One-compound, one-islet: A high-throughput platform for small-molecule discovery

一种化合物，一种胰岛：用于小分子发现的高通量平台

• 批准号: 10356005
• 负责人: ROHIT N. KULKARNI
• 金额: $ 76.11万
• 依托单位: BROAD INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-20 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10356005
• 关键词: Apoptosis; Autoimmune Diabetes; Beta Cell; Biological; Biological Assay; CASP3 gene; Cell Count; Cell Culture Techniques; Cell Line; Cell Proliferation; Cell Survival; Cell physiology; Cells; Cellular biology; Chemicals; Collection; Communities; Cytokine Suppression; Deoxyuridine; Ensure; Fluorescence; Glucose; Goals; Harmine; Human; Image; Inbred NOD Mice; Individual; Inflammatory; Institutes; Insulin-Dependent Diabetes Mellitus; Islet Cell; Label; Measurement; Measures; Methods; Miniaturization; Mission; Monitor; Mus; Natural regeneration; Nonesterified Fatty Acids; Outcome; Performance; Permeability; Phenotype; Physiological; Prodrugs; Proteins; Research Personnel; Resort; Resources; Rodent; S-nitro-N-acetylpenicillamine; Sampling; Ships; Signal Transduction; Specificity; System; Testing; Thymidine; Transplantation; VAMP-2; Vesicle; Zinc; analog; autoimmune pathogenesis; base; cell regeneration; cell type; cost; cytokine; endoplasmic reticulum stress; high throughput screening; in vivo; insulin secretion; islet; novel; response; screening; small molecule; tool

PROJECT SUMMARY Methods to increase or protect beta-cell mass in vivo would have a substantial impact on T1D, where beta cells are selectively targeted for autoimmune destruction. The discovery of novel small molecules represents an attractive opportunity to protect and increase beta-cell mass in vivo, as chemical compounds provide temporal control, tunability, cell permeability, and reversibility. However, current methods to identify new compounds are relatively ad hoc and small in scale. These limitations have led to compromises in the number of compounds that can be screened. Here, we propose to leverage our expertise in chemical and cell biology to develop a high- throughput platform (“one-compound, one-islet”) for small-molecule discovery that enables the use of only a single islet to test each compound. Such an advance will allow us to screen many more compounds with each islet shipment, reducing the number of donors required, and increasing scientific rigor substantially by supporting multiple donors to be tested per compound as well. We will adapt three existing readouts for this purpose. First, we have previously developed a zinc-catalyzed prodrug system for beta cell-selective compound delivery. To measure proliferation, we will extend this synthetic system to the thymidine analog 5-ethynyl-2’-deoxyuridine (EdU). A beta cell-selective EdU will reduce the background from other proliferative cells and enable us to image intact islets after small-molecule treatment. For beta-cell survival, we will measure caspase-3 activation, which is specific to beta cells after cytokine treatment. Finally, to measure insulin secretion, we have developed a novel fluorescence-based assay that will allow us to monitor insulin secretion by imaging islets. The successful outcome of this proposal is a platform of small-molecule assays and chemical tools for understanding and promoting beta-cell regeneration and survival. A key advantage of this proposal is that the phenotypic readouts have already been fully developed, and will benefit tremendously from miniaturization to single-islet format. We are committed to making this platform available to the HIRN community, enabling investigators a) to probe their own compounds in this single-islet format, or b) to use a new readout to evaluate compounds screened in this project.

项目总结 在体内增加或保护β细胞质量的方法将对T1D产生实质性影响，其中β细胞 被选择性地作为自身免疫破坏的目标。新的小分子的发现代表着一种 有吸引力的机会来保护和增加体内的β细胞质量，因为化合物提供了暂时的 可控性、可调性、细胞渗透性和可逆性。然而，目前鉴定新化合物的方法是 相对临时的，规模较小的。这些限制导致了化合物数量的妥协 这是可以筛选的。在这里，我们建议利用我们在化学和细胞生物学方面的专业知识来开发一种高度- 用于小分子发现的吞吐量平台(“一种化合物，一种胰岛”)，它只允许使用 用单个小岛来测试每种化合物。这样的进展将使我们能够筛选出更多的化合物 胰岛运输，减少所需捐赠者的数量，并通过支持以下方式大幅提高科学严谨性 每个化合物也要检测多个捐赠者。为此，我们将调整现有的三个读数。第一, 我们之前已经开发了一种锌催化的前药系统，用于β细胞选择性化合物的递送。至 测量增殖，我们将把这个合成系统扩展到胸腺嘧啶核苷类似物5-乙炔-2‘-脱氧尿苷 (EDU)。对β细胞有选择性的EDU将减少其他增殖细胞的背景，使我们能够 小分子处理后完整的胰岛。对于β细胞的存活，我们将测量caspase-3的激活，这是 在细胞因子治疗后对β细胞是特异的。最后，为了测量胰岛素的分泌，我们开发了一种新的 这将使我们能够通过对胰岛进行成像来监测胰岛素的分泌。成功者 这项提案的成果是一个小分子分析和化学工具的平台，用于理解和 促进贝塔细胞的再生和存活。这一提议的一个关键优势是表型读数 已经完全开发，并将极大地受益于从小型化到单岛格式。我们 致力于将此平台提供给Hirn社区，使调查人员a)能够调查他们的 拥有这种单岛形式的化合物，或b)使用新的读数来评估在此筛选的化合物 项目。