High-Throughput Droplet-Scale Functional Screening of DNA-Encoded Combinatorial Libraries

DNA 编码组合文库的高通量液滴规模功能筛选

• 批准号: 10004373
• 负责人: Brian M Paegel
• 金额: $ 19.39万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2021-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10004373
• 关键词: Academia; Architecture; Automation; Bacteria; Biochemical; Biological Assay; Biological Process; Cathepsins; Cell physiology; Cleaved cell; Code; Collection; Complement; Consumption; DNA; Data; Devices; Dose; Drug Industry; Equipment; Exhibits; FDA approved; Face; Fluorescence; Frequencies; Goals; Government; HIV-1 protease; High-Throughput Nucleotide Sequencing; In Vitro; Incubated; Individual; Industrialization; Informatics; Infrastructure; Label; Laboratories; Libraries; Location; Logistics; Microfluidics; Microscopy; Miniaturization; Mission; Modeling; Molecular; Molecular Bank; Multi-Drug Resistance; Nelfinavir; Optics; Output; Pepstatins; Peptide Hydrolases; Phase; Phenotype; Process; Protease Inhibitor; Proteome; PubChem; Quality Control; Reaction; Reagent; Renin; Resistance; Resources; Robotics; Ships; Solid; Sorting - Cell Movement; Source; Structure; Structure-Activity Relationship; Technology; Translating; Triage; United States National Institutes of Health; Validation; Viral; Viral Cell Proliferation; Virus; base; combinatorial; cost; data acquisition; diphenyl; drug discovery; genome sequencing; high throughput screening; human disease; human pathogen; inhibitor/antagonist; instrument; instrumentation; member; miniaturize; mutant; new therapeutic target; novel therapeutics; operation; pathogen; portability; programs; prototype; reconstitution; response; screening; small molecule; square foot; statine; tool

Project Summary / Abstract The NIH established the Molecular Libraries Program (MLP) and its network of high-throughput screening (HTS) centers to discover probes – highly selective small molecules that modulate cellular function – within the proteomes of humans and pathogens. Probes are not only tools for studying biological function to validate new drug targets, but are also potential leads for new therapeutics. Though successful in its mission to provide HTS resources to academia and having generated hundreds of probes, the MLP is sunsetting and its operational screening centers face the logistic and financial issues that industrial HTS centers have battled for decades (large facilities, costly robotic handling and optical screening equipment upkeep, static compound libraries). Proteome-wide and pathogen-wide probe discovery remains a compelling goal well within reach thanks to distributed and economical genome sequencing technology, which inspires this proposal to develop a similarly distributable molecular screening platform. Combining droplet-scale microfluidic miniaturization and automation with consumable DNA-encoded solid-phase compound libraries comprises a proposed bid to reconstitute the operations of a HTS center in a single benchtop instrument. The device loads compound library beads into picoliter-scale droplets of assay reagent, photochemically cleaves the compound from the bead, incubates the dosed droplets, reads the fluorescence of the incubated droplets, and sorts droplets exhibiting a desired assay fluorescence profile for collection and high-throughput sequencing. The instrument will (1) screen a million compound library in ~6 h, (2) require several square feet of space, (3) consume ~100 µL of assay reagent, and (4) generate dose-response screening data, resulting in massively parallel pan-library structure-activity relationship profiles. This technology will distribute molecular screening, moving it into academic, industrial and government laboratories nationwide, and on a cost scale that will enable discovery of thousands of probes annually.

项目总结/摘要 美国国立卫生研究院建立了分子库计划（MLP）及其高通量筛选网络 (HTS)中心发现探针-高度选择性的小分子，调节细胞功能-内 人类和病原体的蛋白质组。探针不仅是研究生物功能的工具， 新的药物靶点，但也是新疗法的潜在先导。虽然成功地完成了它的使命， HTS资源的学术界和产生了数百个探测器，MLP是日落和其 运行筛选中心面临着工业高温超导中心一直在努力解决的后勤和财务问题 数十年（大型设施，昂贵的机器人处理和光学筛选设备维护，静态复合 图书馆）。蛋白质组范围和病原体范围的探针发现仍然是一个令人信服的目标 由于分布式和经济的基因组测序技术，这激发了这个建议，以开发一个 相似分布的分子筛选平台。结合液滴规模的微流体微型化 和自动化与消费DNA编码的固相化合物库包括一个拟议的出价， 在一台台式仪器中重建HTS中心的操作。该装置装载化合物 将文库珠粒转化为皮升规模的测定试剂液滴，光化学地将化合物从文库珠粒中切割出来。 珠，孵育给药的液滴，读取孵育液滴的荧光，并对液滴进行分类 显示出用于收集和高通量测序的所需测定荧光谱。仪器 将（1）在~6小时内筛选一百万个化合物库，（2）需要几平方英尺的空间，（3）消耗~100 μL测定试剂，和（4）产生剂量-反应筛选数据，产生大规模平行泛文库 构效关系图谱。这项技术将分布分子筛选，将其转移到 全国范围内的学术，工业和政府实验室，并在成本规模，将使发现 每年有数千个探测器