Design, Optimization, and Study of Small Molecules That Target Tau Pre-mRNA and Affect Splicing

Approximately
95% of human genes are alternatively spliced, and
aberrant splicing events can cause disease. One pre-mRNA that is alternatively
spliced and linked to neurodegenerative diseases is tau (microtubule-associated
protein tau), which can cause frontotemporal dementia and parkinsonism
linked to chromosome 17 (FTDP-17) and can contribute to Alzheimer’s
disease. Here, we describe the design of structure-specific lead small
molecules that directly target tau pre-mRNA from sequence. This was
followed by hit expansion and analogue synthesis to further improve
upon these initial lead molecules. The emergent compounds were assessed
for functional activity in a battery of assays, including binding
assays and an assay that mimics molecular recognition of tau pre-mRNA
by a U1 small nuclear ribonucleoprotein (snRNP) splicing factor. Compounds
that emerged from these studies had enhanced potency and selectivity
for the target RNA relative to the initial hits, while also having
significantly improved drug-like properties. The compounds are shown
to directly target tau pre-mRNA in cells, via chemical cross-linking
and isolation by pull-down target profiling, and to rescue disease-relevant
splicing of tau pre-mRNA in a variety of cellular systems, including
primary neurons. More broadly, this study shows that lead, structure-specific
compounds can be designed from sequence and then further optimized
for their physicochemical properties while at the same time enhancing
their activity.

约95%的人类基因存在可变剪接现象，异常剪接事件可引发各类疾病。与神经退行性疾病相关的可变剪接前体mRNA（pre-mRNA）之一为微管相关蛋白tau（microtubule-associated protein tau），其异常剪接可引发17号染色体连锁的额颞叶痴呆与帕金森综合征（FTDP-17），同时参与阿尔茨海默病的病理进程。本研究基于tau前体mRNA的序列信息，设计了可直接靶向该靶点的结构特异性先导小分子化合物。随后通过命中化合物拓展（hit expansion）与类似物合成，对上述初始先导化合物进行了系统性优化。后续通过一系列检测实验对所得化合物的功能活性进行评估，涵盖结合实验，以及模拟U1小核核糖核蛋白（small nuclear ribonucleoprotein, snRNP）剪接因子对tau前体mRNA的分子识别过程的检测实验。相较于初始命中化合物，本研究获得的化合物对靶标RNA的结合效力与选择性均得到显著提升，同时类药性质也得到了明显改善。实验证实，通过化学交联与下拉靶标谱分析技术，这些化合物可在细胞内直接靶向tau前体mRNA，并能在包括原代神经元在内的多种细胞系统中，挽救tau前体mRNA的疾病相关可变剪接异常。从更广泛的研究视角而言，本研究证明，可通过靶标序列信息设计结构特异性先导化合物，并在优化其理化性质的同时同步提升其生物活性。