Activity-Based DNA-Encoded Library Technology

基于活动的 DNA 编码文库技术

• 批准号: 10553645
• 负责人: Brian M Paegel
• 金额: $ 37.8万
• 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/10553645
• 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; Democracy; 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; Protein Synthesis Inhibition; Proteins; Proteome; Ribosomes; Site; Solid; Source; Structure; Surface; System; Technology; Translating; Translations; Vision; 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.

项目概要 绝大多数人类蛋白质组被认为是“不可成药的”。不可成药的蛋白质可能很困难 表达、缺乏表面结合裂口、没有相应的活性测定或其某种组合。 这个概念是当代药物发现的一个主要责任的症状，这需要大量的 投资产生和扩大蛋白质表达或细胞培养，并设计活性测定 每一个新目标。通过直接针对翻译中间体来绕过这些瓶颈是可能的， 或“核糖体新生链”（RNC），其小分子通过选择性抑制蛋白质合成 与 RNC 交互并停止翻译。 RNC 代表了新药物靶点的巨大来源， 遵循成药性规则，但高通量筛选 RNC 靶向的“选择性终止子” 由于标准结构范围有限，“蛋白质合成”（SToPS）完全不成功 复合筛选平台。在之前资助的项目中，我们的仪器和系统 工程实验室开发了固相DNA编码库（DEL）合成方法和微流控 DEL 筛选技术共同在这些大型设备上实现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 DEL 药物样小分子，用于识别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 筛查能力来建立 SToPS 通过两个并行的技术开发计划建立发现平台。第一个是合成的 生物学驱动的微流体液滴规模体外翻译方法来识别小分子 特定目标RNC的STOPS。第二个是聚合物/组织培养工程方法，将探索 翻译停滞的细胞测定，识别 SToPS 原始例子的筛选形式 靶向高胆固醇血症相关蛋白 PCSK9。这两种方法都将受益于基于 DEL 的 化学多样性，可设计用于探索已知引发核糖体结合的化学空间 选择性翻译停滞。细胞 DEL 筛选技术将确保筛选命中具有细胞活性， 更广泛地说，将为药物发现领域提供一种长期寻求的筛选方式。下列的 概念验证 SToPS 屏幕，我们将开发可公开获取的计算工作流程 核糖体分析数据集可预测用于 SToPS 筛选的候选失速位点、解决 CCR5（抗 HIV）和 细菌信号序列作为不可成药靶标的示例。我们设想一个完全即插即用的 将人类基因组序列直接转化为化学探针的化学探针发现策略 探针，从而实现人类基因组计划的最初愿景，消除“不可成药” 药物发现词典。