Abstract
Tumor angiogenesis is mainly regulated by vascular endothelial growth factor (VEGF) produced by cancer cells. It is active on the endothelium via VEGF receptor 2 (VEGFR-2). G-quadruplexes are DNA secondary structures formed by guanine-rich sequences, for example, within gene promoters where they may contribute to transcriptional activity. The proximal promoter of VEGFR-2 contains a G-quadruplex, which has been suggested to interact with small molecules that inhibit VEGFR-2 expression and thereby tumor angiogenesis. However, its structure is not known. Here, we determined its NMR solution structure, which is composed of three stacked G-tetrads containing three syn guanines. The first guanine (G1) is positioned within the central G-tetrad. We also observed that a noncanonical, V-shaped loop spans three G-tetrad planes, including no bridging nucleotides. A long and diagonal loop, which includes six nucleotides, connects reversal double chains. With a melting temperature of 54.51 °C, the scaffold of this quadruplex is stabilized by one G-tetrad plane stacking with one nonstandard bp, G3-C8, whose bases interact with each other through only one hydrogen bond. In summary, the NMR solution structure of the G-quadruplex in the proximal promoter region of the VEGFR-2 gene reported here has uncovered its key features as a potential anticancer drug target.
Highlights
Tumor angiogenesis is mainly regulated by vascular endothelial growth factor (VEGF) produced by cancer cells
VEGF-A is considered to be the most important factor implicated in tumor angiogenesis [13] through binding with high affinity to tyrosine kinase receptors expressed on the surface of endothelial cells such as VEGF receptor 1 (VEGFR-1) and VEGF receptor 2 (VEGFR-2), the receptor functionally more relevant in the transduction of proangiogenic stimuli coming from tumor cells (14 –16)
The interactions between VEGF-A and VEGFR-2 lead to receptor autophosphorylation, and phosphorylated tyrosine residues activate signaling cascades, eventually resulting in cellular processes involved in angiogenesis [17] such as vesicle trafficking, cytoskeleton regulation, microtubule dynamics, cell polarity, and membrane transport [18]
Summary
A putative G-quadruplex structure in the proximal promoter of VEGFR-2 has implications for drug design to inhibit tumor angiogenesis. The NMR solution structure of the G-quadruplex in the proximal promoter region of the VEGFR-2 gene reported here has uncovered its key features as a potential anticancer drug target. A guanine-rich sequence was found within the proximal promoter region of VEGFR-2 and was suggested to form an antiparallel G-quadruplex structure [31]. This G-quadruplex structure can be efficiently stabilized by the small molecule quarfloxin, which has progressed in clinical trials. We report the NMR solution structure of a unique G-quadruplex formed in the proximal promoter region of VEGFR-2 that provides the molecular details of this G-quadruplex and important insights into its loop conformations and interactions with core tetrad structures
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