Background

The transcription-inhibitory G-Quadruplex(Pu27-GQ) at c-MYC promoter is challenging to target due to structural heterogeneity. Nucleoside diphosphate kinase (NM23-H2) specifically binds and unfolds Pu27-GQ to increase c-MYC transcription. Here, we used Inosine 5′-diphosphate (IDP) to disrupt NM23-H2-Pu27-GQ interactions and arrest c-MYC transcription without compromising NM23-H2-mediated kinase properties.

Methods

Site-directed mutagenesis,³¹P‐NMR and STD-NMR studies delineate the epitope of NM23-H2-IDP complex and characterize specific amino acids in NM23-H2 involved in Pu27-GQ and IDP interactions. Immunoprecipitations and phosphohistidine-immunoblots reveal how IDP blocks NM23-H2-Pu27 association to downregulate c-MYC transcription in MDAMB-231 cells exempting NM23-H2-mediated kinase properties.

Results

NMR studies show that IDP binds to the Guanosine diphosphate-binding pocket of NM23-H2 (K_D = 5.0 ± 0.276 μM). Arg88-driven hydrogen bonds to the terminal phosphate of IDP restricts P–O–P bond-rotation increasing its pKa (∆pKa = 0.85 ± 0.0025).9-inosinyl moiety of IDP is stacked over Phe60 phenyl ring driving trans-conformation of inosine and axial geometry of pyrophosphates. Chromatin immunoprecipitations revealed that these interactions rescue NM23-H2-driven Pu27-GQ unfolding, which triggers Nucleolin recruitment and lowers Sp1 occupancy at c-MYC promoter stabilizing Pu27-GQ. This silences c-MYC transcription that reduces c-MYC-Sp1 association amplifying Sp1 recruitment across P21 promoter stimulating P21 transcription and G₂/M arrest.

Conclusions

IDP synergizes the effects of Pu27-GQ-interacting compounds to abrogate c-MYC transcription and induce apoptosis in MDAMB-231 cells by disrupting NM23-H2-Pu27-GQ interactions without affecting NM23-H2-mediated kinase properties.

General significance

Our study provides a pragmatic approach for developing NM23-H2-targeting regulators to rescue NM23-H2 binding at structurally ambiguous Pu27-GQ that synergizes the anti-tumorigenic effects of GQ-based therapeutics with minimized off-target effects.

中文翻译：

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肌苷5'-二磷酸肌醇，从c-MYC G-Quadruplex展开中拯救核苷二磷酸激酶。

背景

由于结构异质性，c-MYC启动子上的转录抑制性G-四联体（Pu27-GQ）具有挑战性。核苷二磷酸激酶（NM23-H2）特异性结合并展开Pu27-GQ以增加c-MYC转录。在这里，我们使用肌苷5'-二磷酸（IDP）来破坏NM23-H2-Pu27-GQ相互作用并阻止c-MYC转录，而不会损害NM23-H2介导的激酶特性。

方法

定点诱变，³¹ P-NMR和STD-NMR研究描绘了NM23-H2-IDP复合物的表位，并表征了参与Pu27-GQ和IDP相互作用的NM23-H2中的特定氨基酸。免疫沉淀和磷酸组氨酸免疫印迹揭示了IDP如何阻止NM23-H2-Pu27缔合以下调MDAMB-231细胞中的c-MYC转录，从而使NM23-H2介导的激酶特性不受限制。

结果

NMR研究表明IDP与NM23-H2的鸟苷二磷酸结合袋结合（K _D  = 5.0±0.276μM）。Arg88驱动的氢键与IDP的末端磷酸结合，限制了P–O–P键的旋转，从而增加了其pKa（∆pKa = 0.85±0.0025）。IDP的9-肌苷部分堆叠在Phe60苯环上，驱动肌苷的反式构象和焦磷酸盐的轴向几何形状。染色质的免疫沉淀显示，这些相互作用可挽救NM23-H2驱动的Pu27-GQ的展开，从而触发Nucleolin募集并降低c-MYC启动子稳定Pu27-GQ的Sp1占有率。这使c-MYC转录沉默，该转录降低了c-MYC-Sp1关联，从而在刺激P21启动子的过程中扩增了Sp1募集。P21转录和G ₂ / M逮捕。

结论

IDP通过破坏NM23-H2-Pu27-GQ相互作用而不影响NM23-H2介导的激酶特性，协同Pu27-GQ相互作用的化合物消除c-MYC转录并诱导MDAMB-231细胞凋亡的作用。

一般意义

我们的研究为开发靶向NM23-H2的调节剂以在结构模糊的Pu27-GQ上抢救NM23-H2结合提供了务实的方法，该协同作用使基于GQ的治疗剂的抗肿瘤发生作用协同作用，并使脱靶作用最小化。