Regulation of c-Kit gene transcription selectively by bisacridine derivative through promoter dual i-motif structures

c-Kit protein is a signal transduction protein involved in multiple signal pathways, which play an important role in a variety of cellular events such as cell proliferation, apoptosis and differentiation. Special DNA secondary structures on the promoter of c-Kit gene, including G-quadruplex and i-motif structures, could act as “molecular switch” for gene transcriptional regulation, which are potentially important target for development of new anti-cancer drugs. We screened and evaluated the effect of compounds on c-Kit through several experiments, including SPR, FRET, CD, MST, NMR, dual-luciferase reporter assay, Western blot, qPCR, immunofluorescence, MTT assay, colony formation, cell scrape, cell apoptosis, cell cycle analysis, and transwell assay. After extensive screening, we found that bisacridine derivative B05 had selective binding and stabilization to dual i-motif structures on c-Kit gene promoter, which could down-regulate c-Kit gene transcription and translation, resulting in inhibition of cell proliferation and metastasis. B05 exhibited potent anti-tumor activity on HGC-27 cells, and strongly suppressed tumor growth in HGC-27 xenograft mice model. B05 could interact with c-Kit promoter dual i-motif structures with excellent selectivity, which make it possible for selective regulation of gene transcription and translation. B05 could be further developed for selective anti-cancer agent targeting c-Kit promoter i-motifs. i-Motifs on different proto-oncogene promoters are diversified, and especially binding of dual i-motifs on the same promoter simultaneously could significantly down-regulate gene transcription with decreased dosage, and therefore increasing the selectivity. This new strategy shed bight light on development of selective DNA-targeting ligands.