A fluorescent HTS assay for phosphohydrolases based on nucleoside 5′-fluorophosphates: its application in screening for inhibitors of mRNA decapping scavenger and PDE-I
Abstract:Several nucleotide-specific phosphohydrolases can cleave P-F bonds in substrate analogues containing a fluorophosphate moiety to release fluoride ions. In this work, by employing a fluoride-sensitive molecular sensor, we harnessed this cleavage reaction to develop a fluorescence assay to screen for phosphohydrolase inhibitors. The assay is rapid, sensitive, and based on simple and synthetically available reagents. The assay was adapted to the high-throughput screening (HTS) format and its utility was demonstra… Show more
“…To gain insight into how the presence of the triazole moiety influences the cap–DcpS interaction, the analogues were also evaluated as hDcpS inhibitors using a recently developed fluorescent HTS assay. 43 The relatively low % inhibition parameters (Fig. S5†) and high IC 50 values (Fig.…”
A different approach for synthesizing 5′ cap mimics to yield a novel class of dinucleotide cap analogues containing a triazole ring within the oligophosphate chain.
“…To gain insight into how the presence of the triazole moiety influences the cap–DcpS interaction, the analogues were also evaluated as hDcpS inhibitors using a recently developed fluorescent HTS assay. 43 The relatively low % inhibition parameters (Fig. S5†) and high IC 50 values (Fig.…”
A different approach for synthesizing 5′ cap mimics to yield a novel class of dinucleotide cap analogues containing a triazole ring within the oligophosphate chain.
“…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 .…”
Section: Nucleotidesmentioning
confidence: 99%
“…[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. [189] Of particular interest wasr eplacement of the 5'-oxygen atom by a sulfur atom, giving rise to relevant molecules that were evaluated fort heir capacity to inhibit the decapping process or increase the translational potential of mRNA.…”
Although the phosphorus atom is found in a variety of oxidation states, most of the phosphorus‐containing molecules of pharmacological importance possess phosphorus in the form of phosphonate or phosphinate functional groups, or in a major oxidation state as a phosphate group. The most common occurrence of phosphorus in drugs is either in prodrugs or in compounds for which the phosphorus atom plays a role in the biological activity, such as in modified nucleotides, in metabolically stable analogues of metabolites bearing phosphate groups, and as bioisosteric analogues of carboxyl groups.
“…As a more sensitive alternative to 19 F NMR experiments, a fluorescent inhibitor screening assay for DcpS based on P–F bond cleavage in m 7 GMPF and the use of a fluoride-sensitive chemosensor for product quantitation has been developed (Fig. 10b) [97]. Fig.…”
Section: Chemically and Enzymatically Labeled Rna Capsmentioning
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 m7G 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 modification of natural RNA properties. Here, we review chemical, enzymatic, and chemoenzymatic approaches that enable preparation of modified cap structures and RNAs carrying such structures, with emphasis on phosphate-modified mRNA cap analogs and their potential applications.
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