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Papers by Marcin Warmiński

SUMMARYPoxviruses encode decapping enzymes that remove the protective 5’ cap from both host and v... more SUMMARYPoxviruses encode decapping enzymes that remove the protective 5’ cap from both host and viral mRNAs to commit transcripts for decay by the cellular exonuclease Xrn1. Decapping by these enzymes is critical for poxvirus pathogenicity by means of simultaneously suppressing host protein synthesis and limiting the accumulation of viral dsRNA, a trigger for antiviral responses. Here we present the first high resolution structural view of the vaccinia virus decapping enzyme D9. This Nudix enzyme contains a novel domain organization in which a three-helix bundle is inserted into the catalytic Nudix domain. The 5’ mRNA cap is positioned in a bipartite active site at the interface of the two domains. Specificity for the methylated guanosine cap is achieved by stacking between conserved aromatic residues in a manner similar to that observed in canonical cap binding proteins VP39, eIF4E, and CBP20 and distinct from eukaryotic decapping enzyme Dcp2.

ACS Chemical Biology, 2021

mRNA-based therapies and vaccines constitute a disruptive technology with the potential to revolu... more 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.

Journal of the American Chemical Society, May 9, 2018

The 5' cap consists of 7-methylguanosine (mG) linked by a 5'-5'-triphosphate bridge t... more The 5' cap consists of 7-methylguanosine (mG) 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 (mGpppG) 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 m...

Bioconjugate chemistry, Jul 19, 2017

mRNA is a template for protein biosynthesis, and consequently mRNA transport, translation, and tu... more mRNA is a template for protein biosynthesis, and consequently mRNA transport, translation, and turnover are key elements in the overall regulation of gene expression. Along with growing interest in the mechanisms regulating mRNA decay and localization, there is an increasing need for tools enabling convenient fluorescent labeling or affinity tagging of mRNA. We report new mRNA 5' cap analog-based tools that enable site-specific labeling of RNA within the cap using N-hydroxysuccinimide (NHS) chemistry. We explored two complementary methods: a co-transcriptional labeling method, in which the label is first attached to a cap analog and then incorporated into RNA by in vitro transcription, and a post-transcriptional labeling method, in which an amino-functionalized cap analog is incorporated into RNA followed by chemical labeling of the resulting transcript. After testing the biochemical properties of RNAs carrying the novel modified cap structures, we demonstrated the utility of fl...

Organic letters, Jan 7, 2017

Commercially available 2'-O-pivaloyloxymethyl (PivOM) phosphoramidites were employed in an SP... more Commercially available 2'-O-pivaloyloxymethyl (PivOM) phosphoramidites were employed in an SPS protocol for RNA 5' azides. The utility of the N3-RNAs in CuAAC and SPAAC was demonstrated by RNA 5' labeling, chemical ligation including fragment joining and cyclization, and bioconjugation. As a result, several new RNA conjugates that may be valuable tools for studies on biological events such as innate immune response (cyclic dinucleotides), post-transcriptional gene regulation (circular RNAs), or mRNA turnover (m(7)G capped RNAs) were obtained.

Nucleic acids research, Jan 28, 2017

Analogues of the mRNA 5΄-cap are useful tools for studying mRNA translation and degradation, with... more Analogues of the mRNA 5΄-cap are useful tools for studying mRNA translation and degradation, with emerging potential applications in novel therapeutic interventions including gene therapy. We report the synthesis of novel mono- and dinucleotide cap analogues containing dihalogenmethylenebisphosphonate moiety (i.e. one of the bridging O atom substituted with CCl2 or CF2) and their properties in the context of cellular translational and decapping machineries, compared to phosphate-unmodified and previously reported CH2-substituted caps. The analogues were bound tightly to eukaryotic translation initiation factor 4E (eIF4E), with CCl2-substituted analogues having the highest affinity. When incorporated into mRNA, the CCl2-substituted dinucleotide most efficiently promoted cap-dependent translation. Moreover, the CCl2-analogues were potent inhibitors of translation in rabbit reticulocyte lysate. The crystal structure of eIF4E in complex with the CCl2-analogue revealed a significantly di...

European Journal of Organic Chemistry, 2015

The Journal of organic chemistry, Jan 27, 2015

To broaden the scope of existing methods based on 19F nucleotide labeling, we developed a new met... more To broaden the scope of existing methods based on 19F nucleotide labeling, we developed a new method for the synthesis of fluorophosphate (oligo)nucleotide analogs containing an O to F substitution at the terminal position of the (oligo)phosphate moiety and evaluated them as tools for 19F 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, m7G), 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 19F NMR hybridization probes. The compounds we...

Bioorganic & Medicinal Chemistry Letters, 2013

Topics in current chemistry (Journal), 2017

The cap is a natural modification present at the 5' ends of eukaryotic messenger RNA (mRNA), ... more The cap is a natural modification present at the 5' ends of eukaryotic messenger RNA (mRNA), which because of its unique structural features, mediates essential biological functions during the process of gene expression. The core structural feature of the mRNA cap is an N7-methylguanosine moiety linked by a 5'-5' triphosphate chain to the first transcribed nucleotide. Interestingly, other RNA 5' end modifications structurally and functionally resembling the m(7)G cap have been discovered in different RNA types and in different organisms. All these structures contain the 'inverted' 5'-5' oligophosphate bridge, which is necessary for interaction with specific proteins and also serves as a cleavage site for phosphohydrolases regulating RNA turnover. Therefore, cap analogs containing oligophosphate chain modifications or carrying spectroscopic labels attached to phosphate moieties serve as attractive molecular tools for studies on RNA metabolism and modif...

Scientific Reports

Fluorescence anisotropy (FA) is a powerful technique for the discovery of protein inhibitors in a... more Fluorescence anisotropy (FA) is a powerful technique for the discovery of protein inhibitors in a high-throughput manner. In this study, we sought to develop new universal FA-based assays for the evaluation of compounds targeting mRNA 5′ cap-binding proteins of therapeutic interest, including eukaryotic translation initiation factor 4E and scavenger decapping enzyme. For this purpose, a library of 19 carboxyfluorescein probes based on 7-methylguanine nucleotides was evaluated as FA probes for these proteins. Optimal probe:protein systems were further investigated in competitive binding experiments and adapted for high-throughput screening. Using a small in-house library of compounds, we verified and confirmed the accuracy of the developed FA assay to study cap-binding protein binders. The applications of the most promising probes were then extended to include evaluation of allosteric inhibitors as well as RNA ligands. From this analysis, we confirmed the utility of the method to stu...