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

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 (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 pOCT4S93 and pOCT4S111, 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.

A review for cell-based screening methods in drug discovery

“…Mohiuddin et al . stably co-expressed target fragments tagged with luminescence probes in HEK-293FT cells and identify five compounds as lead compounds (Mohiuddin et al 2021 ). Fluorescent imaging often requires the removal of background fluorescent signals to obtain robust measurements, which is challenging for high-density microplates.…”

Luminescence Energy Transfer–Based Screening and Target Engagement Approaches for Chemical Biology and Drug Discovery

“…These properties have led to the development of new applications 13 that include assessing gene regulation, 14-16 protein stability, 17 and the evaluation of protein-protein interactions (PPIs) in real time at endogenous expression levels in ways that have not been possible before. 18,19 NanoBRET technology provides an opportunity to design assays for a broad range of molecular interactions. The key to the approach is to bring a suitable acceptor fluorophore into the proximity of NLuc.…”