scholarly journals Small-Molecule Induction Promotes Corneal Endothelial Cell Differentiation From Human iPS Cells

2021 ◽  
Vol 9 ◽  
Author(s):  
Jie Chen ◽  
Qingjian Ou ◽  
Zhe Wang ◽  
Yifan Liu ◽  
Shuqin Hu ◽  
...  
Keyword(s):  
Small Molecules ◽  
Small Molecule ◽  
Corneal Stroma ◽  
Defined Medium ◽  
Efficient Production ◽  
Disease States ◽  
Endothelial Cell Differentiation ◽  
Human Ips Cells ◽  
Small Molecule Screen ◽  
Induced Pluripotent

Purpose: Corneal endothelial cells (CECs) serve as a barrier and foothold for the corneal stroma to maintain the function and transparency of the cornea. Loss of CECs during aging or disease states leads to blindness, and cell replacement therapy using either donated or artificially differentiated CECs remains the only curative approach.Methods: Human induced pluripotent stem cells (hiPSCs) that were cultured in chemically defined medium were induced with dual-SMAD inhibition to differentiate into neural crest cells (NCCs). A small-molecule library was screened to differentiate the NCCs into corneal endothelial-like cells. The characteristics of these cells were identified with real-time PCR and immunofluorescence. Western blotting was applied to detect the signaling pathways and key factors regulated by the small molecules.Results: We developed an effective protocol to differentiate hiPSCs into CECs with defined small molecules. The hiPSC-CECs were characterized by ZO-1, AQP1, Vimentin and Na+/K+-ATPase. Based on our small-molecule screen, we identified a small-molecule combination, A769662 and AT13148, that enabled the most efficient production of CECs. The combination of A769662 and AT13148 upregulated the PKA/AKT signaling pathway, FOXO1 and PITX2 to promote the conversion of NCCs to CECs.Conclusion: We established an efficient small molecule-based method to differentiate hiPSCs into corneal endothelial-like cells, which might facilitate drug discovery and the development of cell-based therapies for corneal diseases.

scholarly journals A novel drug-combination screen in zebrafish identifies epigenetic small molecule candidates for Duchenne muscular dystrophy

2020 ◽  
Author(s):  
Gist H. Farr ◽  
Melanie Morris ◽  
Arianna Gomez ◽  
Thao Pham ◽  
Elizabeth U. Parker ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Small Molecules ◽  
Small Molecule ◽  
Histone Deacetylase Inhibitors ◽  
Neuromuscular Disorder ◽  
Small Molecule Screen ◽  
Chemical Screen ◽  
Novel Drug

SummaryDuchenne muscular dystrophy (DMD) is a severe neuromuscular disorder and is one of the most common muscular dystrophies. There are currently few effective therapies to treat the disease, although many small-molecule approaches are being pursued. Specific histone deacetylase inhibitors (HDACi) can ameliorate DMD phenotypes in mouse and zebrafish animal models and have also shown promise for DMD in clinical trials. However, beyond these HDACi, other classes of epigenetic small molecules have not been broadly and systematically studied for their benefits for DMD. Here, we performed a novel chemical screen of a library of epigenetic compounds using the zebrafish dmd model. We identified candidate pools of epigenetic compounds that improve skeletal muscle structure in dmd zebrafish. We then identified a specific combination of two drugs, oxamflatin and salermide, that significantly rescued dmd zebrafish skeletal muscle degeneration. Furthermore, we validated the effects of oxamflatin and salermide in an independent laboratory. Our results provide novel, effective methods for performing a combination small-molecule screen in zebrafish. Our results also add to the growing evidence that epigenetic small molecules may be promising candidates for treating DMD.

Small molecule targeting of SHIP1 and SHIP2

2020 ◽  
Vol 48 (1) ◽  
pp. 291-300 ◽  
Author(s):  
William G. Kerr ◽  
Chiara Pedicone ◽  
Shawn Dormann ◽  
Angela Pacherille ◽  
John D. Chisholm
Keyword(s):  
Cell Signaling ◽  
Small Molecules ◽  
Cancer Immunotherapy ◽  
Phosphatase Activity ◽  
Small Molecule ◽  
Pi3k Pathway ◽  
Disease States ◽  
Src Homology 2 ◽  
Src Homology ◽  
Unconventional Method

Modulating the activity of the Src Homology 2 (SH2) — containing Inositol 5′-Phosphatase (SHIP) enzyme family with small molecule inhibitors provides a useful and unconventional method of influencing cell signaling in the PI3K pathway. The development of small molecules that selectively target one of the SHIP paralogs (SHIP1 or SHIP2) as well as inhibitors that simultaneously target both enzymes have provided promising data linking the phosphatase activity of the SHIP enzymes to disorders and disease states that are in dire need of new therapeutic targets. These include cancer, immunotherapy, diabetes, obesity, and Alzheimer's disease. In this mini-review, we will provide a brief overview of research in these areas that support targeting SHIP1, SHIP2 or both enzymes for therapeutic purposes.

A Small Molecule – Protein Hybrid for Voltage Imaging via Quenching of Bioluminescence

2020 ◽  
Author(s):  
Brittany Benlian ◽  
Pavel Klier ◽  
Kayli Martinez ◽  
Marie Schwinn ◽  
Thomas Kirkland ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Membrane Potential ◽  
Small Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Live Cells ◽  
Potential Oscillations ◽  
Excitation Light ◽  
Voltage Imaging ◽  
Induced Pluripotent

<p>We report a small molecule enzyme pair for optical voltage sensing via quenching of bioluminescence. This <u>Q</u>uenching <u>B</u>ioluminescent V<u>olt</u>age Indicator, or Q-BOLT, pairs the dark absorbing, voltage-sensitive dipicrylamine with membrane-localized bioluminescence from the luciferase NanoLuc (NLuc). As a result, bioluminescence is quenched through resonance energy transfer (QRET) as a function of membrane potential. Fusion of HaloTag to NLuc creates a two-acceptor bioluminescence resonance energy transfer (BRET) system when a tetramethylrhodamine (TMR) HaloTag ligand is ligated to HaloTag. In this mode, Q-BOLT is capable of providing direct visualization of changes in membrane potential in live cells via three distinct readouts: change in QRET, BRET, and the ratio between bioluminescence emission and BRET. Q-BOLT can provide up to a 29% change in bioluminescence (ΔBL/BL) and >100% ΔBRET/BRET per 100 mV change in HEK 293T cells, without the need for excitation light. In cardiac monolayers derived from human induced pluripotent stem cells (hiPSC), Q-BOLT readily reports on membrane potential oscillations. Q-BOLT is the first example of a hybrid small molecule – protein voltage indicator that does not require excitation light and may be useful in contexts where excitation light is limiting.</p> <p> </p>

A Small Molecule – Protein Hybrid for Voltage Imaging via Quenching of Bioluminescence

2020 ◽  
Author(s):  
Brittany Benlian ◽  
Pavel Klier ◽  
Kayli Martinez ◽  
Marie Schwinn ◽  
Thomas Kirkland ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Membrane Potential ◽  
Small Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Live Cells ◽  
Potential Oscillations ◽  
Excitation Light ◽  
Voltage Imaging ◽  
Induced Pluripotent

<p>We report a small molecule enzyme pair for optical voltage sensing via quenching of bioluminescence. This <u>Q</u>uenching <u>B</u>ioluminescent V<u>olt</u>age Indicator, or Q-BOLT, pairs the dark absorbing, voltage-sensitive dipicrylamine with membrane-localized bioluminescence from the luciferase NanoLuc (NLuc). As a result, bioluminescence is quenched through resonance energy transfer (QRET) as a function of membrane potential. Fusion of HaloTag to NLuc creates a two-acceptor bioluminescence resonance energy transfer (BRET) system when a tetramethylrhodamine (TMR) HaloTag ligand is ligated to HaloTag. In this mode, Q-BOLT is capable of providing direct visualization of changes in membrane potential in live cells via three distinct readouts: change in QRET, BRET, and the ratio between bioluminescence emission and BRET. Q-BOLT can provide up to a 29% change in bioluminescence (ΔBL/BL) and >100% ΔBRET/BRET per 100 mV change in HEK 293T cells, without the need for excitation light. In cardiac monolayers derived from human induced pluripotent stem cells (hiPSC), Q-BOLT readily reports on membrane potential oscillations. Q-BOLT is the first example of a hybrid small molecule – protein voltage indicator that does not require excitation light and may be useful in contexts where excitation light is limiting.</p> <p> </p>

scholarly journals STEM-20. A CANCER STEM CELL-SELECTIVE APOPTOSIS-INDUCING SMALL MOLECULE FOR THE TREATMENT OF GBM

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii200-ii200
Author(s):  
Stephen Skirboll ◽  
Natasha Lucki ◽  
Genaro Villa ◽  
Naja Vergani ◽  
Michael Bollong ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cancer Stem Cells ◽  
Small Molecules ◽  
Small Molecule ◽  
Large Scale ◽  
Critical Role ◽  
Tumor Formation ◽  
Cell Type ◽  
Caspase 1 ◽  
Activation Assay

Abstract INTRODUCTION Glioblastoma multiforme (GBM) is the most aggressive form of primary brain cancer. A subpopulation of multipotent cells termed GBM cancer stem cells (CSCs) play a critical role in tumor initiation and maintenance, drug resistance, and recurrence following surgery. New therapeutic strategies for the treatment of GBM have recently focused on targeting CSCs. Here we have used an unbiased large-scale screening approach to identify drug-like small molecules that induce apoptosis in GBM CSCs in a cell type-selective manner. METHODS A luciferase-based survival assay of patient-derived GBM CSC lines was established to perform a large-scale screen of ∼one million drug-like small molecules with the goal of identifying novel compounds that are selectively toxic to chemoresistant GBM CSCs. Compounds found to kill GBM CSC lines as compared to control cell types were further characterized. A caspase activation assay was used to evaluate the mechanism of induced cell death. A xenograft animal model using patient-derived GBM CSCs was employed to test the leading candidate for suppression of in vivo tumor formation. RESULTS We identified a small molecule, termed RIPGBM, from the cell-based chemical screen that induces apoptosis in primary patient-derived GBM CSC cultures. The cell type-dependent selectivity of RIPGBM appears to arise at least in part from redox-dependent formation of a proapoptotic derivative, termed cRIPGBM, in GBM CSCs. cRIPGBM induces caspase 1-dependent apoptosis by binding to receptor-interacting protein kinase 2 (RIPK2) and acting as a molecular switch, which reduces the formation of a prosurvival RIPK2/TAK1 complex and increases the formation of a proapoptotic RIPK2/caspase 1 complex. In an intracranial GBM xenograft mouse model, RIPGBM was found to significantly suppress tumor formation. CONCLUSIONS Our chemical genetics-based approach has identified a small molecule drug candidate and a potential drug target that selectively targets cancer stem cells and provides an approach for the treatment of GBMs.

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