Activity-Based DNA-Encoded Library Technology

基于活动的 DNA 编码文库技术

• 批准号: 10380694
• 负责人: Brian M Paegel
• 金额: $ 37.87万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10380694
• 关键词: Antibiotics; Antineoplastic Agents; Antiviral Agents; Binding; Biological Assay; Bypass; CCR5 gene; Cell Culture Techniques; Cells; Cellular Assay; Chemicals; Cholesterol; Collection; DNA; Data Set; Disease; Drug Targeting; Engineering; Ensure; Funding; Genes; Genome; HIV; Human; Human Genome; Human Genome Project; In Vitro; Investments; Laboratories; Libraries; Manuals; Medicine; Methods; Microfluidics; Modality; Names; Peptide Signal Sequences; Pharmaceutical Preparations; Phase; Play; Polymers; Process; Protein Biosynthesis; Proteins; Proteome; Ribosomes; Site; Solid; Source; Structure; Surface; System; Technology; Translating; Translations; Vision; base; cellular engineering; clinically relevant; design; drug discovery; experimental study; high throughput screening; human disease; hypercholesterolemia; instrumentation; new therapeutic target; novel; novel therapeutic intervention; programs; protein expression; ribosome profiling; scale up; screening; small molecule; synthetic biology; technology development; tissue culture; translation assay

Project Summary The vast majority of the human proteome is considered “undruggable.” Undruggable proteins may be difficult to express, lack surface binding clefts, do not have corresponding activity assays, or some combination thereof. This concept is symptomatic of a major liability of contemporary drug discovery, which requires significant investment to generate and scale up protein expression or cell culture and engineering an activity assay for every new target. It may be possible to bypass these bottlenecks by directly targeting translation intermediates, or “ribosome nascent chains” (RNCs), with small molecules that selectively inhibit protein synthesis by interacting with an RNC and stalling translation. RNCs represent a vast source of new drug targets that do not follow the rules of druggability, but high-throughput screens for RNC-targeting “Selective Terminators of Protein Synthesis” (SToPS) have been roundly unsuccessful due to the limited scope of structures in standard compound screening decks. During the previously funded project, our instrumentation and systems engineering laboratory developed solid-phase DNA-encoded library (DEL) synthesis methods and microfluidic DEL screening technology that collectively enabled unprecedented activity-based screens on these large collections of novel chemical matter. We demonstrated that this platform can efficiently search DELs of drug-like small molecules to identify novel bioactive molecules for several clinically relevant drug targets. The proposed MIRA program will leverage our activity-based DEL screening capabilities to establish a SToPS discovery platform through two parallel technology development initiatives. The first is a synthetic biology-driven microfluidic droplet-scale in vitro translation-based approach to identifying small molecule SToPS of a specific target RNC. The second is a polymer/tissue culture engineering approach that will explore cellular assays of translation stalling, the screening format that identified the original examples of SToPS targeting the hypercholesterolemia-associated protein, PCSK9. Both approaches will benefit from DEL-based chemical diversity, which can be designed to explore chemical space known to elicit ribosome binding and selective translation stalling. Cellular DEL screening technology will ensure that screening hits are cell active, and more broadly will deliver a long-sought screening modality to the field of drug discovery. Following proof-of-concept SToPS screens, we will develop computational workflows that mine publicly available ribosome profiling data sets to predict candidate stall sites for SToPS screening, tackling CCR5 (anti-HIV) and the bacterial signal sequence as examples of undruggable targets. We envision a completely plug-and-play chemical probe discovery strategy for translating human genome sequence directly into SToPS as chemical probes, thereby fulfilling the original vision of the Human Genome Project and eliminating “undruggable” from the drug discovery lexicon.

项目摘要 绝大多数人蛋白质组都被认为是“不可能的”。不良蛋白质可能很困难 表达缺乏表面结合裂缝，没有相应的活性评估或某些组合。 这个概念是当代药物发现的重大责任的症状，这需要重大 投资以生成和扩展蛋白质表达或细胞培养和工程的活动分析 每个新目标。可以通过直接瞄准翻译中间体，绕过这些瓶颈， 或“核糖体新生链”（RNC），其小分子有选择地抑制蛋白质合成 与RNC和失速翻译相互作用。 RNC代表了不可能的新药物的广泛来源 遵循可药用的规则，但高通量屏幕用于RNC靶向“选择性终止者” 蛋白质合成”（Stop）由于标准中结构的范围有限而彻底失败 复合筛选甲板。在先前资助的项目中，我们的仪器和系统 工程实验室开发的固相DNA编码库（DEL）合成方法和微流体 Del筛选技术在这些大型大型上启用了前所未有的基于活动的屏幕 新型化学物质的集合。我们证明了这个平台可以有效地搜索 药物样的小分子以鉴定几种临床相关药物靶标的新型生物活性分子。这 拟议的MIRA计划将利用我们基于活动的DEL筛查功能来建立停留 通过两个并行技术开发计划的发现平台。第一个是合成的 生物学驱动的微流体液滴尺度基于体外翻译的方法来识别小分子 特定目标RNC的停止。第二个是聚合物/组织培养工程方法，将探索 翻译失速的蜂窝刺激，筛选格式，确定了停止的原始示例 靶向高胆固醇血症相关的蛋白PCSK9。两种方法都将从基于DEL的 化学多样性，旨在探索已知的化学空间，以引起核糖体结合和 选择性翻译失速。蜂窝DEL筛选技术将确保筛选命中是活跃的， 更广泛地将为毒品发​​现领域提供长期筛查的方式。下列的 概念证明停止屏幕，我们将开发公开可用的计算工作流程 核糖体分析数据集可预测候选摊位站点的停止筛查，解决CCR5（反HIV）和 细菌信号序列是不良目标的示例。我们设想一个完全的插件 化学探针发现策略，用于将人类基因组序列直接转化为stops作为化学 问题，从而实现人类基因组项目的最初愿景，并从 药物发现词典。