scholarly journals Identification of Celastrol as a Novel YAP-TEAD Inhibitor for Cancer Therapy by High Throughput Screening with Ultrasensitive YAP/TAZ–TEAD Biosensors

Cancers ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 1596 ◽  
Author(s):  
Kazem Nouri ◽  
Taha Azad ◽  
Min Ling ◽  
Helena J. Janse van Rensburg ◽  
Alexander Pipchuk ◽  
...  
Keyword(s):  
Cancer Therapy ◽  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Hippo Pathway ◽  
Cancer Cell Proliferation ◽  
Protein Protein Interaction ◽  
The Hippo Pathway ◽  
Future Work

The Hippo pathway has emerged as a key signaling pathway that regulates a broad range of biological functions, and dysregulation of the Hippo pathway is a feature of a variety of cancers. Given this, some have suggested that disrupting the interaction of the Hippo core component YAP and its paralog TAZ with transcriptional factor TEAD may be an effective strategy for cancer therapy. However, there are currently no clinically available drugs targeting the YAP/TAZ–TEAD interaction for cancer treatment. To facilitate screens for small molecule compounds that disrupt the YAP–TEAD interaction, we have developed the first ultra-bright NanoLuc biosensor to quantify YAP/TAZ–TEAD protein–protein interaction (PPI) both in living cells and also in vitro using biosensor fusion proteins purified from bacteria. Using this biosensor, we have performed an in vitro high throughput screen (HTS) of small molecule compounds and have identified and validated the drug Celastrol as a novel inhibitor of YAP/TAZ–TEAD interaction. We have also demonstrated that Celastrol can inhibit cancer cell proliferation, transformation, and cell migration. In this study, we describe a new inhibitor of the YAP/TAZ–TEAD interaction warranting further investigation and offer a novel biosensor tool for the discovery of other new Hippo-targeting drugs in future work.

scholarly journals Development of a Novel High-Throughput Screen and Identification of Small-Molecule Inhibitors of the Gα–RGS17 Protein–Protein Interaction Using AlphaScreen

2011 ◽  
Vol 16 (8) ◽  
pp. 869-877 ◽  
Author(s):  
Duncan I. Mackie ◽  
David L. Roman
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
Protein Interaction ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Screening Method ◽  
Fold Increase ◽  
Rgs Proteins ◽  
High Throughput Screen ◽  
Protein Protein Interaction

In this study, the authors used AlphaScreen technology to develop a high-throughput screening method for interrogating small-molecule libraries for inhibitors of the Gαo–RGS17 interaction. RGS17 is implicated in the growth, proliferation, metastasis, and the migration of prostate and lung cancers. RGS17 is upregulated in lung and prostate tumors up to a 13-fold increase over patient-matched normal tissues. Studies show RGS17 knockdown inhibits colony formation and decreases tumorigenesis in nude mice. The screen in this study uses a measurement of the Gαo–RGS17 protein–protein interaction, with an excellent Z score exceeding 0.73, a signal-to-noise ratio >70, and a screening time of 1100 compounds per hour. The authors screened the NCI Diversity Set II and determined 35 initial hits, of which 16 were confirmed after screening against controls. The 16 compounds exhibited IC50 <10 µM in dose–response experiments. Four exhibited IC50 values <6 µM while inhibiting the Gαo–RGS17 interaction >50% when compared to a biotinylated glutathione-S-transferase control. This report describes the first high-throughput screen for RGS17 inhibitors, as well as a novel paradigm adaptable to many other RGS proteins, which are emerging as attractive drug targets for modulating G-protein-coupled receptor signaling.

Abstract 2474: Potent small molecule TEAD inhibitors targeting the Hippo pathway exhibit antiproliferation in vitro and anti-tumor effect in vivo

2020 ◽  
Author(s):  
Ben Amidon ◽  
Sakeena Syed ◽  
Jill Cavanaugh ◽  
Hyejin Frosch ◽  
Prabitha Natarajan ◽  
...  
Keyword(s):  
Small Molecule ◽  
Hippo Pathway ◽  
The Hippo Pathway

scholarly journals A High-Throughput Assay to Identify Small-Molecule Modulators of Alternative Pre-mRNA Splicing

2012 ◽  
Vol 18 (2) ◽  
pp. 180-190 ◽  
Author(s):  
Ahmet Dirim Arslan ◽  
Xiaolong He ◽  
Minxiu Wang ◽  
Emily Rumschlag-Booms ◽  
Lijun Rong ◽  
...  
Keyword(s):  
Cancer Therapy ◽  
Small Molecule ◽  
High Throughput ◽  
Messenger Rna ◽  
High Throughput Screening ◽  
Mrna Splicing ◽  
Polypyrimidine Tract Binding Protein ◽  
High Throughput Screening Assay ◽  
Small Molecule Modulators ◽  
High Throughput Assay

Alternative splicing (AS) is an efficient mechanism that involves the generation of transcriptome and protein diversity from a single gene. Defects in pre–messenger RNA (mRNA) splicing are an important cause of numerous diseases, including cancer. AS of pre-mRNA as a target for cancer therapy has not been well studied. We have reported previously that a splicing factor, polypyrimidine tract-binding protein (PTB), is overexpressed in ovarian tumors compared with matched normal controls, and knockdown of PTB expression by short-hairpin RNA impairs ovarian tumor cell growth, colony formation, and invasiveness. Given the complexity of PTB’s molecular functions, a chemical method for controlling PTB activity might provide a therapeutic and experimental tool. However, no commercially available PTB inhibitors have yet been described. To expand our ability to find novel inhibitors, we developed a robust, fluorometric, cell-based high-throughput screening assay in 96-well plates that reports on the splicing activity of PTB. In an attempt to use the cells for large-scale chemical screens to identify PTB modulators, we established cell lines stably expressing the reporter gene. Our results suggest that this high-throughput assay could be used to identify small-molecule modulators of PTB activity. Based on these findings and the role that upregulated PTB has on cell proliferation and malignant properties of tumors, targeting PTB for inhibition with small molecules offers a promising strategy for cancer therapy.

scholarly journals A High-Throughput Screening Method for Small-Molecule Inhibitors of the Aberrant Mutant SOD1 and Dynein Complex Interaction

2011 ◽  
Vol 17 (3) ◽  
pp. 314-326 ◽  
Author(s):  
Xiaohu Tang ◽  
Kathleen I. Seyb ◽  
Mickey Huang ◽  
Eli R. Schuman ◽  
Ping Shi ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
Protein Interaction ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Small Molecule Inhibitors ◽  
Screening Method ◽  
Live Cells ◽  
Mutant Sod1 ◽  
Protein Protein Interaction

Aberrant protein-protein interactions are attractive drug targets in a variety of neurodegenerative diseases due to the common pathology of accumulation of protein aggregates. In amyotrophic lateral sclerosis, mutations in SOD1 cause the formation of aggregates and inclusions that may sequester other proteins and disrupt cellular processes. It has been demonstrated that mutant SOD1, but not wild-type SOD1, interacts with the axonal transport motor dynein and that this interaction contributes to motor neuron cell death, suggesting that disrupting this interaction may be a potential therapeutic target. However, it can be challenging to configure a high-throughput screening (HTS)–compatible assay to detect inhibitors of a protein-protein interaction. Here we describe the development and challenges of an HTS for small-molecule inhibitors of the mutant SOD1-dynein interaction. We demonstrate that the interaction can be formed by coexpressing the A4V mutant SOD1 and dynein intermediate complex in cells and that this interaction can be disrupted by compounds added to the cell lysates. Finally, we show that some of the compounds identified from a pilot screen to inhibit the protein-protein interaction with this method specifically disrupt the interaction between the dynein complex and mtSOD1 but not the dynein complex itself when applied to live cells.

scholarly journals Identifying small molecule probes of ENTPD5 through high throughput screening

2018 ◽  
Author(s):  
Matthew A Durst ◽  
Kiira Ratia ◽  
Arnon Lavie
Keyword(s):  
Cancer Cell ◽  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Small Molecule Inhibitors ◽  
Molecular Probes ◽  
Protein Glycosylation ◽  
Cancer Cell Proliferation ◽  
The Novel ◽  
Small Molecule Probes

Ectonucleoside Triphosphate Diphosphohydrolase 5 (ENTPD5) has been shown to be important in maintaining cellular function in cancer, and its expression is upregulated through multiple, unique pathways in certain cancers, including laryngeal, glioblastoma multiforme, breast, testicular, and prostate. ENTPD5 supports cancer growth by promoting the import of UDP-glucose, a metabolite used for protein glycosylation and hence proper glycoprotein folding, into the ER by providing the counter molecule, UMP, to the ER antiporter. Despite its cancer-supporting function, no small molecule inhibitors of ENTPD5 are commercially available, and few studies have been performed in tissue culture to understand the effects of chemical inhibition of ENTPD5. We performed a high-throughput screen (HTS) of 21,120 compounds to identify small molecule inhibitors of ENPTD5 activity. Two hits were identified, and we performed a structure activity relationship (SAR) screen around these hits. Further validation of these probes were done in an orthogonal assay and then assayed in cell culture to assess their effect on prostate cancer cell lines. Notably, treatment with the novel ENTPD5 inhibitor reduced the amount of glycoprotein produced in treated cells, consistent with the hypothesis that ENTPD5 is important for glycoprotein folding. This work serves as an important step in designing new molecular probes for ENTPD5 as well as further probing the utility of targeting ENTPD5 to combat cancer cell proliferation.

scholarly journals Using Biosensors to Study Protein–Protein Interaction in the Hippo Pathway

2021 ◽  
Vol 9 ◽  
Author(s):  
Alexander Pipchuk ◽  
Xiaolong Yang
Keyword(s):  
Protein Interactions ◽  
High Throughput Screening ◽  
Cellular Proliferation ◽  
Affinity Purification ◽  
Hippo Pathway ◽  
High Sensitivity ◽  
Live Cells ◽  
Hippo Signaling ◽  
The Hippo Pathway

The Hippo signaling network is dependent on protein–protein interactions (PPIs) as a mechanism of signal transduction to regulate organ size, cellular proliferation and differentiation, tumorigenesis, and other cellular processes. Current efforts aim to resolve the complex regulation of upstream Hippo components or focus on identifying targeted drugs for use in cancer therapy. Despite extensive characterization of the Hippo pathway interactome by affinity purification mass spectrometry (AP-MS) and other methodologies, previous research methods have not been sufficient to achieve these aims. In this review, we describe several recent studies that make use of luciferase-based biosensors as a new approach to study the Hippo Pathway. These biosensors serve as powerful tools with which to study PPIs both in vitro using purified biosensor proteins, and in real time in live cells. Notably, luciferase biosensors have excellent sensitivity and have been used to screen for upstream kinase regulators of the Hippo pathway. Furthermore, the high sensitivity and stability of these biosensors enables their application in high throughput screening for Hippo-targeted chemotherapeutics. Finally, we describe the strengths and weaknesses of this method relative to AP-MS and discuss potential future directions for using biosensors to study Hippo signaling.

scholarly journals A high-throughput screening method for evolving a demethylase enzyme with improved and new functionalities

2020 ◽  
Author(s):  
Yuru Wang ◽  
Christopher D Katanski ◽  
Christopher Watkins ◽  
Jessica N Pan ◽  
Qing Dai ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Screening Method ◽  
Targeted Mutagenesis ◽  
Rna Repair ◽  
Dna And Rna ◽  
Modified Dna ◽  
Dna Substrates ◽  
Trna Sequencing

Abstract AlkB is a DNA/RNA repair enzyme that removes base alkylations such as N1-methyladenosine (m1A) or N3-methylcytosine (m3C) from DNA and RNA. The AlkB enzyme has been used as a critical tool to facilitate tRNA sequencing and identification of mRNA modifications. As a tool, AlkB mutants with better reactivity and new functionalities are highly desired; however, previous identification of such AlkB mutants was based on the classical approach of targeted mutagenesis. Here, we introduce a high-throughput screening method to evaluate libraries of AlkB variants for demethylation activity on RNA and DNA substrates. This method is based on a fluorogenic RNA aptamer with an internal modified RNA/DNA residue which can block reverse transcription or introduce mutations leading to loss of fluorescence inherent in the cDNA product. Demethylation by an AlkB variant eliminates the blockage or mutation thereby restores the fluorescence signals. We applied our screening method to sites D135 and R210 in the Escherichia coli AlkB protein and identified a variant with improved activity beyond a previously known hyperactive mutant toward N1-methylguanosine (m1G) in RNA. We also applied our method to O6-methylguanosine (O6mG) modified DNA substrates and identified candidate AlkB variants with demethylating activity. Our study provides a high-throughput screening method for in vitro evolution of any demethylase enzyme.

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