Structural insight into the bulge-containing KRAS oncogene promoter G-quadruplex bound to berberine and coptisine

Abstract

KRAS is one of the most highly mutated oncoproteins, which is overexpressed in various human cancers and implicated in poor survival. The G-quadruplex formed in KRAS oncogene promoter (KRAS-G4) is a transcriptional modulator and amenable to small molecule targeting. However, no available KRAS-G4-ligand complex structure has yet been determined, which seriously hinders the structure-based rational design of KRAS-G4 targeting drugs. In this study, we report the NMR solution structures of a bulge-containing KRAS-G4 bound to berberine and coptisine, respectively. The determined complex structure shows a 2:1 binding stoichiometry with each compound recruiting the adjacent flacking adenine residue to form a “quasi-triad plane” that stacks over the two external G-tetrads. The binding involves both π-stacking and electrostatic interactions. Moreover, berberine and coptisine significantly lowered the KRAS mRNA levels in cancer cells. Our study thus provides molecular details of ligand interactions with KRAS-G4 and is beneficial for the design of specific KRAS-G4-interactive drugs.

Structure of the Major G-Quadruplex in the Human EGFR Oncogene Promoter Adopts a Unique Folding Topology with a Distinctive Snap-Back Loop

“…We thus collected a set of 2D NMR spectra for Pu26m1T DNA ( EGFR -G4), including NOESY, DQF-COSY, and 1 H- 13 C HSQC, at different mixing times and temperatures in a K + -containing solution (Figure and Figures S3–S7). The complete assignment of imino, aromatic, and sugar resonances in Pu26m1T DNA was accomplished using well-established protocols ,, that followed a parallel G4 topology , (Figure and Table S2). The through-space H1–H8 NOE cross-peaks determined the formation of a core of three G-tetrad planes: G4–G8–G12–G16, G5–G9–G13–G17, and G6–G24–G14–G18 (Figures b and b).…”

“…The G4 structure formed in the DNA template strand can stall the DNA polymerase in synthesizing the complementary strand DNA (Figure a) . In order to determine whether the snap-back EGFR -G4 can form in a longer DNA context and fulfill potential biological activity, we performed a DNA polymerase stop assay using a wild-type EGFR -G4-containing DNA template (Figure a).…”

“…The presence of a G4-stabilizing ligand has been shown to increase the formation of G4-mediated stalled products. , To investigate this, we utilized pyridostatin (PDS), a well-known G4-stabilizing compound, in our DNA polymerase stop assay. The binding activity of PDS to EGFR -G4 was first confirmed through CD and NMR titration experiments, which demonstrated that PDS is a potent binder to EGFR -G4 with a Δ T m of 25 °C while keeping the G4 topology unchanged (Figures S12 and S13).…”

“…Compared to EGFR TKIs for direct oncoprotein binding, targeting DNA G-quadruplex (G4) structures at oncogene proximal promoters for transcription inhibition represents an alternative approach for selective EGFR downregulation. − G4s are four-stranded nucleic acid secondary structures formed by the stacking of two or more G-tetrads with Hoogsteen hydrogen bonds and stabilized by K + or Na + that centrally coordinated with O6 of the tetrad guanines. − DNA G4 structures have been visualized in human cells, and endogenous G4s are enriched in the proximal promoters of highly expressed cancer genes. − The genome functions of G4s are implicated in gene transcription, replication, translation, genome instability, and epigenetic regulation. ,− Stabilizing DNA G4 structures at MYC , , KRAS , , c- KIT , , VEGF, , PDGFR- β, − and many other oncogene promoters ,− with G4-specific small molecules leads to oncogene transcription repression and, subsequently, cancer cell death, suggesting that proximal promoter G4s are a promising biomolecular target for cancer therapy. ,, …”

Effects of Molecular Crowding on the Structure, Stability, and Interaction with Ligands of G-quadruplexes

“…Hoogsteen hydrogen bonds stabilize the G4 structures in the presence of monovalent cations such as K + and Na + . − G4-forming sequences are widely distributed in the genome, and G-rich DNA sequences always exist in eukaryotic telomeres and promoter gene regions such as c-MYC , c-KIT , KRAS , BCl2 , VEGF , etc. The formation and unwinding of G4 structures significantly impact several biological events, including gene replication, recombination, and translation. − Small molecules targeting the telomeric G4 can inhibit telomere lengthening by affecting telomerase activity, leading to the inhibition of tumor cell growth. , In addition, small molecules can act on the promoter region of proto-oncogenes, inhibiting the binding of proteins involved in regulating transcription to the promoter region and thereby suppressing gene expression. Therefore, G4s on proto-oncogenes can also be used as targets for screening antitumor drugs. − …”