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Development of Cell-based High Throughput Luminescence Assay for Drug Discovery in Inhibiting OCT4 and DNA-PKcs Interaction
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  • Ismail Mohiuddin,
  • Sung-Jen Wei,
  • In-Hyoung Yang,
  • Gloria Martinez,
  • Shengping Yang,
  • Eun Jeong Cho,
  • Kevin Dalby,
  • Min Kang
Ismail Mohiuddin
Texas Tech University Health Sciences Center
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Sung-Jen Wei
Texas Tech University Health Sciences Center
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In-Hyoung Yang
Texas Tech University Health Sciences Center
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Gloria Martinez
Texas Tech University Health Sciences Center
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Shengping Yang
Pennington Biomedical Research Center
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Eun Jeong Cho
The University of Texas at Austin College of Pharmacy
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Kevin Dalby
The University of Texas at Austin College of Pharmacy
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Min Kang
Texas Tech University Health Sciences Center
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Abstract

Amplification-independent c-MYC overexpression is suggested in multiple cancers. Targeting c-MYC activity has therapeutic potential, but efforts thus far have been mostly unsuccessful. To find a druggable target to modulate c-MYC activity in cancer, we identified two kinases, MAPKAPK2 and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which phosphorylate the S111 and the S93 residues of OCT4, respectively, to transcriptionally activate c-MYC. Using these observations, we present here a novel cell-based luminescence assay to identify compounds that inhibit the interaction between DNA-PKcs and OCT4. After screening approximately 80,000 compounds, we identified 56 compounds (“hits”) that inhibited the luminescence reaction. Using a custom antibody specific for pOCT4S93, the “hits” were validated for their effect on OCT4 phosphorylation and activation. Seven compounds were selected for the second step of validation, which focused on the interaction between kinase and substrate. After further characterization, we identified two compounds that significantly impaired the ability of DNA-PKcs to bind to and phosphorylate OCT4. The compounds demonstrate a significant ability to kill cancer cells in the nanomolar range. In conclusion, we developed a cell-based luminescence assay to identify novel inhibitors targeting c-MYC transcriptional activation, and have found two compounds that may function as lead compounds for further development.

Peer review status:POSTED

20 Oct 2020Submitted to Biotechnology and Bioengineering
20 Oct 2020Assigned to Editor
20 Oct 2020Submission Checks Completed