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: 10450745
• 负责人: ROHIT N. KULKARNI
• 金额: $ 74.66万
• 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/10450745
• 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.

项目概要 增加或保护体内 β 细胞量的方法将对 T1D 产生重大影响，其中 β 细胞 选择性地针对自身免疫破坏。新型小分子的发现代表了 保护和增加体内 β 细胞质量的诱人机会，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 相对临时性且规模较小。这些限制导致了化合物数量的妥协 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 用于小分子发现的吞吐量平台（“单一化合物，单一胰岛”），仅使用 单个胰岛来测试每种化合物。这样的进步将使我们能够筛选更多的化合物 胰岛运输，减少所需捐赠者的数量，并通过支持大幅提高科学严谨性 每种化合物也需要测试多个供体。为此，我们将调整三个现有读数。第一的， 我们之前开发了一种用于β细胞选择性化合物递送的锌催化前药系统。到 为了测量增殖，我们将把这个合成系统扩展到胸苷类似物 5-乙炔基-2'-脱氧尿苷 （教育大学）。 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 caspase-3 激活，这 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，我们开发了一种新型的 基于荧光的测定使我们能够通过对胰岛进行成像来监测胰岛素分泌。成功者 该提案的成果是一个小分子测定和化学工具平台，用于理解和 促进β细胞再生和存活。该提案的一个关键优点是表型读数 已经完全开发出来，并将从小型化到单岛格式中受益匪浅。我们 致力于向 HIRN 社区提供该平台，使调查人员能够 a) 探究他们的 自己的单胰岛格式化合物，或 b) 使用新的读数来评估在此筛选的化合物 项目。

项目成果

Phenotypic Screening for Small Molecules that Protect β-Cells from Glucolipotoxicity.

• DOI: 10.1021/acschembio.2c00052
• 发表时间: 2022-05-20
• 期刊: ACS CHEMICAL BIOLOGY
• 影响因子: 4
• 作者: Small, Jonnell C.;Joblin-Mills, Aidan;Carbone, Kaycee;Kost-Alimova, Maria;Ayukawa, Kumiko;Khodier, Carol;Dancik, Vlado;Clemons, Paul A.;Munkacsi, Andrew B.;Wagner, Bridget K.
• 通讯作者: Wagner, Bridget K.