+ Site Statistics
+ Search Articles
+ PDF Full Text Service
How our service works
Request PDF Full Text
+ Follow Us
Follow on Facebook
Follow on Twitter
Follow on LinkedIn
+ Subscribe to Site Feeds
Most Shared
PDF Full Text
+ Translate
+ Recently Requested

New fluorescence-based high-throughput screening assay for small molecule inhibitors of tyrosyl-DNA phosphodiesterase 2 (TDP2)

New fluorescence-based high-throughput screening assay for small molecule inhibitors of tyrosyl-DNA phosphodiesterase 2 (TDP2)

European Journal of Pharmaceutical Sciences 118: 67-79

Tyrosyl-DNA phosphodiesterase 2 (TDP2) repairs topoisomerase II (TOP2) mediated DNA damages and causes resistance to TOP2-targeted cancer therapy. Inhibiting TDP2 could sensitize cancer cells toward TOP2 inhibitors. However, potent TDP2 inhibitors with favorable physicochemical properties are not yet reported. Therefore, there is a need to search for novel molecular scaffolds capable of inhibiting TDP2. We report herein a new simple, robust, homogenous mix-and-read fluorescence biochemical assay based using humanized zebrafish TDP2 (14M_zTDP2), which provides biochemical and molecular structure basis for TDP2 inhibitor discovery. The assay was validated by screening a preselected library of 1600 compounds (Z' ≥ 0.72) in a 384-well format, and by running in parallel gel-based assays with fluorescent DNA substrates. This library was curated via virtual high throughput screening (vHTS) of 460,000 compounds from Chembridge Library, using the crystal structure of the novel surrogate protein 14M_zTDP2. From this primary screening, we selected the best 32 compounds (2% of the library) to further assess their TDP2 inhibition potential, leading to the IC50 determination of 10 compounds. Based on the dose-response curve profile, pan-assay interference compounds (PAINS) structure identification, physicochemical properties and efficiency parameters, two hit compounds, 11a and 19a, were tested using a novel secondary fluorescence gel-based assay. Preliminary structure-activity relationship (SAR) studies identified guanidine derivative 12a as an improved hit with a 6.4-fold increase in potency over the original HTS hit 11a. This study highlights the importance of the development of combination approaches (biochemistry, crystallography and high throughput screening) for the discovery of TDP2 inhibitors.

Please choose payment method:

(PDF emailed within 0-6 h: $19.90)

Accession: 065351405

Download citation: RISBibTeXText

PMID: 29574079

DOI: 10.1016/j.ejps.2018.03.021

Related references

New fluorescence-based high-throughput screening assay for small molecule inhibitors of tyrosyl-DNA phosphodiesterase 2 (TDP2). European Journal of Pharmaceutical Sciences 118: 67-79, 2018

High-throughput screening for small-molecule inhibitors of LARG-stimulated RhoA nucleotide binding via a novel fluorescence polarization assay. Journal of Biomolecular Screening 14(2): 161-172, 2009

Deazaflavin Inhibitors of Tyrosyl-DNA Phosphodiesterase 2 (TDP2) Specific for the Human Enzyme and Active against Cellular TDP2. Acs Chemical Biology 11(7): 1925-1933, 2016

A cell-based high-throughput assay for the screening of small-molecule inhibitors of p53-MDM2 interaction. Journal of Biomolecular Screening 16(4): 450-456, 2011

Synthesis and Biological Evaluation of the First Triple Inhibitors of Human Topoisomerase 1, Tyrosyl-DNA Phosphodiesterase 1 (Tdp1), and Tyrosyl-DNA Phosphodiesterase 2 (Tdp2). Journal of Medicinal Chemistry 60(8): 3275-3288, 2017

An RNAi-based high-throughput screening assay to identify small molecule inhibitors of hepatitis B virus replication. Journal of Biological Chemistry 292(30): 12577-12588, 2017

The identification of novel small-molecule inhibitors targeting WDR5-MLL1 interaction through fluorescence polarization based high-throughput screening. Bioorganic and Medicinal Chemistry Letters 29(4): 638-645, 2019

Identification of small molecule inhibitors of the mitotic kinase haspin by high-throughput screening using a homogeneous time-resolved fluorescence resonance energy transfer assay. Journal of Biomolecular Screening 13(10): 1025-1034, 2008

High-throughput compatible fluorescence resonance energy transfer-based assay to identify small molecule inhibitors of AMSH deubiquitinase activity. Analytical Biochemistry 440(1): 71-77, 2013

Discovery of acetyl-coenzyme A carboxylase 2 inhibitors: comparison of a fluorescence intensity-based phosphate assay and a fluorescence polarization-based ADP Assay for high-throughput screening. Assay and Drug Development Technologies 5(2): 225-235, 2007

High-throughput fluorescence assay for small-molecule inhibitors of autophagins/Atg4. Journal of Biomolecular Screening 16(2): 174-182, 2011

Identification of phosphotyrosine mimetic inhibitors of human tyrosyl-DNA phosphodiesterase I by a novel AlphaScreen high-throughput assay. Molecular Cancer Therapeutics 8(1): 240-248, 2009

A high throughput screening assay system for the identification of small molecule inhibitors of gsp. Plos one 9(3): E90766, 2014

Development of a high-throughput screening assay for the discovery of small-molecule SecA inhibitors. Analytical Biochemistry 413(2): 90-96, 2011

High-throughput screening assay for identification of small molecule inhibitors of Aurora2/STK15 kinase. Journal of Biomolecular Screening 9(5): 391-397, 2004