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.

Fig. 1. KRAS-G4 and its interaction with berberine and coptisine.

a Schematic of the human KRAS gene promoter and the formation of a KRAS-G4 as well as its complex with small molecules (ligands). The G4-forming region of the NHE sequence is shown. The G-tracts implicated in KRAS-G4 formation are marked in red and mutations in cyan, respectively. b Chemical structures of berberine and coptisine with numbering. c 1D ¹H NMR titration of Pu24m1 DNA with berberine and coptisine, respectively, with complete imino proton assignment. Conditions: 150 μM DNA, pH 7, 50 mM K⁺ solution, 25 °C, DMSO-d₆ < 1.5%.

Fig. 2. Biophysical characterization of berberine and coptisine binding to the KRAS-G4.

a CD thermal melting curves and CD spectra of Pu24m1 DNA with berberine and coptisine, respectively. Conditions: 20 μM DNA, pH 7, 15 mM K⁺ solution. b Fluorescence intensity change of berberine and coptisine upon titration with Pu24m1 DNA, respectively. Conditions: 0.2 μM compound, pH 7, 50 mM K⁺ solution. c The determined ΔT_m and K_d values of berberine and coptisine to the Pu24m1 DNA. The melting temperature (T_m) was obtained at the intersection between the median of the fitted baselines and the melting curve. The dissociation constant (K_d) was calculated by data fitting with a 2:1 binding equation. The experiments were run in duplicate.

Fig. 3. 2D NMR spectra of the free KRAS-G4.

a The H1–H1 and H1–H8 regions, and b H1′-H6/H8 region from the 2D-NOESY spectra of Pu24m1 DNA in H₂O with sequential assignment pathway. Missing connectivity is marked with red asterisks. The cross-peak assigned to the minor species is labeled in blue. c H6 − C6/H8−C8 cross-peaks of all bases (black label) and the adenine H2-C2 contacts (red label) with assignments for the Pu24m1 DNA by HSQC experiments. d The assigned three G-tetrad planes of KRAS-G4 by NMR experiments. Conditions: 1.5 mM DNA, pH 7, 50 mM K⁺ solution, 25 °C, DMSO-d₆ < 3.5%.

Fig. 4. NMR solution structures of KRAS-G4 and its complexes with berberine and coptisine.

Superposition of the ten lowest energy NMR structures of the free KRAS-G4 (a), berberine-KRAS-G4 (b), and coptisine-KRAS-G4 (c) by NOE-restrained structure calculations. Yellow, berberine; green, coptisine; cyan, flanking guanine; gray, tetrad guanine; magenta, adenine; blue, thymine; orange, cytosine.

Fig. 5. Solution structure details of KRAS-G4 and its complexes with berberine and coptisine.

Cartoon representation and 5′-end and 3′-end top views of the free KRAS-G4 (a), the berberine-KRAS-G4 (b), and the coptisine-KRAS-G4 (c) complexes (Protein Data Bank IDs: 7X8N, 7X8M, and 7X8O). Yellow, berberine; green, coptisine; cyan, flanking guanine; gray, tetrad guanine; magenta, adenine; blue, thymine; orange, cytosine. Potential hydrogen bonds are shown as dashed lines.

Fig. 6. 2D NMR spectra of KRAS–G4 in complex with berberine and coptisine.

Select regions of the 2D-NOESY spectra of 3:1 berberine-KRAS-G4 (a) and 2.5 :1 coptisine-KRAS-G4 (b) complexes in H₂O showing intermolecular cross-peaks between compound and DNA imino protons. Conditions: 1.5 mM Pu24m1 DNA, pH 7, 50 mM K⁺ solution, 35 °C, DMSO-d₆ < 3.5%.

Fig. 7. Functional characterization of KRAS-G4.

a Taq DNA polymerase stop assay using a DNA template containing a wt human KRAS-G4 forming sequence shows that berberine and coptisine can stabilize the KRAS-G4 for replication inhibition. b qRT-PCR results show that berberine (24 µM) and coptisine (10 µM) lower KRAS mRNA levels in both H460 and A549 cancer cells for 24 h. DMSO (<0.1 %) was used as the negative control (no inhibition, 100%). The relative KRAS mRNA levels were normalized with GAPDH. The experiments were run in triplicate. Data are presented as mean values ± SD. P values (**P < 0.01, ***P < 0.001, ****P < 0.0001) were determined by one-way ANOVA with post hoc Dunnett, relative to DMSO control.

Fig. 8. Suggested modifications of berberine enabling additional interaction with the KRAS-G4.

The black circles indicate the positions to be modified by introducing side chains for groove interactions. The black arrows show the grooves at 5′- and 3′-sites where the attached side chain will locate. The red circle suggests the positions to introduce hydrogen bonds between berberine and thymine T10. The KRAS-G4 is shown in cartoon representation. Cyan, guanine; magenta, adenine; blue, thymine; orange, cytosine; yellow, berberine.