A Bioluminescent Assay for Direct Measurement of Sirtuin Activity in Cancer Cells

直接测量癌细胞中 Sirtuin 活性的生物发光测定法

• 批准号: 10272784
• 负责人: Michael W. Deininger
• 金额: $ 30.95万
• 依托单位: VERSITI WISCONSIN, INC.
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-10 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10272784
• 关键词: Acylation; Affinity; Animal Cancer Model; Animal Model; Animals; Apoptosis; Binding; Biological; Biological Assay; Biology; Bioluminescence; C-terminal; CRISPR/Cas technology; Cancer Model; Cell Line; Cell model; Cell physiology; Cell-Free System; Cells; Cellular Assay; Chemicals; Complement; Data; Energy Metabolism; Engineering; Environment; Enzymes; Epigenetic Process; Exhibits; Family; Generations; Hematopoietic; Histones; Human; Imaging Device; Immunodeficient Mouse; In Vitro; Knock-out; Luciferases; Lysine; Magnetic Resonance Imaging; Malignant Neoplasms; Measurement; Measures; Metabolic; Methods; Mitochondria; Modeling; Modification; Molecular Target; Morphologic artifacts; N-terminal; Niacinamide; Nicotinamide adenine dinucleotide; Nuclear Localization Signal; O-Acetyl-ADP-Ribose; Peptides; Performance; Pre-Clinical Model; Process; Proteins; Reporter; Reporting; Resistance; SIRT1 gene; Signal Transduction; Sirtuins; Solid; System; Testing; Time; Translations; Tumor Volume; Variant; acyl group; base; bioluminescence imaging; cancer cell; chemotherapy; clinical translation; complement system; deacylation; design; detection assay; drug development; drug discovery; enzyme activity; high throughput screening; imaging system; in vitro Assay; in vitro activity; in vivo; in vivo imaging; inhibitor/antagonist; knock-down; luminescence; molecular imaging; neoplastic cell; prevent; senescence; small hairpin RNA; subcutaneous; transcription factor; tumor progression; tumorigenesis

A bioluminescent assay for direct measurement of sirtuin activity in cancer cells Sirtuins are nicotinamide adenine dinucleotide (NAD+)-dependent lysine deacylases that act on histones, transcription factors, epigenetic regulators and metabolic enzymes to regulate key cellular functions such as apoptosis, senescence and energy metabolism. Depending on the cellular context, sirtuins can promote or inhibit oncogenesis, defining a critical need for methods capable of interrogating sirtuin activity in intact cells and animal models. However currently available sirtuin assays are confined to cell free systems, are prone to artifacts, and have limited sensitivity and dynamic range. The lack of assays that provide a reliable, direct and real-time readout of sirtuin activity in vitro and in vivo poses a major barrier to drug development and biological studies. To overcome the limitations of current assays we propose luciferase-based assay that reports in real- time sirtuin activity in cell free and cellular assays through generation of a bioluminescence signal by conversion of furimazine to furimamide. Our design is based on a split-NanoLuc complementation system that consists of a truncated catalytically inactive N-terminal moiety (termed 11S), which is activated by complementation with a high affinity 11-aa C-terminal peptide containing two lysine residues (K8,K9). Our premise is that acylation or other modifications of the lysine residues within the high affinity C-terminal complementation peptide inhibit its binding to the N-terminal NanoLuc moiety, preventing activation; active sirtuin enzyme removes the acyl groups, allowing for luciferase complementation and generation of a luminescence signal. Our preliminary data using SIRT5 enzyme demonstrate a robust reporter signal in cell free assays, and suggest that measurement of intracellular sirtuin activity is feasible. In Aim 1 we will adapt the split-NanoLuc assay for measuring sirtuin deacylase activity in cell free systems and determine which lysine modifications within the C-terminal complementation peptide block binding to the large NanoLuc fragment, and are removed by sirtuins 1 – 7. We will optimize assay conditions, and delineate performance parameters (sensitivity, selectivity, dynamic range, coefficient of variation). In Aim 2 we will adapt the split-NanoLuc assay for measurement of SIRT5 activity in live cells and in vivo. We will generate SIRT5 reporter cell lines, targeting the truncated NanoLuc to specific cellular compartments. We will optimize conditions for quantitative bioluminescence measurement in live cancer cells and in vivo, using CRISPR/Cas9, shRNA and inhibitors to modulate SIRT5 activity. Our design has critical advantages over current approaches: (i) Potential adaptability to any type of acyl modification; (ii) High signal- to-background ratio; (iii) Avoidance of artifact-prone chemically modified probes; (iv) Adaptability to measurement of enzyme activity in live cells and animal models. Successful completion of this project will result in the establishment of a versatile and sensitive assay for detection of sirtuin deacylase enzymatic activity in cell free systems, live cancer cells and animal models of cancer, overcoming a major barrier in the field.

直接测定癌细胞中sirtuin活性的生物发光法 Sirtuins是作用于组蛋白的依赖于烟酰胺腺嘌呤二核苷酸(NAD+)的赖氨酸脱酰酶， 转录因子、表观遗传调节因子和代谢酶来调节关键的细胞功能，如 细胞凋亡、衰老和能量代谢。根据细胞环境的不同，sirtuins可以促进或抑制 肿瘤发生，确定了对能够询问完整细胞和动物中sirtuin活性的方法的迫切需要 模特们。然而，目前可用的sirtuin检测仅限于无细胞系统，容易出现伪影，并且 具有有限的灵敏度和动态范围。缺乏能够提供可靠、直接和实时读数的分析方法 在体外和体内，sirtuin活性的缺乏对药物开发和生物学研究构成了主要障碍。 为了克服目前检测方法的局限性，我们提出了基于荧光素酶的检测方法。 通过转化产生生物发光信号在无细胞和细胞检测中的时间sirtuin活性 从呋喃咪嗪到呋喃咪胺。我们的设计基于Split-NanoLuc互补系统，该系统包括一个 被截断的催化失活的N-末端部分(称为11S)，通过与a互补而激活 含有两个赖氨酸残基(K8，K9)的高亲和力11-AA C端肽。我们的前提是酰化或 高亲和力C末端互补肽中赖氨酸残基的其他修饰抑制其 结合到N-末端的NanoLuc部分，防止激活；活性的sirtuin酶去除酰基， 允许荧光素酶的互补和发光信号的产生。我们的初步数据使用 SIRT5酶在无细胞分析中显示出一个强大报告信号，并建议测量 胞内sirtuin活性是可行的。在目标1中，我们将采用Split-NanoLuc法来测量sirtuin 在无细胞系统中的脱酰基酶活性，并确定C末端的哪些赖氨酸修饰 互补多肽阻断与大的NanoLuc片段的结合，并被sirtuins 1-7去除。 将优化分析条件，并描述性能参数(灵敏度、选择性、动态范围、 变异系数)。在目标2中，我们将采用Split-NanoLuc法来测量活体中SIRT5的活性 细胞和体内。我们将产生SIRT5报告细胞系，靶向特定细胞的截短的NanoLuc 车厢。我们将优化活癌细胞生物发光定量测量的条件 在体内，使用CRISPR/Cas9、shRNA和抑制剂来调节SIRT5的活性。我们的设计具有关键的 与目前方法相比的优势：(I)对任何类型的酰基修饰的潜在适应性；(Ii)高信号- 背景比；(3)避免易产生伪影的化学修饰探针；(4)适应 活细胞和动物模型中酶活性的测量。该项目的成功完成将导致 建立一种通用、灵敏的检测细胞中sirtuin脱酰基酶活性的方法 自由系统、活癌细胞和癌症动物模型，克服了该领域的一大障碍。