High-Throughput Screening for Small Molecule Modulators of FGFR2-IIIb Pre-mRNA Splicing

2012 ◽  
pp. 127-138
Author(s):  
Erik S. Anderson ◽  
Peter Stoilov ◽  
Robert Damoiseaux ◽  
Douglas L. Black
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Mrna Splicing ◽  
Small Molecule Modulators

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 Identification of Small Molecule Modulators of Diguanylate Cyclase by FRET-based High-Throughput-Screening

2018 ◽  
Author(s):  
Matthias Christen ◽  
Cassandra Kamischke ◽  
Hemantha D. Kulasekara ◽  
Kathleen C. Olivas ◽  
Bridget R. Kulasekara ◽  
...  
Keyword(s):  
Small Molecules ◽  
Small Molecule ◽  
High Throughput ◽  
Biofilm Formation ◽  
High Throughput Screening ◽  
Diguanylate Cyclase ◽  
Chronic Infections ◽  
Structure Activity ◽  
Diguanylate Cyclases ◽  
Small Molecule Modulators

The bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP) is a key regulator of cellular motility, the cell cycle, and biofilm formation with its resultant antibiotic tolerance, which may make chronic infections difficult to treat. Therefore, diguanylate cyclases, which regulate the spatiotemporal production of c-di-GMP, may be attractive drug targets to control biofilm formation that is part of chronic infections. In this paper, we present a FRET-based biochemical high-throughput screening approach coupled with detailed structure-activity studies to identify synthetic small molecule modulators of the diguanylate cyclase, DgcA, from Caulobacter crescentus. We identified a set of 7 small molecules that in the low µM range regulate DgcA enzymatic activity. Subsequent structure activity studies on selected scaffolds revealed a remarkable diversity of modulatory behaviors, including slight chemical substitutions that revert the effects from allosteric enzyme inhibition to activation. The compounds identified represent novel chemotypes and are potentially developable into chemical genetic tools for the dissection of c-di-GMP signaling networks and alteration of c-di-GMP associated phenotypes. In sum, our studies underline the importance for detailed mechanism of action studies for inhibitors of c-di-GMP signaling and demonstrate the complex interplay between synthetic small molecules and the regulatory mechanisms that control the activity of diguanylate cyclases.

scholarly journals Identification of Small‐Molecule Modulators of Diguanylate Cyclase by FRET‐Based High‐Throughput Screening

ChemBioChem ◽  
2018 ◽  
Vol 20 (3) ◽  
pp. 394-407 ◽  
Author(s):  
Matthias Christen ◽  
Cassandra Kamischke ◽  
Hemantha D. Kulasekara ◽  
Kathleen C. Olivas ◽  
Bridget R. Kulasekara ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Diguanylate Cyclase ◽  
Small Molecule Modulators

High‐throughput screening reveals small molecule modulators inhibitory to Acidovorax citrulli

2020 ◽  
Vol 69 (5) ◽  
pp. 818-826
Author(s):  
Yu Lu ◽  
Loïc Deblais ◽  
Gireesh Rajashekara ◽  
Sally A. Miller ◽  
Yorsa A. Helmy ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Acidovorax Citrulli ◽  
Small Molecule Modulators

scholarly journals A Fluorescence Polarization-Based High-Throughput Screen to Identify the First Small-Molecule Modulators of the Human Adenylyltransferase HYPE/FICD

2020 ◽  
Vol 21 (19) ◽  
pp. 7128
Author(s):  
Ali Camara ◽  
Alyssa George ◽  
Evan Hebner ◽  
Anika Mahmood ◽  
Jashun Paluru ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
Fluorescence Polarization ◽  
High Throughput Screening ◽  
Regulatory Mechanism ◽  
Dose Dependency ◽  
Chemical Libraries ◽  
Cellular Events ◽  
Small Molecule Modulators ◽  
Simultaneous Identification

The covalent transfer of the AMP portion of ATP onto a target protein—termed adenylylation or AMPylation—by the human Fic protein HYPE/FICD has recently garnered attention as a key regulatory mechanism in endoplasmic reticulum homeostasis, neurodegeneration, and neurogenesis. As a central player in such critical cellular events, high-throughput screening (HTS) efforts targeting HYPE-mediated AMPylation warrant investigation. Herein, we present a dual HTS assay for the simultaneous identification of small-molecule activators and inhibitors of HYPE AMPylation. Employing the fluorescence polarization of an ATP analog fluorophore—Fl-ATP—we developed and optimized an efficient, robust assay that monitors HYPE autoAMPylation and is amenable to automated, high-throughput processing of diverse chemical libraries. Challenging our pilot screen with compounds from the LOPAC, Spectrum, MEGx, and NATx libraries yielded 0.3% and 1% hit rates for HYPE activators and inhibitors, respectively. Further, these hits were assessed for dose-dependency and validated via orthogonal biochemical AMPylation assays. We thus present a high-quality HTS assay suitable for tracking HYPE’s enzymatic activity, and the resultant first small-molecule manipulators of HYPE-promoted autoAMPylation.

scholarly journals Physiologically relevant orthogonal assays for the discovery of small-molecule modulators of WIP1 phosphatase in high-throughput screens

2019 ◽  
Vol 294 (46) ◽  
pp. 17654-17668 ◽  
Author(s):  
Victor Clausse ◽  
Dingyin Tao ◽  
Subrata Debnath ◽  
Yuhong Fang ◽  
Harichandra D. Tagad ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Binding Studies ◽  
Wip1 Phosphatase ◽  
Activity Measurements ◽  
Attractive Therapeutic Target ◽  
Small Molecule Modulator ◽  
Small Molecule Modulators ◽  
High Throughput Screens

WT P53-Induced Phosphatase 1 (WIP1) is a member of the magnesium-dependent serine/threonine protein phosphatase (PPM) family and is induced by P53 in response to DNA damage. In several human cancers, the WIP1 protein is overexpressed, which is generally associated with a worse prognosis. Although WIP1 is an attractive therapeutic target, no potent, selective, and bioactive small-molecule modulator with favorable pharmacokinetics has been reported. Phosphatase enzymes are among the most challenging targets for small molecules because of the difficulty of achieving both modulator selectivity and bioavailability. Another major obstacle has been the availability of robust and physiologically relevant phosphatase assays that are suitable for high-throughput screening. Here, we describe orthogonal biochemical WIP1 activity assays that utilize phosphopeptides from native WIP1 substrates. We optimized an MS assay to quantify the enzymatically dephosphorylated peptide reaction product in a 384-well format. Additionally, a red-shifted fluorescence assay was optimized in a 1,536-well format to enable real-time WIP1 activity measurements through the detection of the orthogonal reaction product, Pi. We validated these two optimized assays by quantitative high-throughput screening against the National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection and used secondary assays to confirm and evaluate inhibitors identified in the primary screen. Five inhibitors were further tested with an orthogonal WIP1 activity assay and surface plasmon resonance binding studies. Our results validate the application of miniaturized physiologically relevant and orthogonal WIP1 activity assays to discover small-molecule modulators from high-throughput screens.

scholarly journals Screening for Small-Molecule Modulators of Long Noncoding RNA-Protein Interactions Using AlphaScreen

2015 ◽  
Vol 20 (9) ◽  
pp. 1132-1141 ◽  
Author(s):  
Roya Pedram Fatemi ◽  
Sultan Salah-Uddin ◽  
Farzaneh Modarresi ◽  
Nathalie Khoury ◽  
Claes Wahlestedt ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
Protein Interactions ◽  
High Throughput Screening ◽  
Noncoding Rna ◽  
Target Genes ◽  
Regulatory Function ◽  
Downstream Target ◽  
Small Molecule Modulators ◽  
In Cells

Long non–protein coding RNAs (lncRNAs) are an important class of molecules that help orchestrate key cellular events. Although their functional roles in cells are not well understood, thousands of lncRNAs and a number of possible mechanisms by which they act have been reported. LncRNAs can exert their regulatory function in cells by interacting with epigenetic enzymes. In this study, we developed a tool to study lncRNA-protein interactions for high-throughput screening of small-molecule modulators using AlphaScreen technology. We tested the interaction of two lncRNAs: brain-derived neurotrophic factor antisense ( BDNF-AS) and Hox transcript antisense RNA ( HOTAIR), with Enhancer of zeste homolog 2 (EZH2), a histone methyltransferase against a phytochemical library, to look for small-molecule inhibitors that can alter the expression of downstream target genes. We identified ellipticine, a compound that up-regulates BDNF transcription. Our study shows the feasibility of using high-throughput screening to identify modulators of lncRNA-protein interactions and paves the road for targeting lncRNAs that are dysregulated in human disorders using small-molecule therapies.

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