Targeting the c-Kit Promoter G-quadruplexes with 6-Substituted Indenoisoquinolines

Abstract

Herein, we demonstrate the design, synthesis, biophysical properties, and preliminary biological evaluation of 6-substituted indenoisoquinolines as a new class of G-quadruplex stabilizing small molecule ligands. We have synthesized 6-substituted indenoisoquinolines 1a-e in two steps from commercially available starting materials with excellent yields. The G-quadruplex stabilization potential of indenoisoquinolines 1a-e was evaluated by fluorescence resonance energy transfer-melting analysis, which showed that indenoisoquinolines show a high level of stabilization of various G-quadruplex DNA structures. Indenoisoquinolines demonstrated potent inhibition of cell growth in the GIST882 patient-derived gastrointestinal stromal tumor cell line, accompanied by inhibition of both c-Kit transcription and KIT oncoprotein levels.KEYWORDS DNA, quadruplex, c-kit regulation, inhibition, ligand G -quadruplexes constitute a structural form of DNA that is distinct from the double helix. 1 They are formed from particular guanine-rich nucleic acid sequences in which guanines form planar quartets stabilized using the Watson-Crick and Hoogsteen edges to form hydrogen bonds. Their occurrence has been demonstrated within ciliate telomeres. 2,3 In addition, G-quadruplex sequence motifs (G 3þ N 1-7 G 3þ N 1-7 G 3þ N 1-7 G 3þ ) are found at many positions within genomes, 4,5 with a notable increase in density in close vicinity to transcriptional start sites, highly suggestive of a biological function in gene regulation. 5,6 NMR spectroscopy and X-ray crystallographical studies have shown that these guanine-rich sequences can fold into G-quadruplex structures in vitro. 7,8 Their unique structural features and possible biological functions make them attractive targets for drug design. 9,10 Genomic locations that are known to possess quadruplex structures include the immunoglobulin switch region and the promoters of numerous proto-oncogenes, such as c-MYC, 11 VEGF, 12 BCL-2, 13 and K-RAS. 14 Two quadruplex-forming motifs have been identified in the c-Kit promoter (c-Kit 1 15,16 and c-Kit 2 17,18 ). Here, we present a new class of quadruplex stabilizing compounds that have been used to explore relationships between quadruplexes and their biological functions.c-Kit is a proto-oncogene that codes for a 145-160 kDa protein belonging to the receptor tyrosine kinase (RTK) family. The KIT protein regulates signal transduction cascades that control cell growth and differentiation. 19 c-Kit expression has been shown to control the growth of various cell lines, including gastrointestinal stromal tumors (GIST), hematopoietic cells, small cell lung cancer, and colorectal cancer. [20][21][22][23] Gain-of-function mutations in the KIT protein are found in various highly malignant human cancers, especially GIST. Gleevec (imatinib mesylate), a small molecule antagonist originally developed to treat chronic myelogenous leukemia (CML), acts by maintaining the bcr-abl kinase in an inactive conformation. Gleevec is also the mainstay of target therapy ...

The C-Kit Receptor-Mediated Signal Transduction and Tumor-Related Diseases

“…To date, the research on the small molecule ligands targeting the c-kit gene promoter region attracts more attention. For example it was reported that 6-substituted indenoisoquinoline 51 (Fig. 5A), benzo[α]phenoxazine derivatives 52 (Fig.…”

Synthesis and Evaluation of Quinazolone Derivatives as a New Class of c-KIT G-Quadruplex Binding Ligands

“…15,16 These rigid ligands comprise a planar, aromatic core with the capability of stacking on the G-quartet. Some flexible ligands with unfused aromatic scaffolds, such as bisindole carboxamides and diarylethynyl amides, 20,21 are also found to bind to the c-KIT G-quadruplex DNA in vitro.…”

“…14,15 The unfused aromatic ring system seems too feasible and nonplanar to effectively stack on the G-quartet and stabilize the G-quadurplex DNA. In addition, Figure 2.…”

Mechanism of the Antiproliferative Activity of Some Naphthalene Diimide G-Quadruplex Ligands

“…In theory, oncogenes associated with all six hallmarks of cancer (Hanahan and Weinberg, 2011) can be targeted with G-quadruplex ligands. G-quadruplex ligands can inhibit c-kit expression and activity in a c-kitexpressing gastrointestinal stromal tumor cell line (Gunaratnam et al, 2009;Bejugam et al, 2010;McLuckie et al, 2011). The G-quadruplex ligand quarfloxin decreases RNA polymerase I transcription by inhibiting nucleolin binding to ribosomal DNA G-quadruplexes in the nucleoli (Drygin et al, 2011).…”