A desthiobiotin labelled NAD+ analogue to uncover Poly(ADP-ribose) polymerase 1 protein targets.

ADP-ribosylation is a post-translational modification catalyzed by the enzyme family of polyadenosine diphosphate (ADP)-ribose) polymerases (PARPs). This enzymatic process involves the transfer of single or multiple ADP-ribose molecules onto proteins, utilizing nicotinamide adenine dinucleotide (NAD+ ) as a substrate. It, thus, plays a pivotal role in regulating various biological processes. Unveiling PARP-selective protein targets is crucial for a better understanding of their biological functions. Nonetheless, this task proves challenging due to overlapping targets shared among PARP family members. Therefore, we applied the "bump-and-hole" strategy to modify the nicotinamide binding site of PARP1 by introducing a hydrophobic pocket ("hole"). This PARP1-mutant binds an orthogonal NAD+ (Et-DTB-NAD+ ) containing an ethyl group ("bump") at the nicotinamide moiety. Furthermore, we added a desthiobiotin (DTB) tag directly to the adenosine moiety, enabling affinity enrichment of ADP-ribosylated proteins. Employing this approach, we successfully identified protein targets modified by PARP1 in cell lysate. This strategy expands the arsenal of chemically modified NAD+ analogs available for studying ADP-ribosylation, providing a powerful tool to study these critical post-translational modifications.

Ebselen derivatives inhibit SARS-CoV-2 replication by inhibition of its essential proteins: PLpro and Mpro proteases, and nsp14 guanine N7-methyltransferase.

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.

Fluorescence Anisotropy Assay with Guanine Nucleotides Provides Access to Functional Analysis of Gαi1 Proteins.

Gα proteins as part of heterotrimeric G proteins are molecular switches essential for G protein-coupled receptor- mediated intracellular signaling. The role of the Gα subunits has been examined for decades with various guanine nucleotides to elucidate the activation mechanism and Gα protein-dependent signal transduction. Several approaches describe fluorescent ligands mimicking the GTP function, yet lack the efficient estimation of the proteins' GTP binding activity and the fraction of active protein. Herein, we report the development of a reliable fluorescence anisotropy-based method to determine the affinity of ligands at the GTP-binding site and to quantify the fraction of active Gαi1 protein. An advanced bacterial expression protocol was applied to produce active human Gαi1 protein, whose GTP binding capability was determined with novel fluorescently labeled guanine nucleotides acting as high-affinity Gαi1 binders compared to the commonly used BODIPY FL GTPγS. This study thus contributes a new method for future investigations of the characterization of Gαi and other Gα protein subunits, exploring their corresponding signal transduction systems and potential for biomedical applications.

2'-O-Methylation of the second transcribed nucleotide within the mRNA 5' cap impacts the protein production level in a cell-specific manner and contributes to RNA immune evasion.

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.

Fluorescence-Based Activity Screening Assay Reveals Small Molecule Inhibitors of Vaccinia Virus mRNA Decapping Enzyme D9.

Vaccinia virus (VACV) represents a family of poxviruses, which possess their own decapping machinery as a part of their strategy to eliminate host mRNAs and evade the innate immune response. D9 is one of the two encoded VACV decapping enzymes that is responsible for cap removal from the 5' end of both host mRNA transcripts and viral double-stranded RNAs. Little is known about the structural requirements for D9 inhibition by small molecules. Here, we identified a minimal D9 substrate and used it to develop a real-time fluorescence assay for inhibitor discovery and characterization. We screened a panel of nucleotide-derived substrate analogues and pharmacologically active candidates to identify several compounds with nano- and low micromolar IC50 values. m7GpppCH2p was the most potent nucleotide inhibitor (IC50 ∼ 0.08 µM), and seliciclib and CP-100356 were the most potent drug-like compounds (IC50 0.57 and 2.7 µM, respectively). The hits identified through screening inhibited D9-catalyzed decapping of 26 nt RNA substrates but were not active toward VACV D10 or human decapping enzyme, Dcp1/2. The inhibition mode for one of the compounds (CP-100356) was elucidated based on the X-ray cocrystal structure, opening the possibility for structure-based design of novel D9 inhibitors and binding probes.

Cellular delivery of dinucleotides by conjugation with small molecules: targeting translation initiation for anticancer applications.

Targeting cap-dependent translation initiation is one of the experimental approaches that could lead to the development of novel anti-cancer therapies. Synthetic dinucleoside 5',5'-triphosphates cap analogs are potent antagonists of eukaryotic translation initiation factor 4E (eIF4E) in vitro and could counteract elevated levels of eIF4E in cancer cells; however, transformation of these compounds into therapeutic agents remains challenging - they do not easily penetrate into cells and are susceptible to enzymatic cleavage. Here, we tested the potential of several small molecule ligands - folic acid, biotin, glucose, and cholesterol - to deliver both hydrolyzable and cleavage-resistant cap analogs into cells. A broad structure-activity relationship (SAR) study using model fluorescent probes and cap-ligand conjugates showed that cholesterol greatly facilitates uptake of cap analogs without disturbing the interactions with eIF4E. The most potent cholesterol conjugate identified showed apoptosis-mediated cytotoxicity towards cancer cells.

Enzymatic Assays to Explore Viral mRNA Capping Machinery.

In eukaryotes, mRNA is modified by the addition of the 7-methylguanosine (m7 G) 5' cap to protect mRNA from premature degradation, thereby enhancing translation and enabling differentiation between self (endogenous) and non-self RNAs (e. g., viral ones). Viruses often develop their own mRNA capping pathways to augment the expression of their proteins and escape host innate immune response. Insights into this capping system may provide new ideas for therapeutic interventions and facilitate drug discovery, e. g., against viruses that cause pandemic outbreaks, such as beta-coronaviruses SARS-CoV (2002), MARS-CoV (2012), and the most recent SARS-CoV-2. Thus, proper methods for the screening of large compound libraries are required to identify lead structures that could serve as a basis for rational antiviral drug design. This review summarizes the methods that allow the monitoring of the activity and inhibition of enzymes involved in mRNA capping.

Identification and evaluation of potential SARS-CoV-2 antiviral agents targeting mRNA cap guanine N7-Methyltransferase.

SARS-CoV-2, the cause of the currently ongoing COVID-19 pandemic, encodes its own mRNA capping machinery. Insights into this capping system may provide new ideas for therapeutic interventions and drug discovery. In this work, we employ a previously developed Py-FLINT screening approach to study the inhibitory effects of compounds against the cap guanine N7-methyltransferase enzyme, which is involved in SARS-CoV-2 mRNA capping. We screened five commercially available libraries (7039 compounds in total) to identify 83 inhibitors with IC50 < 50 µM, which were further validated using RP HPLC and dot blot assays. Novel fluorescence anisotropy binding assays were developed to examine the targeted binding site. The inhibitor structures were analyzed for structure-activity relationships in order to define common structural patterns. Finally, the most potent inhibitors were tested for antiviral activity on SARS-CoV-2 in a cell based assay.

Evaluation of carboxyfluorescein-labeled 7-methylguanine nucleotides as probes for studying cap-binding proteins by fluorescence anisotropy.

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 study small molecule ligands and evaluate differently 5' capped RNAs.

Structural Insights into the Interaction of Clinically Relevant Phosphorothioate mRNA Cap Analogs with Translation Initiation Factor 4E Reveal Stabilization via Electrostatic Thio-Effect.

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 ß-phosphate (ß-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 ß-O-to-S or -Se substitution on the interaction with eIF4E. We determined the SP/RP configurations of ß-S-ARCA and related compounds and obtained structural insights into the binding. Unexpectedly, in both stereoisomers, the ß-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 ß-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.

Exploring tryptamine conjugates as pronucleotides of phosphate-modified 7-methylguanine nucleotides targeting cap-dependent translation.

Eukaryotic translation initiation factor 4E (eIF4E) is overexpressed in many cancers deregulating translational control of the cell cycle. mRNA 5' cap analogs targeting eIF4E are small molecules with the potential to counteract elevated levels of eIF4E in cancer cells. However, the practical utility of typical cap analogs is limited because of their reduced cell membrane permeability. Transforming the active analogs into their pronucleotide derivatives is a promising approach to overcome this obstacle. 7-Benzylguanosine monophosphate (bn7GMP) is a cap analog that has been successfully transformed into a cell-penetrating pronucleotide by conjugation of the phosphate moiety with tryptamine. In this work, we explored whether a similar strategy is applicable to other cap analogs, particularly phosphate-modified 7-methylguanine nucleotides. We report the synthesis of six new tryptamine conjugates containing N7-methylguanosine mono- and diphosphate and their analogs modified with thiophosphate moiety. These new potential pronucleotides and the expected products of their activation were characterized by biophysical and biochemical methods to determine their affinity towards eIF4E, their ability to inhibit translation in vitro, their susceptibility to enzymatic degradation and their turnover in cell extract. The results suggest that compounds containing the thiophosphate moiety may act as pronucleotides that release low but sustainable concentrations of 7-methylguanosine 5'-phosphorothioate (m7GMPS), which is a translation inhibitor with in vitro potency higher than bn7GMP.

Direct High-Throughput Screening Assay for mRNA Cap Guanine-N7 Methyltransferase Activity.

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.

N1-Propargylguanosine Modified mRNA Cap Analogs: Synthesis, Reactivity, and Applications to the Study of Cap-Binding Proteins.

The mRNA 5' cap consists of N7-methylguanosine bound by a 5',5'-triphosphate bridge to the first nucleotide of the transcript. The cap interacts with various specific proteins and participates in all key mRNA-related processes, which may be of therapeutic relevance. There is a growing demand for new biophysical and biochemical methods to study cap-protein interactions and identify the factors which inhibit them. The development of such methods can be aided by the use of properly designed fluorescent molecular probes. Herein, we synthesized a new class of m7Gp3G cap derivatives modified with an alkyne handle at the N1-position of guanosine and, using alkyne-azide cycloaddition, we functionalized them with fluorescent tags to obtain potential probes. The cap derivatives and probes were evaluated in the context of two cap-binding proteins, eukaryotic translation initiation factor (eIF4E) and decapping scavenger (DcpS). Biochemical and biophysical studies revealed that N1-propargyl moiety did not significantly disturb cap-protein interaction. The fluorescent properties of the probes turned out to be in line with microscale thermophoresis (MST)-based binding assays.

Fluorescent Turn-On Probes for the Development of Binding and Hydrolytic Activity Assays for mRNA Cap-Recognizing Proteins.

The m7 G cap is a unique nucleotide structure at the 5'-end of all eukaryotic mRNAs. The cap specifically interacts with numerous cellular proteins and participates in biological processes that are essential for cell growth and function. To provide small molecular probes to study important cap-recognizing proteins, we synthesized m7 G nucleotides labeled with fluorescent tags via the terminal phosph(on)ate group and studied how their emission properties changed upon protein binding or enzymatic cleavage. Only the pyrene-labeled compounds behaved as sensitive turn-on probes. A pyrene-labeled m7 GTP analogue showed up to eightfold enhanced fluorescence emission upon binding to eukaryotic translation initiation factor 4E (eIF4E) and over 30-fold enhancement upon cleavage by decapping scavenger (DcpS) enzyme. These observations served as the basis for developing binding- and hydrolytic-activity assays. The assay utility was validated with previously characterized libraries of eIF4E ligands and DcpS inhibitors. The DcpS assay was also applied to study hydrolytic activity and inhibition of endogenous enzyme in cytoplasmic extracts from HeLa and HEK cells.

Exploring the potential of phosphotriazole 5' mRNA cap analogues as efficient translation initiators.

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.

ExciTides: NTP-derived probes for monitoring pyrophosphatase activity based on excimer-to-monomer transitions.

We describe a new type of nucleotide-derived fluorescent probe designed for monitoring pyrophosphatase activity based on excimer-to-monomer transitions, called ExciTide. The nucleotides were designed with two self-interacting dye moieties and synthesised using copper-catalysed azide-alkyne cycloaddition click chemistry. We applied these probes for enzyme activity monitoring and inhibitor evaluation. Some of the probes permeated into living cells, yielding interesting prospects for future applications.

7-Methylguanosine monophosphate analogues with 5'-(1,2,3-triazoyl) moiety: Synthesis and evaluation as the inhibitors of cNIIIB nucleotidase.

The hydrolysis of nucleoside 5'-monophosphates to the corresponding nucleosides and inorganic phosphate is catalysed by 5'-nucleotidases, thereby contributing to the control of endogenous nucleotide turnover and affecting the fate of exogenously delivered nucleotide- and nucleoside-derived therapeutics in cells. A recently identified nucleotidase cNIIIB shows preference towards 7-methylguanosine monophosphate (m7GMP) as a substrate, which suggests its potential involvement in mRNA degradation. However, the extent of biological functions and the significance of cNIIIB remains to be elucidated. Here, we synthesised a series of m7GMP analogues carrying a 1,2,3-triazole moiety at the 5' position as the potential inhibitors of human cNIIIB. The compounds were synthesised by using the copper-catalysed azide-alkyne cycloaddition (CuAAC) between 5'-azido-5'-deoxy-7-methylguanosine and different phosphate or phosphonate derivatives carrying terminal alkyne. The analogues were evaluated as cNIIIB inhibitors using HPLC and malachite green assays, demonstrating that compound 1a, carrying a 1,2,3-triazoylphosphonate moiety, inhibits cNIIIB activity at micromolar concentrations (IC50 87.8 ±â€¯7.5 µM), while other analogues showed no activity. In addition, compound 1d was identified as an artifical substrate for HscNIIIB. Further characterization of inhibitor 1a revealed that it is poorly recognised by other m7G-binding proteins, eIF4E and DcpS, indicating its selectivity towards cNIIIB. The first inhibitor (1a) and unnatural substrate (1d) of cNIIIB, identified here, can be used as molecular probes for the elucidation of biological roles of cNIIIB, including the verification of its proposed function in mRNA metabolism.

Acetylpyrene-labelled 7-methylguanine nucleotides: unusual fluorescence properties and application to decapping scavenger activity monitoring.

7-Methylguanosine (m(7)G) nucleotides labelled with acetylpyrene (AcPy) were synthesized as fluorescent mRNA 5' end (cap) analogues. The unique fluorescent properties of m(7)G-AcPy conjugates, different from G-AcPy, can be applied to studying various mRNA cap-related processes including the evaluation of putative inhibitors of DcpS enzyme-a therapeutic target in neuromuscular diseases.

Synthesis of fluorophosphate nucleotide analogues and their characterization as tools for ¹9F NMR studies.

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).