Splice sensors for cancer drug discovery

用于癌症药物发现的拼接传感器

基本信息

批准号：
9200532
负责人：
Balajee Somalinga
金额：
$ 22.43万
依托单位：
LUCERNA, INC.
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-07-05 至 2018-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9200532
关键词：
Affect Antineoplastic Agents Apoptosis Basic Cancer Research Basic Science Binding Biological Assay Biosensor Buffers Cancer Cell Growth Cancer cell line Cell Count Cell Proliferation Cells Chemicals Cisplatin Clinical Color Cultured Cells Defect Dependence Development Disease Drug Industry Drug Targeting Embryo Exhibits Fluorescence Goals Guidelines In Vitro Left Ligands Link Marketing Measures Messenger RNA Metabolic Methods Monitor Nature Oxidative Phosphorylation Performance Pharmaceutical Preparations Phase Play Preclinical Drug Evaluation Process Property Protein Isoforms Protein Kinase Protein Splicing Pyruvate Kinase RNA RNA Splicing RNA-Binding Proteins Reading Research Personnel Role Sampling Scientist Sensitivity and Specificity Signal Transduction Sodium Chloride Specificity Spinach - dietary System Technology Therapeutic Intervention Time Transcription Coactivator Validation Warburg Effect Work Xenograft procedure aerobic glycolysis angiogenesis anti-cancer therapeutic aptamer assay development base cancer cell commercialization design docetaxel drug candidate drug discovery fluorophore gemcitabine glucose metabolism high throughput screening improved inhibitor/antagonist knock-down mRNA Expression mRNA Precursor neoplastic cell non-oncogenic novel novel strategies novel therapeutics paralogous gene prevent prototype public health relevance research study sensor small molecule tumor tumorigenesis tumorigenic

项目摘要

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.

概括：尽管有令人信服的证据，但靶向丙酮酸激酶仍然是癌症药物发现的一个挑战。强调丙酮酸激酶同工型 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的发现工作重点是开发能够抑制 PKM2 的化合物。活性（激活剂）或非活性状态（抑制剂），要么陷入效力或特异性问题，要么已经离开 PKM2 的非糖酵解致瘤活性基本上未受影响，因此迫切需要能够发挥作用的药物。阻断 PKM2 的糖酵解和非糖酵解活性。异核 RNA 结合蛋白 (hnRNP) 和剪接因子参与 PKM2 亚型的形成一种靶向癌细胞的新方法是抑制 PKM2 mRNA 剪接以促进剪接。这会导致非致癌 PKM1 同工型，尽管这会阻止糖酵解和非糖酵解。由于缺乏 PKM2 的功能，针对 PKM2 剪接的发现工作并不存在。快速、简单且兼容 HTS 的熔接传感平台技术当前可用的方法。缓慢、费力或复杂，而且它们不允许直接监测内源剪接。在这里，我们提出了一种强大的、HTS 兼容的混合读取剪接传感器检测方法。基于经过验证的“菠菜”荧光生物传感器技术。这种拼接传感器将允许直接连接。剪接传感器将在体外细胞实验中监测内源性 PKM2 mRNA 水平。荧光强度与细胞内PKM2 mRNA的量成正比，不受其他干扰mRNA的影响例如 Lucerna 开发的 PKM1 或 PKM pre-mRNA 原型 PKM1 和 PKM2 剪接传感器。科学家们已经证明了这一概念的可行性，确立了其特殊性，并证实它是在这个项目中，我们将另外构建基于 PKM 的传感器，允许同时（多路复用）。监测同一样本中的 PKM1 和 PKM2 RNA 我们将优化原型传感器和传感器。用于 HTS 的多重传感器并开发条件测定，使其表现出高荧光、灵敏度、特异性和宽动态范围，同时显示最小的背景信号，更重要的是，这种新的 HTS。兼容的方法将直接作用于细胞裂解物，并使研究人员能够首次测量在第一阶段项目结束时，我们将以快速、可靠的方式检测内源 PKM2 mRNA 水平。将此拼接传感器商业化为检测套件在项目的下一阶段，我们将验证拼接传感器。通过进行试点验证药物筛选来进行基于 HTS 的药物发现分析。基于体外细胞的剪接传感器测定将使制药业能够开发出阻断最后，我们将利用剪接传感器平台来研究 PKM2 糖酵解和非糖酵解致瘤活性。开发一套检测方法，能够对其他疾病中的异常剪接进行药理学靶向。