Synthesis, properties, and biological activity of boranophosphate analogs of the mRNA cap: versatile tools for manipulation of therapeutically relevant cap-dependent processes

Abstract

Modified mRNA cap analogs aid in the study of mRNA-related processes and may enable creation of novel therapeutic interventions. We report the synthesis and properties of 11 dinucleotide cap analogs bearing a single boranophosphate modification at either the α-, β- or γ-position of the 5′,5′-triphosphate chain. The compounds can potentially serve either as inhibitors of translation in cancer cells or reagents for increasing expression of therapeutic proteins in vivo from exogenous mRNAs. The BH3-analogs were tested as substrates and binding partners for two major cytoplasmic cap-binding proteins, DcpS, a decapping pyrophosphatase, and eIF4E, a translation initiation factor. The susceptibility to DcpS was different between BH3-analogs and the corresponding analogs containing S instead of BH3 (S-analogs). Depending on its placement, the boranophosphate group weakened the interaction with DcpS but stabilized the interaction with eIF4E. The first of the properties makes the BH3-analogs more stable and the second, more potent as inhibitors of protein biosynthesis. Protein expression in dendritic cells was 2.2- and 1.7-fold higher for mRNAs capped with m27,2′-OGppBH3pG D1 and m27,2′-OGppBH3pG D2, respectively, than for in vitro transcribed mRNA capped with m27,3′-OGpppG. Higher expression of cancer antigens would make mRNAs containing m27,2′-OGppBH3pG D1 and m27,2′-OGppBH3pG D2 favorable for anticancer immunization.

Two-headed tetraphosphate cap analogs are inhibitors of the Dcp1/2 RNA decapping complex

“…Our data suggest that 12b competes for the active site of Dcp2 and may act as a tightly bound mimic of the 5 ′ cap, paving the way for future structural studies and the development of potent, selective inhibitors of Dcp2. Observations made during our initial screening of the synthetic nucleotide library for Dcp2 inhibition are consistent with earlier reports concerning the influences of structural alterations of nucleotide phosphate chains on cap-protein interactions (Kowalska et al 2008(Kowalska et al , 2014Rydzik et al 2012). We find that the introduction of charge-retaining nonbridging modifications (S − , BH 3 − ) into cap analogs seemed to generally have higher impact on Dcp2 decapping activity than the bridging (CH 2 and NH) modifications.…”

Applications of Phosphate Modification and Labeling to Study (m)RNA Caps

“…Such an approach provides a relatively simple way to obtain a wide scope of chemically modified oligophosphate mono-and diesters (Fig. 7), including phosphorothioates [79][80][81], seleno- [82], borano- [83], and fluorophosphates [84], as well as phosphoramidates [85], C-phosphonates [86], and bisphosphonates [87]. P-imidazolides were shown to react readily with other inorganic nucleophiles such as fluorides [84] and sulfates [88] to yield fluorophosphates and phosphosulfates, respectively.…”

“…The first analogs, which combined these two features, were compounds carrying a non-bridging O-S substitution at the beta position of the triphosphate chain, referred to as b-S-ARCAs [108]. Later, it was found that similar properties could be conferred to mRNA by an O-to-BH 3 substitution [65,83]. Recent studies have shown that applying two O-S substitutions at two adjacent phosphate moieties in a tri-or tetraphosphate bridge may provide biological properties superior to b-S-ARCA [81].…”

Phosphate-modified analogues of m 7 GTP and m 7 Gppppm 7 G—Synthesis and biochemical properties

“…5, 6 We have recently shown that some chemical modifications of the oligophosphate chain within the cap structure can increase the affinity to eIF4E and confer resistance towards DcpS. 1,[7][8][9] For instance, replacement of one of the c-oxygens in m 7 Gp c p b p a G or a-oxygens in m 7 GTP with either BH 3 or S generally stabilises the complex with eIF4E 10,11 and significantly decreases susceptibility to hydrolysis by DcpS. Inhibition studies in RRL (Rabbit Reticulocyte Lysate) have shown that a combination of these two features results in compounds that are potent and stable inhibitors of cap-dependent translation.…”

“…Inhibition studies in RRL (Rabbit Reticulocyte Lysate) have shown that a combination of these two features results in compounds that are potent and stable inhibitors of cap-dependent translation. 8,[10][11][12] In contrast to mononucleotide cap analogues, dinucleotide analogues can be incorporated in vitro into mRNA by bacterial 13 or viral 14 RNA polymerases. Such capped transcripts can find applications in biochemistry and gene therapy.…”