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

项目摘要 微流体技术和一般的小型化是增强化学反应器性能的有力工具。 分析系统。优点包括提高分析速度、高通量并行进样 处理，并大幅减少样品，试剂和功耗。因此，微流体系统 是许多“下一代”分离、质谱和DNA测序的核心技术 系统.在过去的十年里，我获得了微流体和微流体技术的理论和实践专业知识， 化学分析技术的发展，并来到斯克里普斯研究所（TSRI）追求 应用驱动的研究。到目前为止，这项研究主要集中在使用我已经掌握的技能， 获得了工程师的下一代小型化液滴为基础的平台，用于化合物筛选。这 一个集成的系统分配了数十万个DNA编码的一珠一化合物（OBOC）， 将组合文库珠粒放入皮升规模的液滴中，用UV光精确地给每个液滴定量， 从珠表面分离化合物，进行测定孵育，并定量测定命中读数 识别和分类。筛选具有> 105个成员的OBOC文库仅需要数小时和< 200 µL的 测定试剂。 在过去的两年里，我已经确定了我的职业目标，但我也意识到，我目前还不具备 技能和科学知识来实现它们。我的主要目标是将微流体液滴- 基于化合物筛选技术，我帮助发展成为一家初创公司。这家公司将 专注于针对病毒和细菌靶标筛选DNA编码的化合物文库。穷人的回报 在这些治疗领域的投资迫使大型制药公司从该领域撤资， 小型化高通量筛选（HTS）技术可以使这种努力再次具有成本效益， 为急需药物的疾病重新注入活力。 K25职业发展奖是一个很好的工具，以补充我在微流体方面的专业知识， 分析化学，分子生物学，有机化学和分析开发培训， 这是引领独立小分子发现工作的先决条件。为了掌握这些技能，我建议 为了产生DNA编码的OBOC组合文库，探索ADEP 1支架的多样化， 对革兰氏阳性菌具有抗生素活性的酰基缩酚酸肽天然产物，并筛选其对革兰氏阳性菌和 生物化学活性和全细胞活性。正交试验筛选大型相关化合物库 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。 为抗感染药物的发现搭建了一个框架。 我选择了Brian M. Mogel（TSRI）和Tom Kodadek（TSRI）作为这个项目的导师和共同导师。 教授Replogel将为我提供分子生物学和分析开发技术方面的广泛培训， Kodadek教授是一个宝贵的资源，他将指导OBOC库的设计和合成。TSRI 为项目执行提供了理想的环境：TSRI拥有大型HTS设施， 筛查和检测开发，TSRI的传染病部门拥有众多世界一流的 研究人员和蛋白质组学、基因组学和基于细胞的筛选核心设施可提供支助。 该提案整合了DNA编码固相合成（DESPS）和液滴 微流体技术，形成一个可分配的，高效的药物发现平台。我会生成一个组合的 包含> 200，000个ADEP类似物的文库，其包含天然和非天然构建块。单体 评价偶联反应的DNA相容性、产物产率和纯度。我将开发一个 使用体外转录检测ADEP介导的其靶ClpP活化的生化活性测定 以及ClpP和测定探针GFP的翻译（IVTT）。用于> 106液滴反应的足够蛋白质可 可以快速、廉价地产生，并且不需要细胞培养。将筛选ADEP模拟OBOC文库 使用开发的测定法测定皮升级液滴中的ClpP活性。我也会筛选ADEP 在全细胞珠扩散测定中，将文库用于活性。单独合并每个筛选的命中微珠， 它们的DNA编码标签被扩增，它们的结构通过下一代测序被阐明。点击率将是 在基于微孔板的生化活性测定和全细胞活力测定中重新合成并验证。 最后，将根据活动和每个屏幕的点击之间的比较SAR对点击进行排名， 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.