Key result
β-S-ARCA diastereomers demonstrated higher affinity for eIF4E than unmodified caps, driven by electrostatic interactions between the negatively charged S/Se atom and positively charged amino acids.
Why the study?
The molecular basis for the beneficial thio-effect of antireverse cap analog diastereomers with an O-to-S substitution within the beta-phosphate remained unclear.
The study reveals the structural basis for the 'thio-effect' in mRNA cap analogs, showing that electrostatic interactions stabilize binding to eIF4E, which can guide the design of better mRNA therapies.
Modified cap analogs may boost mRNA translation; leaves open cardiovascular therapeutic applications pending clinical validation.
High Resolution Image Download MS PowerPoint Slide 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.
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Warmiński et al. (2021) studied this question. β-S-ARCA diastereomers vs. unmodified caps was evaluated on Binding affinity and structural interaction with eIF4E. β-S-ARCA diastereomers demonstrated higher affinity for eIF4E than unmodified caps, driven by electrostatic interactions between the negatively charged S/Se atom and positively charged amino acids.