School of Pharmacy, University of Wisconsin-Madison, Madison, Wisconsin 53705, United States.
McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, Wisconsin 53705, United States.
ACS Chem Biol. 2020 Jun 19;15(6):1487-1496. doi: 10.1021/acschembio.0c00140. Epub 2020 Apr 16.
Proteolysis targeting chimeras (PROTACs) have emerged as useful chemical probes and potential therapeutics by taking advantage of the ubiquitin-proteasome system to degrade intracellular disease-associated proteins. PROTACs are heterobifunctional molecules composed of a target protein ligand, E3 ubiquitin ligase ligand, and a linker between them. The generation of efficient PROTACs requires screening of many parameters, especially the lengths and types of the linkers. We report our proof-of-concept study using a two-stage strategy to facilitate the development of PROTACs against the estrogen receptor (ER). In stage one, a library of close to 100 PROTACs was synthesized by simply mixing a library of ERα ligands containing a hydrazide functional group at different positions with a preassembled library of E3 ligase ligands bearing different types and lengths of linkers with a terminal aldehyde group in a 1:1 ratio. Cell-based screening occurred without further purification, because the formation of the acylhydrazone linkage is highly efficient and produces water as the only byproduct. Compound A3 was the most potent ER degrader in two ER+ cell lines (DC= ∼ 10 nM, = ≥ 95%). Stage two involved transformation to a more stable amide linker to generate a more drug-like molecule. The new compound, AM-A3, showed comparable biological activity (DC = 1.1 nM, = 98%) and induced potent antiproliferation (IC= 13.2 nM, = 69%) in MCF-7. This proof-of -concept study demonstrates that the two-stage strategy can significantly facilitate the development of PROTACs against ER without the tedious process of making large numbers of PROTACs one by one. It has the potential to be expanded to many other targets.
蛋白水解靶向嵌合体（PROTACs）利用泛素-蛋白酶体系统降解细胞内与疾病相关的蛋白，已成为有用的化学探针和潜在的治疗药物。PROTACs 是由靶蛋白配体、E3 泛素连接酶配体和它们之间的连接子组成的杂双功能分子。高效 PROTACs 的产生需要筛选许多参数，尤其是连接子的长度和类型。我们报告了使用两阶段策略来促进针对雌激素受体（ER）的 PROTACs 开发的概念验证研究。在第一阶段，通过简单地将包含酰腙官能团的 ERα 配体库与带有末端醛基的预先组装的 E3 连接酶配体库以 1:1 的比例混合，合成了近 100 个 PROTACs 的库。不需要进一步纯化就可以进行基于细胞的筛选，因为酰腙键的形成非常高效，只产生水作为唯一的副产物。在两种 ER+细胞系中，化合物 A3 是最有效的 ER 降解剂（DC=∼10 nM，=≥95%）。第二阶段涉及转化为更稳定的酰胺连接子以生成更具类药性的分子。新化合物 AM-A3 在 MCF-7 中表现出相当的生物学活性（DC=1.1 nM，=98%）和强烈的增殖抑制作用（IC=13.2 nM，=69%）。这项概念验证研究表明，两阶段策略可以显著促进针对 ER 的 PROTACs 的开发，而无需繁琐的逐个制作大量 PROTACs 的过程。它有可能扩展到许多其他靶标。
