DNA-encoded libraries have emerged as a widely used resource for the discovery of bioactive small molecules, and offer substantial advantages compared with conventional small-molecule libraries. Here, we have developed and streamlined multiple fundamental aspects of DNA-encoded and DNA-templated library synthesis methodology, including computational identification and experimental validation of a 20 × 20 × 20 × 80 set of orthogonal codons, chemical and computational tools for enhancing the structural diversity and drug-likeness of library members, a highly efficient polymerase-mediated template library assembly strategy, and library isolation and purification methods. We have integrated these improved methods to produce a second-generation DNA-templated library of 256,000 small-molecule macrocycles with improved drug-like physical properties. In vitro selection of this library for insulin-degrading enzyme affinity resulted in novel insulin-degrading enzyme inhibitors, including one of unusual potency and novel macrocycle stereochemistry (IC₅₀ = 40 nM). Collectively, these developments enable DNA-templated small-molecule libraries to serve as more powerful, accessible, streamlined and cost-effective tools for bioactive small-molecule discovery.

DNA 编码文库已成为发现生物活性小分子的一种广泛使用的资源，与传统的小分子文库相比具有显着优势。在这里，我们开发并简化了 DNA 编码和 DNA 模板库合成方法的多个基本方面，包括 20 × 20 × 20 × 80 正交密码子集的计算识别和实验验证，用于增强结构的化学和计算工具文库成员的多样性和药物相似性、高效聚合酶介导的模板文库组装策略以及文库分离和纯化方法。我们整合了这些改进的方法，以产生具有改进的药物样物理特性的 256,000 个小分子大环的第二代 DNA 模板库。₅₀  = 40 纳米）。总的来说，这些发展使 DNA 模板化小分子文库能够成为更强大、更易于访问、更精简和更具成本效益的生物活性小分子发现工具。