Microfluidic High-Throughput Droplet-Scale Screening of DNA-Encoded Compound Libraries for Activators of the Bacterial Target ClpP

项目摘要 微流控技术和微型化技术是提高化学品性能的有力工具 分析系统。优势包括分析速度的提高、高通量的并行采样 加工，并大幅减少样品、试剂和电力消耗。因此，微流控系统 是许多“下一代”分离、质谱学和DNA测序的核心技术 系统。在过去的十年里，我在微流体和 化学分析技术的发展，并来到斯克里普斯研究所(TSRI)追求 以应用为导向的研究。到目前为止，这项研究主要集中在使用我已经掌握的技能 被收购以设计下一代基于液滴的化合物筛选平台。这 集成系统分发数十万个DNA编码的一珠一化合物(OBOC) 组合库珠子形成皮升尺度的液滴，精确地用紫外光照射每个液滴以释放 来自微珠表面的化合物，执行检测孵化，并定量检测HIT的检测读数 识别和分类。筛选拥有&gt；105名成员的OBOC库只需要几个小时和&lt；200µL 化验试剂。 在过去的两年里，我确定了我的职业目标，但我也意识到，我目前还没有 完成这些任务所需的技能和科学知识。我的主要目标是将微流控液滴- 基于化合物筛选技术，我曾帮助将其发展成为一家初创公司。这家公司将 重点筛选针对病毒和细菌靶标的DNA编码化合物文库。穷人又回来了 对这类治疗领域的投资迫使大型制药公司退出该领域，但 小型化的高通量筛选(HTS)技术可以使这种努力再次具有成本效益和 为迫切需要的疾病重振药品供应渠道。 K25职业发展奖是一个极好的工具，可以补充我在微流体和 具有分子生物学、有机化学和分析开发方面的培训的分析化学 引领独立小分子发现努力的先决条件。为了获得这些技能，我建议 为了生成一个DNA编码的OBOC组合库，以探索ADEP 1支架的多样性，一个 对革兰氏阳性菌具有抗菌活性的酰基多肽天然产物，并对其进行筛选 生化活性和全细胞活性。用正交法筛选大量相关化合物文库 模式应该提供对“渗透规则”的洞察，这可以为新型合成材料的设计提供信息。 抗感染发现的脚手架。 我选择了Brian M Paegel(TSRI)和Tom Kodadek(TSRI)作为这个项目的导师和共同导师。 佩格尔教授将为我提供分子生物学和测试开发技术方面的广泛培训， Kodadek教授是一位宝贵的资源，他将指导OBOC库的设计和合成。TSRI 为项目执行提供理想的环境：TSRI拥有一个大型HTS设施，专家在 筛查和化验的发展，TSRI的传染病部门拥有众多世界级的 研究人员，以及蛋白质组学、基因组学和基于细胞的筛查核心设施可供支持。 这一建议集成了DNA编码固相合成(DESPS)和液滴的关键进展 微流控形成可分布的、高效的药物发现平台。我将生成一个组合 &gt；200,000个ADEP模拟物的库，包含自然和非自然构建块。单体 将对偶联反应进行DNA兼容性、产品产率和纯度的评估。我将开发一种 利用体外转录检测ADEP介导的靶向ClpP激活的生化活性测定 以及ClpP和检测探针GFP的翻译(IVTT)。足够进行&gt；106液滴反应的蛋白质可以 快速、廉价且不需要细胞培养。将筛选ADEP模拟OBOC库 使用所开发的方法在皮升尺度的液滴中检测对ClpP的活性。我还会放映ADEP 全细胞微珠扩散法活性文库。另外，来自每个屏幕的热门珠子将被汇集在一起， 它们的DNA编码标签被扩增，它们的结构通过下一代测序得到阐明。点击率将会是 在基于微孔板的生化活性检测和全细胞活性检测中重新合成和验证。 最后，点击将根据活跃度和每个屏幕的点击之间的比较SAR进行排名 确定有助于细胞渗透的物理化学参数。

Project Summary Microfluidics, and miniaturization in general, is a powerful tool to enhance the performance of chemical analysis systems. Advantages include an increase in analysis speed, high-throughput parallel sample processing, and drastic reductions in sample, reagent, and power consumption. As such, microfluidic systems are the core technologies in many “next-generation” separation, mass spectrometry, and DNA sequencing systems. Over the past decade, I have acquired theoretical and practical expertise in both microfluidic and chemical analysis technology development and came to The Scripps Research Institute (TSRI) to pursue application-driven research. To date, this research has largely focused on using the skills that I've already acquired to engineer a next-generation miniaturized droplet-based platform for compound screening. This integrated system distributes hundreds of thousands of DNA-encoded one-bead-one-compound (OBOC) combinatorial library beads into picoliter-scale droplets, precisely doses each droplet with UV light to liberate compound from the bead surface, performs assay incubation, and quantitates assay readout for hit identification and sorting. Screening an OBOC library with > 105 members requires only hours and < 200 µL of assay reagents. Over the past two years, I have identified goals for my career, but I also realize that I do not currently possess the skills and scientific knowledge to accomplish them. My main goal is to transition the microfluidic droplet- based compound screening technology that I have helped develop into a start-up company. This company will focus on screening DNA-encoded compound libraries against viral and bacterial targets. The poor return on investment in such therapeutic areas has forced large pharmaceutical companies to divest from the space, but miniaturized high-throughput screening (HTS) technology can make such efforts cost-effective again and reinvigorate the drug pipeline for diseases that desperately need it. The K25 career development award is an excellent vehicle to supplement my expertise in microfluidics and analytical chemistry with training in molecular biology, organic chemistry, and assay development that is prerequisite for spearheading independent small molecule discovery efforts. To acquire these skills, I propose to generate a DNA-encoded OBOC combinatorial library that explores diversification of the ADEP 1 scaffold, an acyldepsipeptide natural product with antibiotic activity against Gram-positive bacteria, and screen it for both biochemical activity and whole-cell activity. Screening a large library of related compounds in orthogonal modes should provide insight into “rules of penetration,” which can inform the design of novel synthetic scaffolds for anti-infective discovery. I have selected Brian M Paegel (TSRI) and Tom Kodadek (TSRI) as my mentor and co-mentor for this project. Prof. Paegel will provide me with extensive training in molecular biology and assay development techniques, and Prof. Kodadek is an invaluable resource who will guide design and synthesis of the OBOC library. TSRI provides an ideal environment for the project execution: TSRI houses a large HTS facility with experts in screening and assay development, TSRI's Infectious Diseases department has numerous world-class researchers, and proteomics, genomics, and cell-based screening core facilities are available for support. This proposal integrates key advances in DNA-encoded solid-phase synthesis (DESPS) and droplet microfluidics to form a distributable, highly efficient drug discovery platform. I will generate a combinatorial library of > 200,000 ADEP analogs that incorporates both natural and unnatural building blocks. Monomer coupling reactions will be evaluated for DNA compatibility, product yield, and purity. I will develop a biochemical activity assay that detects ADEP-mediated activation of its target ClpP using in vitro transcription and translation (IVTT) of both ClpP and the assay probe, GFP. Sufficient protein for > 106 droplet reactions can be generated quickly, inexpensively, and without cell culture. The ADEP analog OBOC library will be screened for activity against ClpP in picoliter-scale droplets using the developed assay. I will also screen the ADEP library for activity in a whole-cell bead diffusion assay. Separately, hit beads from each screen will be pooled, their DNA encoding tags amplified and their structures elucidated via next-generation sequencing. Hits will be resynthesized and validated in microplate-based biochemical activity assays and whole-cell viability assays. Lastly, hits will be ranked according to activity and comparative SAR between the hits for each screen should identify physicochemical parameters that aid in cell penetration.