Activity-Based DNA-Encoded Library Technology

项目摘要 人类蛋白质组的绝大多数被认为是“不可药用的”。不可药用的蛋白质可能很难 表达、缺乏表面结合裂缝、不具有相应的活性测定或其某种组合。 这一概念是当代药物发现的一个主要缺陷的症状，这需要大量的研究。 投资以产生和扩大蛋白质表达或细胞培养，并工程化活性测定， 每一个新目标通过直接靶向翻译中间体， 或“核糖体新生链”（RNCs），具有通过以下方式选择性抑制蛋白质合成的小分子： 与RNC交互并停止翻译。RNCs代表了一个巨大的新药物靶点来源， 遵循药物的规则，但高通量筛选RNC靶向的“选择性终止剂”， 蛋白质合成”（SToPS）由于标准中结构的有限范围而全面不成功 复合屏蔽甲板在以前资助的项目中，我们的仪器和系统 工程实验室开发了固相DNA编码库（DEL）合成方法和微流体 DEL筛选技术，共同实现了前所未有的基于活动的屏幕上，这些大型 新的化学物质的集合。我们证明了该平台可以有效地搜索 药物样小分子，以确定新的生物活性分子的几个临床相关的药物靶标。的 拟议的MIRA计划将利用我们基于活动的DEL筛选能力，建立一个SToPS 通过两项并行的技术开发计划，开发了一个新的发现平台。第一种是合成的 基于生物学驱动的微流控液滴规模的体外鉴定小分子的方法 特定目标RNC的SToPS。第二种是聚合物/组织培养工程方法， 翻译停滞的细胞分析，识别SToPS原始实例的筛选格式 靶向高胆固醇血症相关蛋白PCSK 9。这两种方法都将受益于基于DEL的 化学多样性，其可以被设计为探索已知引发核糖体结合的化学空间， 选择性翻译停顿。细胞DEL筛选技术将确保筛选命中是细胞活性的， 更广泛地说，它将为药物发现领域提供一种长期寻求的筛选模式。以下 概念验证SToPS屏幕，我们将开发计算工作流程， 核糖体分析数据集，以预测SToPS筛选的候选失速位点，处理CCR 5（抗HIV）， 细菌信号序列作为不可药靶的例子。我们设想一个完全即插即用的 用于将人类基因组序列直接翻译成SToPS作为化学探针的化学探针发现策略 探针，从而实现了人类基因组计划的最初愿景，并消除了“不可药”， 药物发现词典

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.