PO.CH01.01 · 化学
用于靶向蛋白降解的FBXW7结合剂的开发
Development of FBXW7 binders for targeted protein degradation
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
蛋白质-蛋白质相互作用（PPI）在人类细胞几乎所有生理过程中发挥着重要作用。诱导或增强特定PPI的邻近以实现靶向蛋白降解（TPD）已受到广泛关注。由诱导蛋白邻近引起的TPD通常通过两类小分子实现：分子胶和蛋白水解靶向嵌合体（PROTAC）。这两类都利用泛素介导的蛋白水解系统进行降解，其中E3连接酶在底物识别中发挥关键作用。尽管人类系统中有超过600种E3连接酶，但仅有不到10%被用于TPD研究。在此，我们报道了一组针对FBXW7的潜在结合剂的发现，FBXW7是一种E3连接酶和关键的肿瘤抑制因子，此前尚未发现其结合剂。我们的项目旨在发现FBXW7的强效小分子结合剂，可用作PROTAC设计的新弹头，用于TPD并最终抑制多种癌症。利用我们的高通量筛选技术——小分子微阵列（SMM），我们在筛选65,000个"类药"小分子文库后成功鉴定出3组（共44个）可能与FBXW7结合的化合物。这是通过将这些小分子直接打印到玻璃芯片上，与内部纯化的His标签FBXW7孵育，然后用荧光偶联抗体识别来实现的。随后我们使用nanoDSF进行二次检测，其中一组中的多个小分子诱导FBXW7熔点变化超过0.3℃，表明具有结合活性。有趣的是，这些小分子具有非常相似的化学结构，彼此仅在一个六元环上的一个官能团上有所差异。随后的热位移检测通过Western印迹显示，这些小分子候选物以剂量依赖方式稳定FBXW7。迄今为止，使用ITC显示我们的先导化合物对FBXW7的结合亲和力为0.3μM。合成并测试这些结合剂用于一组概念验证PROTAC的尝试正在进行中。总之，我们的发现鉴定出一组针对FBXW7的新潜在结合剂，扩展了PROTAC设计的工具箱。我们还证明了SMM作为探索更多E3连接酶结合剂的可行方法学的优势，这将极大地有利于开发用于癌症治疗的TPD工具。
查看英文原文 English abstract
Protein-protein interactions (PPIs) play essential roles in almost all the physiological processes in human cells. Inducing or enhancing the proximity of specific PPIs for targeted protein degradation (TPD) has received much attention. TPD caused by induced protein proximity is usually achieved by two categories of small molecules: molecular glues and proteolysis targeting chimeras (PROTACs). Both categories utilize the ubiquitin-mediated proteolysis system for degradation, in which E3 ligases play vital roles in substrate recognition. Despite the fact that there are over 600 E3 ligases in the human system, only less than 10% are investigated for TPD. Here we report the discovery of a set of potential binders for FBXW7, an E3 ligase and key tumor suppressor for which no previous binders have been found. Our project aims to discover potent small-molecule binders to FBXW7, which can be used as new warheads for PROTAC design for TPD and eventually suppression of various cancers. Taking advantage of our high-throughput screening technology, Small Molecule Microarrays (SMMs), we successfully identified 3 groups of compounds (44 total) that potentially bind to FBXW7 after screening our 65,000 ‘drug-like' small molecule library. This is done by printing these small molecules directly onto glass chips and incubating with in-house purified His-tag FBXW7, and then recognizing with fluorescence-conjugated antibody. We then performed secondary assays using nanoDSF, in which multiple small molecules from one of the groups induced over 0.3 ℃ change in the melting point of FBXW7, indicating binding activity. Interestingly, these small molecules share very similar chemical structures, which differ from others by merely one functional group on one of the 6-membered rings. Following thermo shift assay indicates that these small molecule candidates stabilize FBXW7 in a dose-dependent manner shown by western blot. So far, our leading compound is shown to have a binding affinity of 0.3μM to FBXW7 using ITC. Attempts to synthesize and test these binders for a set of proof-of-concept PROTACs are in progress. In summary, our findings identify a new set of potential binders to FBXW7, which expands the toolbox for PROTAC design. We also prove the strength of SMM as a viable methodology for the exploration of more E3 ligase binders, which will greatly benefit the development of tools in TPD for cancer therapeutics.
利益披露 Disclosure
S. Guo, None..
B. Leifer, None.