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Updated: Feb 17, 2026
High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes
Published on: March 3, 2015
High-Throughput Screening Approach for Identifying Compounds That Inhibit Nonhomologous End Joining
Andrea L Bredemeyer1, Bruce S Edwards2, Mark K Haynes2
11 Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA.
Abstract:
DNA double-strand breaks (DSBs) are repaired primarily by homologous recombination (HR) or nonhomologous end joining (NHEJ). Compounds that modulate HR have shown promise as cancer therapeutics. The V(D)J recombination reaction, which assembles antigen receptor genes in lymphocytes, is initiated by the introduction of DNA DSBs at two recombining gene segments by the RAG endonuclease, followed by the NHEJ-mediated repair of these DSBs. Here, using HyperCyt automated flow cytometry, we develop a robust high-throughput screening (HTS) assay for NHEJ that utilizes engineered pre-B-cell lines where the V(D)J recombination reaction can be induced and monitored at a single-cell level. This approach, novel in processing four 384-well plates at a time in parallel, was used to screen the National Cancer Institute NeXT library to identify compounds that inhibit V(D)J recombination and NHEJ. Assessment of cell light scattering characteristics at the primary HTS stage (83,536 compounds) enabled elimination of 60% of apparent hits as false positives. Although all the active compounds that we identified had an inhibitory effect on RAG cleavage, we have established this as an approach that could identify compounds that inhibit RAG cleavage or NHEJ using new chemical libraries.
Insights
Researchers developed a high-throughput screening assay to identify compounds inhibiting nonhomologous end joining (NHEJ), a DNA repair pathway crucial in V(D)J recombination. This assay successfully screened over 83,000 compounds, identifying potential inhibitors of DNA repair mechanisms.
Area of Science:
- Molecular Biology
- Genetics
- Immunology
Background:
- DNA double-strand breaks (DSBs) are critical DNA lesions repaired by homologous recombination (HR) or nonhomologous end joining (NHEJ).
- Modulators of HR show therapeutic potential in cancer treatment.
- The V(D)J recombination process in lymphocytes, essential for adaptive immunity, involves RAG endonuclease-initiated DSBs followed by NHEJ repair.
Purpose of the Study:
- To develop a robust high-throughput screening (HTS) assay for identifying inhibitors of nonhomologous end joining (NHEJ).
- To screen a large chemical library for compounds that modulate the V(D)J recombination pathway.
Main Methods:
- Utilized engineered pre-B-cell lines for single-cell level monitoring of V(D)J recombination.
- Implemented HyperCyt automated flow cytometry for parallel processing of 384-well plates.
- Incorporated cell light scattering analysis to filter false positives during HTS.
Main Results:
- Screened 83,536 compounds from the National Cancer Institute NeXT library.
- Eliminated 60% of apparent hits as false positives through light scattering assessment.
- Identified active compounds that inhibit RAG cleavage, a key step in V(D)J recombination.
Conclusions:
- Established a novel HTS approach for identifying inhibitors of NHEJ and RAG cleavage.
- The assay is adaptable for screening new chemical libraries to find modulators of DNA repair pathways.
- This method provides a platform for discovering novel therapeutic agents targeting DNA repair mechanisms.
