Splice sensors for cancer drug discovery

摘要： 靶向丙酮酸激酶在抗癌药物发现中仍然是一个挑战。尽管有令人信服的证据 强调了丙酮酸激酶亚型M2(PKM2)在癌细胞生长和增殖中的重要性， 针对PKM2糖酵解活性的药物发现工作尚未产生成功的候选药物 临床应用。发现工作的重点是开发化合物，使PKM2在 活性(激活剂)或非活性状态(抑制剂)，要么深陷效力或特异性问题，要么已经离开 PKM2的非糖酵解致瘤活性基本未受影响。人们迫切需要一种能够 阻断PKM2的糖酵解和非糖酵解活性。最近的证据表明，各种 异核RNA结合蛋白(HnRNPs)和剪接因子参与了PKM2亚型的形成 在癌细胞中。靶向癌细胞的一种新方法是抑制PKM2 mRNA的剪接，而不是剪接 这导致了非致癌的PKM1亚型。尽管这会阻止糖酵解和非糖酵解 PKM2的功能，针对PKM2剪接的发现努力是不存在的。这种匮乏是由于缺乏商品。 快速、简单且与HTS兼容的拼接传感平台技术。目前可用的方法 是缓慢的、费力的或复杂的。此外，它们不允许直接监控内源性剪接 在细胞中形成的信使核糖核酸。在这里，我们提出了一种稳健的、与HTS兼容的混合读取剪接传感器检测方法 是基于一种经过验证的“菠菜”荧光生物传感器技术。这种拼接传感器将允许直接 在基于细胞的体外实验中监测内源性PKM2的mRNA水平。拼接传感器将产生 荧光与细胞内PKM2的mRNA量成正比，不受其他干扰mRNAs的影响 如PKM1或PKM Pre-mRNA。Lucerna公司开发的PKM1和PKM2剪接传感器原型 科学家们已经证明了这一概念的可行性，建立了它的特殊性，并证实了它是 可调的。在这个项目中，我们还将构建基于PKM的传感器，允许同时(多路传输) 监测同一样本中PKM1和PKM2的RNA。我们将对原型传感器和 用于HTS的多路传感器，并开发测试条件，以使它们表现出高荧光、灵敏度 特异性和较宽的动态范围，同时显示最小的背景信号。更重要的是，这款新的HTS Compatible方法将直接在细胞裂解物上工作，并使研究人员能够首次测量 内源性PKM2信使核糖核酸快速、可靠地表达。在第一阶段项目结束时，我们将 将这种拼接传感器作为检测试剂盒进行商业化。在项目的下一阶段，我们将验证拼接传感器 通过进行中试验证药物筛选，对基于HTS的药物发现进行化验。智能手机的HTS改编版 基于细胞的体外剪接传感器分析将使制药业能够开发出阻止 PKM2糖酵解和非糖酵解致癌活性。最后，我们将利用Splice传感器平台 开发一套分析方法，使其他疾病中异常剪接的药物靶向成为可能。

SUMMARY: Targeting pyruvate kinase remains a challenge in cancer drug discovery. Despite compelling evidence highlighting the importance of the pyruvate kinase isoform M2 (PKM2) in cancer cell growth and proliferation, drug discovery efforts targeting the glycolytic activities of PKM2 have yet to yield a successful drug candidate for clinical use. Discovery efforts that are focused on developing chemical compounds that keep PKM2 arrested in an active (activators) or inactive state (inhibitors), are either mired in potency or specificity issues, or have left the non-glycolytic tumorigenic activity of PKM2 largely untouched. There is a critical need for drugs that can block both the glycolytic and non-glycolytic activities of PKM2. Recent evidence has shown that various heteronuclear RNA binding proteins (hnRNPs) and splicing factors are involved in formation of the PKM2 isoform in cancer cells. A novel approach to target cancer cells is to suppress PKM2 mRNA splicing in favor of splicing that leads to the non-oncogenic PKM1 isoform. Although this would prevent both glycolytic and non-glycolytic functions of PKM2, discovery efforts targeting PKM2 splicing are non-existent. This paucity is due to lack of good splice sensing platform technologies that are fast, simple, and HTS compatible. The currently available methods are slow, laborious, or complicated. Moreover, they do not allow direct monitoring of endogenously spliced mRNA that forms in cells. Here, we propose a robust, HTS compatible, mix-and-read splice sensor assay that is based on a proven “Spinach” fluorescence biosensor technology. This splice sensor would allow direct monitoring of endogenous PKM2 mRNA levels in in vitro cell-based experiments. The splice sensor will produce fluorescence proportional to the amount of PKM2 mRNA in cells and not be affected by other interfering mRNA such as PKM1 or the PKM pre-mRNA. Prototype PKM1 and PKM2 splice sensors developed by Lucerna scientists have demonstrated the feasibility of this concept, established its specificity, and confirmed that it is tunable. In this project, we will additionally construct PKM-based sensors that allow simultaneous (multiplexed) monitoring of PKM1 and PKM2 RNA in the same sample. We will optimize both the prototype sensors and the multiplexed sensors for HTS and develop assay conditions such that they exhibit high fluorescence, sensitivity, specificity and broad dynamic range while showing minimal background signal. More importantly, this new HTS compatible method will work directly on cell lysates and enable researchers for the first time to measure the endogenous PKM2 mRNA levels in a fast, and reliable way. At the end of this phase I project, we will commercialize this splice sensor as assay kits. In the next phase of the project, we will validate the splice sensor assay for HTS-based drug discovery by performing a pilot validation drug screen. The HTS adaptation of the in vitro cell-based splice sensor assay will enable the pharmaceutical industry to develop new drugs that block the PKM2 glycolytic and non-glycolytic tumorigenic activities. Lastly, we will leverage the splice sensor platform to develop a suite of assays that would enable the pharmacologic targeting of aberrant splicing in other diseases.