【Angew.Chem.】中国药科大学王凯波、孔令义、卞金磊联手湖南大学郑克威|突破“不可成药”靶点！科学家设计新型平行 G-四联体稳定剂，实现 20 倍抗癌活性提升#

Identification of dehydroevodiamine (DEE) as a distinct KRAS-G4-binding ligand. (a) Structure of the human KRAS gene promoter (left). The guanine runs involved in forming the major KRAS-G4 are highlighted in gray. The folding topology of KRAS-G4 adopted by the Pu24m1 sequence (right). (b) Thermal stabilization (Δ_T_m) of KRAS-G4 induced by 445 compounds, measured via FRET-melting assay. The data shown are the average values from the three independent experiments. DEE is marked in orange. Conditions: 200 nM labeled DNA, 10 µM compounds, 100 mM K+, pH 7. (c) The botanical origin and chemical structure of DEE. (d) 1D 1H-NMR spectra showing the imino region of KRAS-G4 upon incremental addition of DEE. Conditions: 150 µM DNA, 50 mM K+, pH 7, 25°C. (e) Select regions of 2D-NOESY spectra of the 2:1 KRAS–G4–DEE complex showing intermolecular cross-peaks between DEE and KRAS-G4 imino protons. Conditions: 1.6 mM DNA, 10 mM K+, pH 7, 25°C. (f) Predicted binding mode of DEE to KRAS-G4 (PDB ID: 7X8O): overall binding pose (left) and detailed interaction view (right).#

Design and evaluation of DEE derivatives targeting the KRAS-G4. (a) Rational design strategy for DEE derivatives. Small substituents (e.g., fluoro, methoxy) were introduced at the two circled positions on the DEE scaffold to modulate electronic properties and steric bulk. Diverse side chains were introduced at the circled and arrowed sites on the DEE to enhance the binding affinity. (b) 1D 1H-NMR spectra showing the imino region of KRAS-G4 upon addition of 3 equivalents of DEE derivatives. Conditions: 150 µM DNA, 50 mM K+, pH 7, 25°C. (c) CD spectra (left) and CD thermal melting curves (right) of KRAS-G4 with and without compounds 7i and 7f. Conditions: 20 µM DNA, 80 µM compound, 15 mM K+, pH 7. (d) The Δ_T_m values of DEE derivatives against KRAS-G4 and antiproliferative activity of the selected derivatives. Cells were incubated with different concentrations of corresponding compounds for 3 days, with berberine as positive control. IC50 values were given as the mean ± SD (n = 3). NT means not tested.#

为了阐明这种活性激增的底层结构奥秘，研究团队利用二维核磁共振波谱法（2D NMR）成功解析了 KRAS–G4–7i 结合复合物的高分辨率溶液结构（PDB ID: 22YQ）。结构分析显示，该复合物呈现出前所未见的双重结合模式：化合物 7i 的芳香核心在 5′-端和 3′-端与外层的 G-四联体平面进行强烈的 π–π 轴向堆积，与此同时，其精心设计的侧链定向投射进由回路和四联体核心构成的沟槽区域。在 5′-端，7i 的亚氨基与 G13 的磷酸基团形成了精确的氢键；在 3′-端，其甲氧基和羟基则分别与 A23 和 G11 建立了稳固的氢键网络。这种芳香核外层堆积与侧链特异性沟槽结合的协同效应，赋予了 7i 极高的靶向选择性，使其能够选择性地结合平行拓扑结构的 G4，而对其他构型的 G4 或发卡 DNA 几乎不产生作用。

Binding selectivity of compound 7i and structure-activity relationship analysis. (a) The binding affinity of the selected compounds for KRAS-G4. (b) The selectivity of compound 7i toward different G4s and hairpin DNA. ND means not determined. (c) Structure-activity relationship analysis of DEE derivatives.#

The NMR spectra of the KRAS–G4–7i complex. (a) The H1−H8 (top) and H1−H1 (bottom) regions and (b) H1′−H6/H8 region of the KRAS–G4–7i complex in 10 mM K+ buffer from the 2D-NOESY spectrum with a sequential assignment pathway at 25°C. The missing connectivities are marked with asterisks. (c) 1D 1H-NMR spectra showing the imino region of the KRAS–G4–7i complex with the assignment at 15°C, 25°C, and 35°C. The G-tetrad imino proton signals at the 5′-end, middle, and 3′-end are marked in blue, black, and red, respectively. (d) Selected regions of 2D-NOESY spectra (35°C) of the 1:2 KRAS–G4–7i complex in H2O showing intermolecular cross-peaks between 7i and KRAS-G4 imino protons. Conditions: 1.6 mM DNA, 10 mM K+, pH 7. (e) Model diagram of the KRAS–G4–7i complex.#

NMR solution structures of the KRAS–G4–7i complex. (a) A representative refined KRAS–G4–7i complex structure is shown in a surface view. (b) Superposition of the 10 lowest-energy NMR solution structures of the KRAS–G4–7i complex. (c) A refined KRAS–G4–7i structure is depicted in cartoon representation (PDB ID: 22YQ). (d, e) Top and side views of the 5′-end and 3′-end of the KRAS–G4–7i complex. Orange, 7i; gray, guanine; violet, adenine; tv_blue, thymine; pale yellow, cytosine. Potential hydrogen bonds are shown as dashed lines.#

在细胞及生物学功能验证层面，DNA 聚合酶阻断实验（DPSA）率先证实了 7i 能够在长链基因组 DNA 序列中浓度依赖性地诱导并稳定 KRAS-G4 结构的形成。随后的定量逆转录 PCR（qRT-PCR）结果表明，7i 处理不仅显著下调了结直肠癌细胞株中 KRAS 的 mRNA 转录水平，还同步抑制了 BLM、NEIL3 和 hTERT 等多个富含 G4 启动子基因的表达。细胞免疫荧光实验进一步显示，7i 导致细胞核内 G4 特异性肽（G4P）信号大幅增强，并诱发了高密度的 γH2AX 染色质病灶，这直接证明该化合物在全基因组范围内实现了 G4 的广泛稳定，进而引发了严重的复制应激及 DNA 双链断裂。最终，在更贴近临床的患者来源结直肠癌肿瘤类器官（PDO）模型中，7i 以 4.5 µM 的半数抑制浓度（IC50）展现出强大的肿瘤生长抑制和诱导细胞死亡的能力，并再度证实了其在三维组织水平上对 KRAS 的转录抑制功效。

Effects of compound 7i on KRAS-G4-mediated biological function. (a) DNA polymerase stop assay under different concentrations of K+ and 7i (in a 5 mM K+-containing solution). (b) The KRAS mRNA levels with and without 7i treatment in HT29 and HCT116 cells for 8 h. DMSO (< 0.1%) was used as the negative control. The experiments were run in triplicate. Data are presented as mean values ± SD. (c) Representative immunofluorescence images (63×, left) of HT29 cells after treatment with 7i (10 µM) for 8 h and quantitative analysis histogram (right). (d) Representative images of colorectal cancer organoids after treatment with 7i (10 µM) for 7 days. DMSO (< 0.1%) was used as the negative control. (e) The growth area and (f) Relative organoid sizes after treatment with increasing concentrations of compound 7i. (g) The KRAS mRNA levels with 7i treatment in tumor organoids for 8 h. DMSO (< 0.1%) was used as the negative control. The experiments were run in quadruplicate. Data are presented as mean values ± SD. P values (**p < 0.01, ***p < 0.001, ****p < 0.0001) were determined relative to negative control using two-tailed Student’s t-test.#