mRNA-based therapies and vaccines constitute a disruptive technology with the potential to revolutionize modern medicine. Chemically modified 5′ cap structures have provided access to mRNAs with superior translational properties that could benefit the currently flourishing mRNA field. Prime examples of compounds that enhance mRNA properties are antireverse cap analog diastereomers that contain an O-to-S substitution within the β-phosphate (β-S-ARCA D1 and D2), where D1 is used in clinically investigated mRNA vaccines. The compounds were previously found to have high affinity for eukaryotic translation initiation factor 4E (eIF4E) and augment translation in vitro and in vivo. However, the molecular basis for the beneficial “thio-effect” remains unclear. Here, we employed multiple biophysical techniques and captured 11 cap analog-eIF4E crystallographic structures to investigate the consequences of the β-O-to-S or -Se substitution on the interaction with eIF4E. We determined the S_P/R_P configurations of β-S-ARCA and related compounds and obtained structural insights into the binding. Unexpectedly, in both stereoisomers, the β-S/Se atom occupies the same binding cavity between Lys162 and Arg157, indicating that the key driving force for complex stabilization is the interaction of negatively charged S/Se with positively charged amino acids. This was observed for all structural variants of the cap and required significantly different conformations of the triphosphate for each diastereomer. This finding explains why both β-S-ARCA diastereomers have higher affinity for eIF4E than unmodified caps. Binding affinities determined for di-, tri-, and oligonucleotide cap analogs suggested that the “thio-effect” was preserved in longer RNAs. Our observations broaden the understanding of thiophosphate biochemistry and enable the rational design of translationally active mRNAs and eIF4E-targeting drugs.

中文翻译：

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对临床相关硫代磷酸酯 mRNA 帽类似物与翻译起始因子 4E 相互作用的结构洞察揭示了通过静电硫效应的稳定性

基于 mRNA 的疗法和疫苗构成了一种颠覆性技术，具有彻底改变现代医学的潜力。化学修饰的 5' 帽结构提供了获得具有卓越翻译特性的 mRNA 的途径，这可能有利于当前蓬勃发展的 mRNA 领域。增强 mRNA 特性的化合物的主要例子是抗反向帽类似物非对映异构体，其在 β-磷酸（β-S-ARCA D1 和 D2）中包含 O-to-S 取代，其中 D1 用于临床研究的 mRNA 疫苗。先前发现这些化合物对真核翻译起始因子 4E (eIF4E) 具有高亲和力并在体外和体内增强翻译. 然而，有益的“硫效应”的分子基础仍不清楚。在这里，我们采用了多种生物物理技术并捕获了 11 个帽类似物-eIF4E 晶体结构，以研究 β-O-to-S 或 -Se 取代对与 eIF4E 相互作用的影响。我们确定了S _P / R _Pβ-S-ARCA 和相关化合物的构型，并获得了对结合的结构见解。出乎意料的是，在两种立体异构体中，β-S/Se 原子占据 Lys162 和 Arg157 之间相同的结合腔，表明复合物稳定的关键驱动力是带负电的 S/Se 与带正电的氨基酸的相互作用。这对于帽的所有结构变体都观察到，并且每个非对映异构体需要显着不同的三磷酸构象。这一发现解释了为什么 β-S-ARCA 非对映异构体对 eIF4E 的亲和力高于未修饰的帽。对二、三和寡核苷酸帽类似物测定的结合亲和力表明，“硫效应”在较长的 RNA 中得以保留。