In mammals, m7G-adjacent nucleotides undergo extensive modifications. Ribose of the first or first and second transcribed nucleotides can be subjected to 2′-O-methylation to form cap1 or cap2, respectively. When the first transcribed nucleotide is 2′-O-methylated adenosine, it can be additionally modified to N6,2′-O-dimethyladenosine (m6Am). Recently, the crucial role of cap1 in distinguishing between ‘self’ and ‘non-self’ in mammalian cells during viral infection was revealed. Here, we attempted to understand the impact of cap methylations on RNA-related processes. Therefore, we synthesized tetranucleotide cap analogues and used them for RNA capping during in vitro transcription. Using this tool, we found that 2′-O-methylation of the second transcribed nucleotide within the mRNA 5′ cap influences protein production levels in a cell-specific manner. This modification can strongly hamper protein biosynthesis or have no influence on protein production levels, depending on the cell line. Interestingly, 2′-O-methylation of the second transcribed nucleotide and the presence of m6Am as the first transcribed nucleotide serve as determinants that define transcripts as ‘self’ and contribute to transcript escape from the host innate immune response. Additionally, cap methylation status does not influence transcript affinity towards translation initiation factor eIF4E or in vitro susceptibility to decapping by DCP2; however, we observe the resistance of cap2-RNA to DXO (decapping exoribonuclease)-mediated decapping and degradation.

To broaden the scope of existing methods based on (19)F nucleotide labeling, we developed a new method for the synthesis of fluorophosphate (oligo)nucleotide analogues containing an O to F substitution at the terminal position of the (oligo)phosphate moiety and evaluated them as tools for (19)F NMR studies. Using three efficient and comprehensive synthetic approaches based on phosphorimidazolide chemistry and tetra-n-butylammonium fluoride, fluoromonophosphate, or fluorophosphate imidazolide as fluorine sources, we prepared over 30 fluorophosphate-containing nucleotides, varying in nucleobase type (A, G, C, U, m(7)G), phosphate chain length (from mono to tetra), and presence of additional phosphate modifications (thio, borano, imido, methylene). Using fluorophosphate imidazolide as fluorophosphorylating reagent for 5'-phosphorylated oligos we also synthesized oligonucleotide 5'-(2-fluorodiphosphates), which are potentially useful as (19)F NMR hybridization probes. The compounds were characterized by (19)F NMR and evaluated as (19)F NMR molecular probes. We found that fluorophosphate nucleotide analogues can be used to monitor activity of enzymes with various specificities and metal ion requirements, including human DcpS enzyme, a therapeutic target for spinal muscular atrophy. The compounds can also serve as reporter ligands for protein binding studies, as exemplified by studying interaction of fluorophosphate mRNA cap analogues with eukaryotic translation initiation factor (eIF4E).

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The m 7 Gc ap is au nique nucleotide structure at the 5'-end of all eukaryotic mRNAs. The cap specificallyinteracts with numerous cellular proteins and participates in biological processes that are essential for cell growth and function. To provide small molecularp robest os tudy important cap-recognizing proteins, we synthesized m 7 Gn ucleotides labeled with fluorescent tags via the terminal phosph(on)ate group and studied how their emission properties changed upon protein binding or enzymaticc leavage. Only the pyrene-labeled compounds behaved as sensitive turn-on probes. Ap yrene-labeled m 7 GTP analogue showed up to eightfold enhanced fluorescencee mission upon binding to eukaryotic translation initiation factor 4E (eIF4E) and over 30-folde nhancementu pon cleavageb yd ecapping scavenger (DcpS) enzyme. These observations serveda st he basis for developing binding-and hydrolytic-activity assays. The assay utility was validated with previously characterizedl ibraries of eIF4E ligands and DcpS inhibitors. The DcpS assay was also appliedtostudy hydrolytic activity and inhibition of endogenous enzyme in cytoplasmic extracts from HeLa and HEK cells.

In eukaryotes, mature mRNA is formed through modifications of precursor mRNA, one of which is 5’ cap biosynthesis, involving RNA cap guanine‐N7 methyltransferase (N7‐MTase). N7‐MTases are also encoded by some eukaryotic viruses and facilitate their replication. N7‐MTase inhibitors have therapeutic potential, but their discovery is difficult because long RNA substrates are usually required for activity. Herein, we report a universal N7‐MTase activity assay based on small‐molecule fluorescent probes. We synthesized 12 fluorescent substrate analogues (GpppA and GpppG derivatives) varying in the dye type, dye attachment site, and linker length. GpppA labeled with pyrene at the 3’‐O position of adenosine acted as an artificial substrate with the properties of a turn‐off probe for all three tested N7‐MTases (human, parasite, and viral). Using this compound, a N7‐MTase inhibitor assay adaptable to high‐throughput screening was developed and used to screen synthetic substrate analogues and a commercial library. Several inhibitors with nanomolar activities were identified.

Augmenting the mRNA translation efficiency and stability by replacing the standard 7-methylguanosine 5'-cap with properly designed analogues is a viable strategy for increasing the in vivo expression of proteins from exogenously delivered mRNA. However, the development of novel cap analogues with superior biological properties is hampered by the challenges associated with the synthesis of such highly modified nucleotides. To provide a simpler alternative to traditional methods for cap analogue preparation, we have recently proposed a click-chemistry-based strategy for the synthesis of dinucleotide cap analogues and identified several triazole-containing compounds with promising biochemical properties. Here, we further explored the concept of CuAAC-mediated cap synthesis by designing and studying 'second generation' triazole-modified caps, which were derived from the most promising 'first generation' compounds by modifying the oligophosphate chain length, altering the position of the triazole moiety, or replacing chemically labile P-N bonds with P-O bonds. The biochemical properties of the new analogues were evaluated by determining their affinity for eIF4E, susceptibility to hDcp2-catalysed decapping, and translation efficiencies in vitro and in cultured cells. The results led to identification of cap analogues that have superior translational properties compared to standard caps and the parent triazole-modified compounds as well as provided directions for future improvements.

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Proteases encoded by SARS-CoV-2 constitute a promising target for new therapies against COVID-19. SARS-CoV-2 main protease (Mpro, 3CLpro) and papain-like protease (PLpro) are responsible for viral polyprotein cleavage—a process crucial for viral survival and replication. Recently it was shown that 2-phenylbenzisoselenazol-3(2H)-one (ebselen), an organoselenium anti-inflammatory small-molecule drug, is a potent, covalent inhibitor of both the proteases and its potency was evaluated in enzymatic and antiviral assays. In this study, we screened a collection of 34 ebselen and ebselen diselenide derivatives for SARS-CoV-2 PLpro and Mpro inhibitors. Our studies revealed that ebselen derivatives are potent inhibitors of both the proteases. We identified three PLpro and four Mpro inhibitors superior to ebselen. Independently, ebselen was shown to inhibit the N7-methyltransferase activity of SARS-CoV-2 nsp14 protein involved in viral RNA cap modification. Hence, selected compounds were also evaluated as nsp14 inhibitors. In the second part of our work, we employed 11 ebselen analogues—bis(2-carbamoylaryl)phenyl diselenides—in biological assays to evaluate their anti-SARS-CoV-2 activity in Vero E6 cells. We present their antiviral and cytoprotective activity and also low cytotoxicity. Our work shows that ebselen, its derivatives, and diselenide analogues constitute a promising platform for development of new antivirals targeting the SARS-CoV-2 virus.