5′-Phosphorothiolate Dinucleotide Cap Analogues: Reagents for Messenger RNA Modification and Potent Small-Molecular Inhibitors of Decapping Enzymes

Abstract

The 5′ cap consists of 7-methyl­guanosine (m7G) linked by a 5′–5′-triphosphate bridge to messenger RNA (mRNA) and acts as the master regulator of mRNA turnover and translation initiation in eukaryotes. Cap analogues that influence mRNA translation and turnover (either as small molecules or as part of an RNA transcript) are valuable tools for studying gene expression, which is often also of therapeutic relevance. Here, we synthesized a series of 15 dinucleotide cap (m7GpppG) analogues containing a 5′-phosphoro­thiolate (5′-PSL) moiety (i.e., an O-to-S substitution within the 5′-phospho­ester) and studied their biological properties in the context of three major cap-binding proteins: translation initiation factor 4E (eIF4E) and two decapping enzymes, DcpS and Dcp2. While the 5′-PSL moiety was neutral or slightly stabilizing for cap interactions with eIF4E, it significantly influenced susceptibility to decapping. Replacing the γ-phospho­ester with the 5′-PSL moiety (γ-PSL) prevented β-γ-pyrophosphate bond cleavage by DcpS and conferred strong inhibitory properties. Combining the γ-PSL moiety with α-PSL and β-phosphoro­thioate (PS) moiety afforded first cap-derived hDcpS inhibitor with low nanomolar potency. Susceptibility to Dcp2 and translational properties were studied after incorporation of the new analogues into mRNA transcripts by RNA polymerase. Transcripts containing the γ-PSL moiety were resistant to cleavage by Dcp2. Surprisingly, superior translational properties were observed for mRNAs containing the α-PSL moiety, which were Dcp2-susceptible. The overall protein expression measured in HeLa cells for this mRNA was comparable to mRNA capped with the translation augmenting β-PS analogue reported previously. Overall, our study highlights 5′-PSL as a synthetically accessible cap modification, which, depending on the substitution site, can either reduce susceptibility to decapping or confer superior translational properties on the mRNA. The 5′-PSL-analogues may find application as reagents for the preparation of efficiently expressed mRNA or for investigation of the role of decapping enzymes in mRNA processing or neuromuscular disorders associated with decapping.

The Role of the Phosphorus Atom in Drug Design

“…Recently, several structurally related analogues of m 7 GpppG (compound 196 ) bearing a 5′‐phosphorothiolate unit were synthesized, and biological activity was evaluated toward three capping enzymes: translation initiation factor 3E, DcpS, and Dcp2. Isosteric analogues of the 5′,5′‐triphosphate bridge to give rise to sulfur‐containing nucleotides turned out to be fascinating molecules as inhibitors of these enzymes . Among them are structural variations at the bridge by replacing an oxygen atom either by a methylene group or by an amino group, or even modifications outside the bridge by replacing the γ‐oxygen atom by sulfur or BH 3 , etc .…”

“…Compound 197 (β‐S‐ARCA) bearing an asymmetric phosphorus atom due to presence of the sulfur atom, exhibited a beneficial effect in the half‐life of cellular mRNA and translation properties . Of particular interest was replacement of the 5′‐oxygen atom by a sulfur atom, giving rise to relevant molecules that were evaluated for their capacity to inhibit the decapping process or increase the translational potential of mRNA . Notably, the position of the sulfur atom (being part of the bridge or bound to a phosphorus atom coming out of the bridge) affected biological activity, exhibiting diverse actions either on translation initiation factor 3E or DcpS and Dcp2 enzymes .…”

“…Notably, the position of the sulfur atom (being part of the bridge or bound to a phosphorus atom coming out of the bridge) affected biological activity, exhibiting diverse actions either on translation initiation factor 3E or DcpS and Dcp2 enzymes . For example, 198 was found to be an effective nanomolar inhibitor of cap‐derived hDspS (Figure ) …”

“…Sulfur-containing nucleotide analogues are quite interesting molecules that target ecto-dinucleotide pyrophosphatase-1 (NPP1) or decapping enzymes,f or example. [187][188][189] NPP1 is a metalloenzyme that catalyzes the hydrolysis of anumber phosphate bonds in nucleotides to give rise to the corresponding 5'-monophosphatenucleotides, as is the case for ATP, its principal substrate, which is convertedi ntoA MP. [190] This enzyme is associated with av ariety of biological eventss uch as bone mineralization, soft-tissue calcification, and tumor cell growth.…”

“…Isosteric analogues of the 5',5'-triphosphate bridge to give rise to sulfur-containing nucleotides turned out to be fascinating molecules as inhibitors of these enzymes. [187] Amongt hem are structuralv ariations at the bridge by replacing an oxygen atom either by am ethylene group or by an amino group, or even modifications outside the bridge by replacing the goxygen atom by sulfur or BH 3 ,e tc. [192] Compound 197 (b-S-ARCA) bearinga na symmetric phosphorus atom due to presence of the sulfur atom, exhibited ab eneficiale ffect in the ChemMedChem 2019, 14,190 -216 www.chemmedchem.org half-life of cellular mRNA and translation properties.…”