The MYC gene encodes for the transcription factor MYC, which is responsible for the regulation of 15-90% of the human genome and has been linked to proliferation, differentiation, apoptosis, and oncogenesis. Importantly, MYC is overexpressed in 20% of all cancers. However, it has proven difficult to develop direct small molecule inhibitors of MYC. An attractive alternative route is the prevention of MYC expression via small molecule-mediated stabilization of the G-quadruplex (G4) present within its promoter region. However, a major barrier in developing biologically active small molecules that bind to nucleic acids has been the identification of selective interactions. Previous approaches have generally yielded pan-quadruplex binding molecules, and strategies to generate selective ligands are lacking. Here, we report an approach for the identification of G4-binding small molecules using small molecule microarrays (SMMs). We use the SMM screening platform to identify a novel G4-binding small molecule (D089). Biophysical techniques, such as surface plasmon resonance (SPR), thermal melt assays, WaterLOGSY NMR, and fluorescence titrations, demonstrate that D089 binds reversibly to the MYC G4 with single digit micromolar affinity, with weaker or no measurable binding to several other G4s. Additionally, D089 inhibits MYC expression in cell models, with minimal impact on the expression of other G4-associated genes, while MYC target genes are downregulated. Development of a synthetic route to D089 has enabled the discovery of an improved analog (DC-34) that has superior binding affinity and efficacy in silencing MYC expression. Furthermore, from a preliminary NMR structure of DC-34 and the MYC G4, we can observe that the MYC G4 undergoes a conformational change to accommodate ligand binding in a way that appears unique for this molecular scaffold. Biochemical and cell-based assays demonstrate that DC-34 effectively silences MYC transcription and translation via a G4-dependent mechanism of action. We demonstrate that the SMM approach can reveal a selective G4 binder for oncogene inhibition. Efforts toward applying SMMs to other G4-associated oncogenes are being pursued to discover new selective binding scaffolds.

Selectively targeting MYC expression with nucleic acid binding small molecules

254th National Meeting (2017)

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