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Many G4 stabilizing compounds are known however, a G4-modulating drug remains elusive. The use of small molecules to stabilize the G4 conformation and consequently decrease MYC expression is an attractive therapeutic goal in cancers where MYC contributes to the oncogenic phenotype. (13) A second, and perhaps more likely, model suggests that G4 formation modulates protein–DNA interactions (for example, with nucleolin (14) or ADAR1 (15)), leading to regulation of transcription. (12) The specific mechanism by which the G4 regulates transcription remains under investigation, though one model that has been put forth is that formation of a G4 in this sequence results in a “kink” in the DNA that prevents the polymerase from continuing along its reading frame, ultimately resulting in down-regulation of the associated gene. (10, 11) MYC Expression is regulated by a 27 base pair (Pu27) sequence, found in the nuclease hypersensitive element III(1) region (NHEIII 1) of the MYC gene, that is known to form a G4. (9) G4s are guanine-rich, noncanonical Hoogsteen-bonded nucleotide structures found in many RNA and DNA sequences ( Figure 1A). (7, 8) One such mechanism is through stabilization of the G-quadruplex (G4) present in the MYC promoter region. (2) However, targeting the MYC protein directly has proven to be difficult due to a lack of well-defined pockets amenable to small molecule binding, (3-6) which makes it desirable to evaluate alternative mechanisms for inhibiting MYC function. The oncogenic transcription factor MYC has a pleiotropic role in a wide range of cell processes (1) and is deregulated in some 70% of human cancers. In addition to providing a novel chemical scaffold that modulates MYC expression through G4 binding, this work suggests that the SMM screening approach may be broadly useful as an approach for the identification of new G4-binding small molecules. As a measure of selectivity, gene expression analysis and qPCR experiments demonstrated that MYC and several MYC target genes were downregulated upon treatment with this compound, while the expression of several other G4-driven genes was not affected. The compound induced G1 arrest and was selectively toxic to MYC-driven cancer cell lines containing the G4 in the promoter but had minimal effects in peripheral blood mononucleocytes or a cell line lacking the G4 in its MYC promoter. Biochemical and cell-based assays demonstrated that the compound effectively silenced MYC transcription and translation via a G4-dependent mechanism of action. Surface plasmon resonance (SPR) and thermal melt assays demonstrated that this molecule binds reversibly to the MYC G4 with single digit micromolar affinity, and with weaker or no measurable binding to other G4s. We use the SMM screening platform to identify a novel G4-binding small molecule that inhibits MYC expression in cell models, with minimal impact on the expression of other G4-associated genes. Here, we report an approach for the identification of G4-binding small molecules using small molecule microarrays (SMMs).
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Although molecules that bind globally to quadruplex DNA and influence gene expression are well-known, the identification of new chemical scaffolds that selectively modulate G4-driven genes remains a challenge. One attractive route to pharmacological inhibition of MYC has been the prevention of its expression through small molecule-mediated stabilization of the G-quadruplex (G4) present in its promoter. However, it has proven difficult to develop small molecule inhibitors of MYC. The transcription factor MYC plays a pivotal role in cancer initiation, progression, and maintenance.