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.
Abstract
Proteases encoded by SARS-CoV-2 constitute a promising target for new therapies against COVID-19. SARS-CoV-2 main protease (M
pro
, 3CL
pro
) and papain-like protease (PL
pro
) are ...responsible for viral polyprotein cleavage—a process crucial for viral survival and replication. Recently it was shown that 2-phenylbenzisoselenazol-3(2
H
)-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 PL
pro
and M
pro
inhibitors. Our studies revealed that ebselen derivatives are potent inhibitors of both the proteases. We identified three PL
pro
and four M
pro
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.
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 m
Gp
G 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.
In eukaryotes, mRNA is modified by the addition of the 7‐methylguanosine (m7G) 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.
To escape recognition by the host innate immune system, viruses often incorporate 5′ caps into their mRNAs. The differences in mRNA capping make this process an interesting therapeutic target. Here, we review methods to explore the enzymes involved in cap biosynthesis. Such methods allow inhibitor selection, facilitating antiviral drug discovery, which is especially important considering the ongoing COVID‐19 pandemic and possible future outbreaks.
Posttranslational modifications (PTMs) greatly enhance the functional diversity of proteins, surpassing the number of gene‐encoded variations. One intriguing PTM is ADP‐ribosylation, which utilizes ...nicotinamide adenine dinucleotide (NAD+) as a substrate and is essential in cell signaling pathways regulating cellular responses. Here, we report the first cell‐permeable NAD+ analogs and demonstrate their utility for investigating cellular ADP‐ribosylation. Using a desthiobiotin‐labelled analog for affinity enrichment of proteins that are ADP‐ribosylated in living cells under oxidative stress, we identified protein targets associated with host‐virus interactions, DNA damage and repair, protein biosynthesis, and ribosome biogenesis. Most of these targets have been noted in various literature sources, highlighting the potential of our probes for cellular ADP‐ribosylome studies.
Abstract RNA 2′-phosphotransferase Tpt1 catalyzes the removal of an internal RNA 2′-PO4 via a two-step mechanism in which: (i) the 2′-PO4 attacks NAD+ C1″ to form an RNA-2′-phospho-(ADP-ribose) ...intermediate and nicotinamide; and (ii) transesterification of the ADP-ribose O2″ to the RNA 2′-phosphodiester yields 2′-OH RNA and ADP-ribose-1″,2″-cyclic phosphate. Although Tpt1 enzymes are prevalent in bacteria, archaea, and eukarya, Tpt1 is uniquely essential in fungi and plants, where it erases the 2′-PO4 mark installed by tRNA ligases during tRNA splicing. To identify a Tpt1 ‘poison’ that arrests the reaction after step 1, we developed a chemical synthesis of 2″OMeNAD+, an analog that cannot, in principle, support step 2 transesterification. We report that 2″OMeNAD+ is an effective step 1 substrate for Runella slithyformis Tpt1 (RslTpt1) in a reaction that generates the normally undetectable RNA-2′-phospho-(ADP-ribose) intermediate in amounts stoichiometric to Tpt1. EMSA assays demonstrate that RslTpt1 remains trapped in a stable complex with the abortive RNA-2′-phospho-(ADP-2″OMe-ribose) intermediate. Although 2″OMeNAD+ establishes the feasibility of poisoning and trapping a Tpt1 enzyme, its application is limited insofar as Tpt1 enzymes from fungal pathogens are unable to utilize this analog for step 1 catalysis. Analogs with smaller 2″-substitutions may prove advantageous in targeting the fungal enzymes.
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.
Unveiling PARP‐selective protein targets is crucial for a better understanding of ADP‐ribosylation. Here, we report a novel chemically modified NAD+ analog which can be applied in the “bump‐and‐hole” strategy for PARP1. The analog bears an ethyl group (“bump”) at the nicotinamide moiety that drives selectivity towards a PARP1 mutant. An affinity tag linked to the adenine moiety enables affinity enrichment of PARP1 specific ADP‐ribosylation targets.
Abstract
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.
The m7G 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 m7G 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 m7GTP 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.
Caps on/off: The m7G cap is a unique nucleotide structure at the 5′‐end of all eukaryotic mRNAs. To provide small molecular probes to study important cap‐recognizing proteins, m7G nucleotides labeled with fluorescent tags were synthesized and changes to their emission properties upon incubation with eIF4E and DcpS proteins were studied (see figure). Pyrene‐labeled compounds were selected as sensitive turn‐on probes and used to develop binding‐ and hydrolytic‐activity assays for studies on eukaryotic translation initiation factor 4E (eIF4E) and decapping scavenger (DcpS) enzyme.
Posttranslational modifications (PTMs) greatly enhance the functional diversity of proteins, surpassing the number of gene‐encoded variations. One intriguing PTM is ADP‐ribosylation, which utilizes ...nicotinamide adenine dinucleotide (NAD+) as a substrate and is essential in cell signaling pathways regulating cellular responses. Here, we report the first cell‐permeable NAD+ analogs and demonstrate their utility for investigating cellular ADP‐ribosylation. Using a desthiobiotin‐labelled analog for affinity enrichment of proteins that are ADP‐ribosylated in living cells under oxidative stress, we identified protein targets associated with host‐virus interactions, DNA damage and repair, protein biosynthesis, and ribosome biogenesis. Most of these targets have been noted in various literature sources, highlighting the potential of our probes for cellular ADP‐ribosylome studies.
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