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“无法下药的”。无法下药的蛋白质可能很难 为了表达，缺乏表面结合裂隙，没有相应的活性测定，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征兆，这需要显著的 投资产生和放大蛋白质表达或细胞培养，并设计一种活性分析 每一个新目标。有可能通过直接瞄准翻译中间体来绕过这些瓶颈， 或“核糖体新生链”(RNC)，具有选择性地抑制蛋白质合成的小分子。 与RNC交互并停止翻译。RNC代表了一个巨大的新药靶点来源，而这些靶点并不 遵循可制药性规则，但针对RNC的高通量筛查-靶向 蛋白质合成“(STOP)由于标准中结构范围的限制而彻底失败 复合筛选甲板。在之前资助的项目中，我们的仪器和系统 工程实验室开发的固相DNA编码库(DEL)合成方法和微流控技术 戴尔筛选技术，共同实现了前所未有的基于活动的屏幕 收集新的化学物质。我们证明了该平台可以高效地搜索DELS 为几个临床相关的药物靶点识别新的生物活性分子的类药物小分子。这个 建议的Mira计划将利用我们基于活动的DEL筛查能力来建立一个站点 发现平台通过两个并行的技术开发举措。第一种是合成的 基于生物驱动的微流控液滴体外翻译识别小分子方法 特定目标RNC的停止。第二个是聚合物/组织培养工程学方法，将探索 翻译停顿的细胞分析，识别停顿的原始例子的筛选格式 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，这可以被设计来探索已知的引起核糖体结合和 选择性翻译停滞。细胞删除筛选技术将确保筛选命中的细胞是活跃的， 更广泛地说，将为药物发现领域提供一种长期寻求的筛选模式。跟随 概念验证停止屏幕，我们将开发可公开挖掘的计算工作流 核糖体分析数据集用于预测停靠站筛查、应对CCR5(抗HIV)和 细菌信号序列作为非药物靶标的例子。我们设想了一个完全即插即用的 将人类基因组序列直接翻译为化学物质的化学探针发现策略 探测，从而实现了人类基因组计划的最初愿景，并从 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.