiated at the Megakaryocyte/Erythroid Progenitor (MEP) stage of differentiation and is not observed in non-erythroid hematopoietic lineages or healthy erythroblasts. We propose that NLK mediates aberrant erythropoiesis in DBA and is a potential target for therapy.

Topics: Anemia, Diamond-Blackfan; Animals; Benzamides; Cell Differentiation; Cell Proliferation; Cells, Cultured; Dioxoles; Disease Models, Animal; Erythropoiesis; Hematopoietic Stem Cells; Humans; Mice; Mice, Transgenic; Mutation; Primary Cell Culture; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Pyrazoles; Quinolines; Ribosomal Proteins; RNA, Small Interfering

Transforming growth factor (TGF)-β stimulates extracellular matrix (ECM) deposition during development of liver fibrosis and cirrhosis, the most important risk factor for the onset of hepatocellular carcinoma. In liver cancer, TGF-β is responsible for a more aggressive and invasive phenotype, orchestrating remodeling of the tumor microenvironment and triggering epithelial-mesenchymal transition of cancer cells. This is the scientific rationale for targeting the TGF-β pathway via a small molecule, galunisertib (intracellular inhibitor of ALK5) in clinical trials to treat liver cancer patients at an advanced disease stage. In this study, the hypothesis that galunisertib modifies the tissue microenvironment via inhibition of the TGF-β pathway is tested in an experimental preclinical model. At the age of 6 months, Abcb4ko mice-a well-established model for chronic liver disease development and progression-are treated twice daily with galunisertib (150 mg/kg) via oral gavage for 14 consecutive days. Two days after the last treatment, blood plasma and livers are harvested for further assessment, including fibrosis scoring and ECM components. The reduction of Smad2 phosphorylation in both parenchymal and non-parenchymal liver cells following galunisertib administration confirms the treatment effectiveness. Damage-related galunisertib does not change cell proliferation, macrophage numbers and leucocyte recruitment. Furthermore, no clear impact on the amount of fibrosis is evident, as documented by PicroSirius red and Gomori-trichome scoring. On the other hand, several fibrogenic genes, e.g., collagens (Col1α1 and Col1α2), Tgf-β1 and Timp1, mRNA levels are significantly downregulated by galunisertib administration when compared to controls. Most interestingly, ECM/stromal components, fibronectin and laminin-332, as well as the carcinogenic β-catenin pathway, are remarkably reduced by galunisertib-treated Abcb5ko mice. In conclusion, TGF-β inhibition by galunisertib interferes, to some extent, with chronic liver progression, not by reducing the stage of liver fibrosis as measured by different scoring systems, but rather by modulating the biochemical composition of the deposited ECM, likely affecting the fate of non-parenchymal cells.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B; ATP-Binding Cassette Sub-Family B Member 4; Disease Models, Animal; Extracellular Matrix; Liver; Liver Cirrhosis; Mice, Inbred BALB C; Mice, Knockout; Pyrazoles; Quinolines; Smad2 Protein; Transforming Growth Factor beta

TGFβ signaling plays a pleotropic role in tumor biology, promoting tumor proliferation, invasion and metastasis, and escape from immune surveillance. Inhibiting TGFβ's immune suppressive effects has become of particular interest as a way to increase the benefit of cancer immunotherapy. Here we utilized preclinical models to explore the impact of the clinical stage TGFβ pathway inhibitor, galunisertib, on anti-tumor immunity at clinically relevant doses.. In vitro treatment with galunisertib reversed TGFβ and regulatory T cell mediated suppression of human T cell proliferation. In vivo treatment of mice with established 4T1-LP tumors resulted in strong dose-dependent anti-tumor activity with close to 100% inhibition of tumor growth and complete regressions upon cessation of treatment in 50% of animals. This effect was CD8+ T cell dependent, and led to increased T cell numbers in treated tumors. Mice with durable regressions rejected tumor rechallenge, demonstrating the establishment of immunological memory. Consequently, mice that rejected immunogenic 4T1-LP tumors were able to resist rechallenge with poorly immunogenic 4 T1 parental cells, suggesting the development of a secondary immune response via antigen spreading as a consequence of effective tumor targeting. Combination of galunisertib with PD-L1 blockade resulted in improved tumor growth inhibition and complete regressions in colon carcinoma models, demonstrating the potential synergy when cotargeting TGFβ and PD-1/PD-L1 pathways. Combination therapy was associated with enhanced anti-tumor immune related gene expression profile that was accelerated compared to anti-PD-L1 monotherapy.. Together these data highlight the ability of galunisertib to modulate T cell immunity and the therapeutic potential of combining galunisertib with current PD-1/L1 immunotherapy.

Topics: Animals; Combined Modality Therapy; Disease Models, Animal; Female; Humans; Immunotherapy; Male; Mice; Pyrazoles; Quinolines; Transforming Growth Factor beta

The vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-β pathways regulate key biological features of glioblastoma. Here we explore whether the TGF-β pathway, which promotes angiogenesis, invasiveness, and immunosuppression, acts as an escape pathway from VEGF inhibition.. The role of the TGF-β pathway in escape from VEGF inhibition was assessed in vitro and in vivo and by gene expression profiling in syngeneic mouse glioma models.. We found that TGF-β is an upstream regulator of VEGF, whereas VEGF pathway activity does not alter the TGF-β pathway in vitro. In vivo, single-agent activity was observed for the VEGF antibody B20-4.1.1 in 3 and for the TGF-β receptor 1 antagonist LY2157299 in 2 of 4 models. Reduction of tumor volume and blood vessel density, but not induction of hypoxia, correlated with benefit from B20-4.1.1. Reduction of phosphorylated (p)SMAD2 by LY2157299 was seen in all models but did not predict survival. Resistance to B20 was associated with anti-angiogenesis escape pathway gene expression, whereas resistance to LY2157299 was associated with different immune response gene signatures in SMA-497 and GL-261 on transcriptomic profiling. The combination of B20 with LY2157299 was ineffective in SMA-497 but provided prolongation of survival in GL-261, associated with early suppression of pSMAD2 in tumor and host immune cells, prolonged suppression of angiogenesis, and delayed accumulation of tumor infiltrating microglia/macrophages.. Our study highlights the biological heterogeneity of murine glioma models and illustrates that cotargeting of the VEGF and TGF-β pathways might lead to improved tumor control only in subsets of glioblastoma.

Topics: Angiogenesis Inhibitors; Animals; Bevacizumab; Brain Neoplasms; Cell Line, Tumor; Cell Survival; Disease Models, Animal; Glioma; Lymphotoxin-alpha; Phosphorylation; Pyrazoles; Quinolines; Signal Transduction; Smad2 Protein; Vascular Endothelial Growth Factor A

The observation that approximately 15% of women with disseminated breast cancer will develop symptomatic brain metastases combined with treatment guidelines discouraging single-agent chemotherapeutic strategies facilitates the desire for novel strategies aimed at outright brain metastasis prevention. Effective and robust preclinical methods to evaluate early-stage metastatic processes, brain metastases burden, and overall mean survival are lacking. Here, we develop a novel method to quantitate early metastatic events (arresting and extravasation) in addition to traditional end time-point parameters such as tumor burden and survival in an experimental mouse model of brain metastases of breast cancer. Using this method, a reduced number of viable brain-seeking metastatic cells (from 3,331 ± 263 cells/brain to 1,079 ± 495 cells/brain) were arrested in brain one week postinjection after TGFβ knockdown. Treatment with a TGFβ receptor inhibitor, galunisertib, reduced the number of arrested cells in brain to 808 ± 82 cells/brain. Furthermore, we observed a reduction in the percentage of extravasated cells (from 63% to 30%) compared with cells remaining intralumenal when TGFβ is knocked down or inhibited with galunisertib (40%). The observed reduction of extravasated metastatic cells in brain translated to smaller and fewer brain metastases and resulted in prolonged mean survival (from 36 days to 62 days). This method opens up potentially new avenues of metastases prevention research by providing critical data important to early brain metastasis of breast cancer events.

Topics: Animals; Blood-Brain Barrier; Brain; Brain Neoplasms; Cell Line, Tumor; Disease Models, Animal; Female; Humans; Mammary Neoplasms, Experimental; Mice; Mice, Nude; Microscopy, Fluorescence; Neoplasm Metastasis; Pyrazoles; Quinolines; Signal Transduction; Transforming Growth Factor beta

The purpose is to investigate the mechanism of subconjunctival fibrosis caused by benzalkonium chloride (BAC), which is the most common preservative in ophthalmic preparations.. The left eyes of male Sprague-Dawley rats were topically treated with 0.01% BAC or PBS twice daily for 1 month. Primary conjunctival fibroblasts (CFs) were exposed for 24 hours to 0.00005% BAC, 0.000075% BAC, 0.000075% BAC + LY2157299 (a selective transforming growth factor β receptor type I inhibitor); 0.000075% BAC + NS-398 (a selective cyclooxygenase-2 inhibitor) and PBS, respectively. The pathological changes of the bulbar conjunctival tissue of rats were examined using hematoxylin-eosin (HE), Van Gieson's (vG), periodic acid-Schiff (PAS) stains, or immunohistochemisty (IHC). The expression of the extracellular matrix (ECM), the transforming growth factor β (TGF-β) signaling pathway-related molecules, and cyclooxygenase-2 (COX-2) in bulbar conjunctival tissues and CFs were detected using Western blot (WB) and quantitative real-time RT-PCR (qRT-PCR).. Rats treated with 0.01% BAC exhibited a slight increase of the fibroblast density and a more compact collagen deposition in the bulbar subepithelial connective tissues in comparison with rats treated with PBS. Western blot and qRT-PCR analyses showed that the expression of ECM, TGF-β signaling pathway-related molecules, and COX-2 were markedly increased in the bulbar conjunctival tissues of rats exposed to 0.01% BAC and in CFs exposed to 0.00005% and 0.000075% BAC. In conjunctival fibroblasts, BAC-induced ECM expression was clearly decreased by LY2157299, while the BAC-induced activation of the TGF-β1/Smad3 signaling pathway was greatly attenuated by NS-398.. Subconjunctival fibrosis BAC-induced is a consequence of excessive ECM production of CFs through the COX-2-modulated activation of a TGF-β1/Smad3 signaling pathway.

Topics: Animals; Benzalkonium Compounds; Biomarkers; Blotting, Western; Conjunctiva; Conjunctival Diseases; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Disease Models, Animal; Extracellular Matrix; Fibroblasts; Fibrosis; Male; Preservatives, Pharmaceutical; Pyrazoles; Quinolines; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta1