The platelet-derived growth factor receptor β (PDGFR-β) signaling pathway is a validated and important target for the treatment of certain malignant and nonmalignant pathologies. We previously identified a G-quadruplex-forming nuclease hypersensitive element (NHE) in the human PDGFR-β promoter that putatively forms four overlapping G-quadruplexes. Therefore, we further investigated the structures and biological roles of the G-quadruplexes and i-motifs in the PDGFR-β NHE with the ultimate goal of demonstrating an alternate and effective strategy for molecularly targeting the PDGFR-β pathway. Significantly, we show that the primary G-quadruplex receptor for repression of PDGFR-β is the 3′-end G-quadruplex, which has a GGA sequence at the 3′-end. Mutation studies using luciferase reporter plasmids highlight a novel set of G-quadruplex point mutations, some of which seem to provide conflicting results on effects on gene expression, prompting further investigation into the effect of these mutations on the i-motif-forming strand. Herein we characterize the formation of an equilibrium between at least two different i-motifs from the cytosine-rich (C-rich) sequence of the PDGFR-β NHE. The apparently conflicting mutation results can be rationalized if we take into account the single base point mutation made in a critical cytosine run in the PDGFR-β NHE that dramatically affects the equilibrium of i-motifs formed from this sequence. We identified a group of ellipticines that targets the G-quadruplexes in the PDGFR-β promoter, and from this series of compounds, we selected the ellipticine analog GSA1129, which selectively targets the 3′-end G-quadruplex, to shift the dynamic equilibrium in the full-length sequence to favor this structure. We also identified a benzothiophene-2-carboxamide (NSC309874) as a PDGFR-β i-motif-interactive compound. In vitro, GSA1129 and NSC309874 downregulate PDGFR-β promoter activity and transcript in the neuroblastoma cell line SK-N-SH at subcytotoxic cell concentrations. GSA1129 also inhibits PDGFR-β-driven cell proliferation and migration. With an established preclinical murine model of acute lung injury, we demonstrate that GSA1129 attenuates endotoxin-mediated acute lung inflammation. Our studies underscore the importance of considering the effects of point mutations on structure formation from the G- and C-rich sequences and provide further evidence for the involvement of both strands and associated structures in the control of gene expression.

中文翻译：

---

血小板衍生的生长因子受体β核心启动子核酸酶超敏元件中重叠的G-四链体和i-基序的后果可以解释突变的意想不到的作用，并为小分子选择性靶向两种结构提供下调基因表达的机会。

血小板衍生的生长因子受体β（PDGFR-β）信号通路是治疗某些恶性和非恶性病理的有效且重要的靶标。我们先前在人PDGFR-β启动子中确定了一个形成G-四链体的核酸酶超敏元件（NHE），该启动子可能形成了四个重叠的G-四链体。因此，我们进一步研究了PDGFR-βNHE中G-四链体和i-基序的结构和生物学作用，其最终目的是证明分子靶向PDGFR-β途径的另一种有效策略。显着地，我们显示了用于阻遏PDGFR-β的主要G-四链体受体是3'-端G-四链体，其在3'-端具有GGA序列。使用萤光素酶报告基因质粒进行的突变研究突出了一组新的G-四链体点突变，其中一些似乎对基因表达的影响提供了矛盾的结果，促使人们进一步研究这些突变对i-基序形成链的影响。本文中，我们描述了PDGFR-βNHE的富含胞嘧啶（富含C）序列中至少两个不同的i-基序之间形成平衡的特征。如果我们考虑到在PDGFR-βNHE中关键胞嘧啶中产生的单个基点突变会显着影响由该序列形成的i-基序的平衡，那么显然矛盾的突变结果就可以合理化。我们鉴定了一组针对PDGFR-β启动子中G-四链体的玫瑰树碱，并从这一系列化合物中选择了玫瑰树碱类似物GSA1129，其选择性地针对3'-端G-四链体，使全长序列的动态平衡发生变化，从而有利于这种结构。我们还确定了苯并噻吩-2-羧酰胺（NSC309874）为PDGFR-βi-基序相互作用化合物。在体外，在亚细胞毒性细胞浓度下，GSA1129和NSC309874下调了神经母细胞瘤细胞系SK-N-SH中PDGFR-β启动子的活性和转录物。GSA1129还抑制PDGFR-β驱动的细胞增殖和迁移。建立了急性肺损伤的临床前鼠模型，我们证明GSA1129减弱了内毒素介导的急性肺部炎症。我们的研究强调了考虑点突变对富含G和C序列的结构形成的影响的重要性，并为链和相关结构参与基因表达控制提供了进一步的证据。