Development of cell-based high throughput luminescence assay for drug discovery in inhibiting OCT4/DNA-PKcs and OCT4-MK2 interactions

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 (MK2) and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which phosphorylate the Ser111 and the Ser93 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 these kinases and OCT4. After screening approximately 80,000 compounds, we identified 56 compounds (hits) that inhibited the luminescence reaction between DNA-PKcs and OCT4, and 65 hits inhibiting the MK2-OCT4 interaction. Using custom antibodies specific for pOCT4(S93) and pOCT4(S111), the hits were validated for their effect on OCT4 phosphorylation and activation. Using a two-step method for validation, we identified two candidate compounds from the DNA-PKcs assay and three from the MK2 assay. All five 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 five compounds that may function as lead compounds for further development.

Automated summary

A novel cell-based luminescence assay was developed to identify inhibitors targeting c-MYC transcriptional activation, leading to the discovery of five potential lead compounds with significant ability to kill cancer cells. Two candidate compounds from the DNA-PKcs assay and three from the MK2 assay were identified through a two-step validation method.