Dataset Information

Design of a Fluorescence Polarization Probe for Enterovirus 2C Proteins.

ABSTRACT: Enteroviruses (EVs), such as EV-D68, EV-A71, and CVB3, cause significant human disease; yet, no antivirals are currently approved. The highly conserved 2C protein, an essential AAA+ ATPase and helicase, is a prime antiviral target; however, it lacks suitable assays for inhibitor screening. Here, we report a fluorescence polarization (FP) assay using a rationally designed probe, Jun14157, which binds a conserved allosteric site in 2C with high affinity. This assay enables the quantitative assessment of binding to diverse 2C inhibitors with high signal-to-background ratios, DMSO tolerance, and a strong correlation between FP K_i and cellular EC₅₀. Using this platform, we validated hits from virtual screening and identified two novel inhibitors, Jun15716 and Jun15799. This FP assay offers a robust and scalable tool for the mechanistic characterization and high-throughput screening of 2C-targeting antivirals.

SUBMITTER: Li K

PROVIDER: S-EPMC12333363 | biostudies-literature | 2025 Jul

REPOSITORIES: biostudies-literature

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Publications

Design of a Fluorescence Polarization Probe for Enterovirus 2C Proteins.

Li Kan K Demssie Hiwot A HA Wang Jun J

Journal of medicinal chemistry 20250621 13

Enteroviruses (EVs), such as EV-D68, EV-A71, and CVB3, cause significant human disease; yet, no antivirals are currently approved. The highly conserved 2C protein, an essential AAA+ ATPase and helicase, is a prime antiviral target; however, it lacks suitable assays for inhibitor screening. Here, we report a fluorescence polarization (FP) assay using a rationally designed probe, <b>Jun14157</b>, which binds a conserved allosteric site in 2C with high affinity. This assay enables the quantitative ...[more]

PMID: 40542722

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Project description:MicroRNAs (miRNAs) have been shown to play an important role in many different cellular, developmental, and physiological processes. Accordingly, numerous methods have been established to identify and quantify miRNAs. The shortness of miRNA sequence results in a high dynamic range of melting temperatures and, moreover, impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here, we present a microarray based approach for global and absolute quantification of miRNAs. The method relies on an equimolar pool of about 1000 synthetic miRNAs of known concentration which is used as an universal reference and labeled and hybridized in a dual colour approach on the same array as the sample of interest. Each single miRNA is quantified with respect to the universal reference outbalancing bias related to sequence, labeling, hybridization or signal detection method. We demonstrate the accuracy of the method by various spike in experiments. Further, we quantified miRNA copy numbers in liver samples and CD34(+)CD133(-) hematopoietic stem cells. Different probe designs were investigated with respect to sensitivity and selectivity of microarray hybridization. An array with 11 different variants of oligonucleotides for miR-122a and miR-16 was produced. 5 µg of respective total RNA was fluorescently labelled by 3’ ligation. Total RNA was hybridized in a dual colour approach to microarrays versus a second labelled synthetic miRNA pool. The synthetic miRNA pool consisted of 5 fmol of each of 816 non redundant miRNAs sequences and miRControl 3 sequences. Local background was subtracted from the signal to obtain the net signal intensity and the mean of the net signal intensities of 4 corresponding spots representing the same miRNA was computed for those spots only which were unflagged (empty spots, poor spots, negative spots) and for which the fluorescent intensity of the miRNAs derived from the samples of interest was two-fold the mean background value (2bkg dataset).

Dataset Information

Design of a Fluorescence Polarization Probe for Enterovirus 2C Proteins.

Publications

Design of a Fluorescence Polarization Probe for Enterovirus 2C Proteins.

Similar Datasets

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