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.

项目摘要 增加或保护体内β细胞群的方法将对T1 D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了 在体内保护和增加β-细胞质量的有吸引力的机会，因为化合物提供了暂时的 控制、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 相对临时的和规模小的。这些限制导致了化合物数量的妥协 可以被筛选。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，开发一种高- 用于小分子发现的通量平台（“一种化合物，一种胰岛”）， 单个胰岛以测试每种化合物。这样的进步将使我们能够筛选更多的化合物， 胰岛运输，减少所需的捐赠者数量，并通过支持大幅提高科学严谨性， 每种化合物也要测试多个供体。为此，我们将调整现有的三个读数。第一、 我们先前已经开发了用于β细胞选择性化合物递送的锌催化的前药系统。到 为了测量增殖，我们将这个合成系统扩展到胸苷类似物5-乙炔基-2 '-脱氧尿苷 （EdU）.β细胞选择性EdU将减少其他增殖细胞的背景，使我们能够成像 小分子处理后的完整胰岛。对于β细胞存活，我们将测量半胱天冬酶-3活化， 对细胞因子处理后的β细胞具有特异性。最后，为了测量胰岛素分泌，我们开发了一种新的 基于荧光的分析，这将使我们能够通过成像胰岛来监测胰岛素分泌。成功 该提案的成果是一个小分子测定和化学工具的平台， 促进β细胞再生和存活。该提议的一个关键优势是， 已经得到了充分的发展，并将大大受益于小型化到单胰岛格式。我们 致力于为HIRN社区提供这个平台，使调查人员能够a）调查他们的 以这种单胰岛形式筛选自己的化合物，或B）使用新的读数来评估在这种单胰岛形式中筛选的化合物， 项目