Abstract 4257: Discovery of small molecules to target DDX5 unfolding of MYC promoter G-quadruplex for MYC inhibition

Abstract c-MYC (MYC) is one of the most deregulated oncogenes in human cancers and encodes a master regulator of cell proliferation. Direct targeting of MYC protein has proven to be challenging due to its disordered structure and lack of clear drug binding pocket. G-quadruplexes (G4s) are globular DNA secondary structures that form in guanine-rich regions of the genome with important regulatory roles. The G4 formed in the proximal promoter of the MYC gene (MycG4) acts as a transcriptional silencer. Our lab has previously discovered the DDX5 helicase, a founding member of the DEAD-box RNA helicase family, actively unfolds the MycG4 and transactivates MYC in human cancer cells. Therefore, the DDX5-MycG4 interaction and unfolding represents a promising target for effective MYC downregulation and anti-cancer therapeutics. Previously, our lab has shown for proof-of-principle that G4-ligand TMPyP4 can inhibit the DDX5-MycG4 interaction and downregulate MYC expression in cancer cells. However, TMPyP4 is not a good drug candidate due to its phototoxicity. Further, no inhibitors are available that directly bind DDX5 and specifically inhibit its G4 unfolding activity. Here, we report a high-throughput screen of ∼20,000 diverse drug-like compounds to identify specific inhibitors of the DDX5-MycG4 interaction for MYC downregulation in human cancers. We adapted a FRET-based G4 unfolding assay for high-throughput screening in a 384-well plate format. Briefly, a 6-fluorescein fluorophore and a Black Hole Quencher 1 FRET pair is attached to the 3’- and 5’-end of the MycG4 DNA sequence, respectively, whose fluorescence is quenched in a formed G4. Unfolding of the G4 by DDX5 increases the fluorophore distance and increases the detected fluorescence. The Z’ score of the adapted FRET assay was ∼0.5 which provides sufficient assay range for screening inhibitors. Compounds that reduce DDX5 unfolding by more than 40% were defined as a hit. Out of 20,000 screened compounds, 31 initial hits were identified. Significantly, we identified five different classes of hit compound chemical structures. Several hits are substantially structurally different from classic G4 interacting compounds, suggesting a different mechanism of action for inhibition of the DDX5 protein-DNA interaction. We conducted hit validation and tested the identified compounds for DDX5 G4 and RNA unfolding, to better understand the mechanisms of action of different compound classes. The hits that specifically inhibit DDX5-MycG4 interaction were tested against MYC expression levels in cancer cells. In summary, we have developed a high-throughput screening method to discover inhibitors of the DDX5-MycG4 interaction and have identified several hit compounds with different structure classes. Our goal is to identify lead compounds to target this interaction for MYC downregulation for future anti-cancer therapeutics. Citation Format: Sarah D. Washburn, Danzhou Yang. Discovery of small molecules to target DDX5 unfolding of MYC promoter G-quadruplex for MYC inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4257.