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Updated: Apr 27, 2026
A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1
Published on: April 16, 2021
Biochemical assays for the discovery of TDP1 inhibitors
Christophe Marchand1, Shar-yin N Huang2, Thomas S Dexheimer3
1Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute; marchanc@mail.nih.gov pommier@nih.gov.
Abstract:
Drug screening against novel targets is warranted to generate biochemical probes and new therapeutic drug leads. TDP1 and TDP2 are two DNA repair enzymes that have yet to be successfully targeted. TDP1 repairs topoisomerase I-, alkylation-, and chain terminator-induced DNA damage, whereas TDP2 repairs topoisomerase II-induced DNA damage. Here, we report the quantitative high-throughput screening (qHTS) of the NIH Molecular Libraries Small Molecule Repository using recombinant human TDP1. We also developed a secondary screening method using a multiple loading gel-based assay where recombinant TDP1 is replaced by whole cell extract (WCE) from genetically engineered DT40 cells. While developing this assay, we determined the importance of buffer conditions for testing TDP1, and most notably the possible interference of phosphate-based buffers. The high specificity of endogenous TDP1 in WCE allowed the evaluation of a large number of hits with up to 600 samples analyzed per gel via multiple loadings. The increased stringency of the WCE assay eliminated a large fraction of the initial hits collected from the qHTS. Finally, inclusion of a TDP2 counter-screening assay allowed the identification of two novel series of selective TDP1 inhibitors.
Insights
Researchers screened for novel drug leads targeting TDP1, a DNA repair enzyme. A new assay identified selective TDP1 inhibitors, advancing drug discovery for DNA repair mechanisms.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Discovery
Background:
- TDP1 and TDP2 are critical DNA repair enzymes that remain untargeted for therapeutic intervention.
- TDP1 resolves DNA damage from topoisomerase I, alkylation, and chain terminators.
- TDP2 repairs DNA damage induced by topoisomerase II.
Purpose of the Study:
- To identify novel biochemical probes and therapeutic drug leads targeting TDP1.
- To develop and validate robust screening assays for TDP1 inhibitors.
- To discover selective TDP1 inhibitors through a counter-screening approach.
Main Methods:
- Quantitative high-throughput screening (qHTS) of the NIH Molecular Libraries Small Molecule Repository against recombinant human TDP1.
- Development of a secondary gel-based assay using whole cell extract (WCE) from engineered DT40 cells to enhance specificity.
- Optimization of buffer conditions, noting potential interference from phosphate buffers.
- Inclusion of a TDP2 counter-screening assay to ensure TDP1 selectivity.
Main Results:
- A qHTS campaign identified initial hits against recombinant TDP1.
- The WCE-based assay demonstrated high specificity and allowed analysis of numerous samples.
- The more stringent WCE assay successfully eliminated false positives from the qHTS.
- Two novel series of selective TDP1 inhibitors were identified through counter-screening against TDP2.
Conclusions:
- The developed screening assays are effective for identifying selective TDP1 inhibitors.
- The study successfully identified novel chemical series with potential as TDP1-targeted therapeutics.
- This work provides a foundation for further development of TDP1-based drug discovery efforts.
