NECEEM-Based technology for selection of drug leads from libraries of DNA-Encoded small molecules

调节蛋白是主要的治疗靶点；寻找与这些蛋白结合的小分子是现代药物发现的主流。从小分子的组合文库中选择蛋白质结合剂是对缓慢而昂贵的合理药物设计的一种有希望的补充。由于这种选择依赖于随机机会，其成功程度随着图书馆多样性的增加而提高。具有几乎无限独特结构的小分子库可以通过分裂和池方法来合成。然而，由于缺乏兼容的结构鉴定工具，从高度多样化的小分子文库中选择蛋白质结合剂是具有挑战性的。最近，通过创建DNA编码小分子库(LDESM)找到了一种解决方案，其中使用DNA标签来识别小分子结构。目前已成功合成了超过百亿种独特化合物的LDESM。在目前使用LDESMS的选择方法中，将目标蛋白固定在表面上，并使该表面暴露于与LDESM接触。在接近结合平衡后，洗掉未与表面结合的化合物，收集与表面结合的化合物，并通过对DNA标签进行测序来鉴定其结构。然而，化合物与表面的非特异性结合极大地影响了这种方法的效率，从而导致了不可接受的高“化学噪声”。我们最近发明了一种基于溶液(而不是基于表面)的方法，用于从高度多样化的文库中选择蛋白质结合体。我们的方法称为平衡混合物的非平衡毛细管电泳法(NECEEM)。我们拥有3项NECEEM在药物开发方面的实际应用专利。在这里，我们建议将NECEEM用于从LDESM中选择蛋白质结合蛋白的应用减少到实践中。所有商业上无法获得的材料，包括激光扫描电子显微镜和靶材，都将由一家工业合作者提供。我们将进行一系列测试，以展示NECEEM在LDESM应用中的出色性能。这项工作对于我们将NECEEM技术商业化的努力是必要的。

Regulatory proteins are major therapeutic targets; finding small molecules which bind those proteins is the mainstream in modern drug discovery. Selection of protein binders from combinatorial libraries of small molecules is a promising addition to slow and expensive rational drug design. Since such selection relies on a random chance, its success improves with increasing diversity of the library. Libraries of small molecules with practically an unlimited number of unique structures can be synthesized by a split-and-pool method. However, selection of protein binders from highly-diverse libraries of small molecules is challenging due to the lack of compatible structure-identification tools. A solution was recently found through the creation of libraries of DNA-encoded small molecules (LDESMs) in which the DNA tag is used to identify the small-molecule structure. LDESMs of more than ten billion unique compounds have been successfully synthesized. In a currently used selection method using LDESMs, a target protein is immobilized on the surface and the surface is exposed to contact with a LDESM. After binding equilibrium is approached, the compounds that do not bind the surface are washed out and the ones that are bound to the surface are collected and their structures are identified through sequencing the DNA tags. The efficiency of this approach is, however, dramatically affected by non-specific binding of compounds to the surface leading to unacceptably-high "chemical noise". We recently invented a solution-based (rather than surface-based) approach for selection of protein binders from highly-diverse libraries. Our approach is called non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM). We hold 3 patents for practical applications of NECEEM for drug development. Here we propose to reduce to practice the application of NECEEM for selection of protein binders from LDESMs. All commercially unavailable materials, including LDESMs and targets, will be provided by an industrial collaborator. We will conduct a series of tests to demonstrate outstanding performance of NECEEM in application to LDESMs. This work is necessary for our efforts to commercialize the NECEEM technology.