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

Anna Wojtczak,Jacek Jemielity,Dorota Kubacka,Pawel J Sikorski,Renata Kasprzyk,Joanna Kowalska,Krystian Ubych,Marcin Warmiński

doi:10.1038/s41598-021-87306-8

Anna Wojtczak, Jacek Jemielity + Show 6 more

Open Access

https://doi.org/10.1038/s41598-021-87306-8

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Abstract

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.

Highlights

A 7-methylguanosine cap structure is present at the 5′ end of eukaryotic mRNA and influences numerous cellular functions related to mRNA m etabolism1. 7-Methylguanosine ( m7G) is a positively charged nucleoside that, together with the negatively charged 5′,5′-triphosphate chain linking it to the first transcribed nucleotide of RNA, creates a unique molecular recognition pattern targeted by specific proteins involved in mRNA turnover[2,3]
decapping scavenger enzyme (DcpS) is a therapeutic target in spinal muscular atrophy (SMA), an autosomal recessive disease caused by deletion or mutational inactivation of the survival motor neuron (SMN) 1 gene[19]
As a starting point for the design of the probes, we used several known cap-derived eIF4E and DcpS binders differing in structural complexity (Fig. 1)

Summary

Introduction

A 7-methylguanosine cap structure is present at the 5′ end of eukaryotic mRNA and influences numerous cellular functions related to mRNA m etabolism1. 7-Methylguanosine ( m7G) is a positively charged nucleoside that, together with the negatively charged 5′,5′-triphosphate chain linking it to the first transcribed nucleotide of RNA, creates a unique molecular recognition pattern targeted by specific proteins involved in mRNA turnover[2,3]. Identifying new high-affinity ligands to limit active pools of eIF4E is the first step towards the development of therapeutic strategies in anticancer t reatment[12,13]. Another cap-binding protein is decapping scavenger enzyme (DcpS), which prevents the accumulation of free cap structures released as a result of 3′-to-5′ mRNA decay, thereby blocking inhibition of proteins crucial for mRNA splicing and translation and avoiding potentially toxic e ffects[14]. A commonly used technique to study interactions between cap-binding proteins and ligands is fluorescence quenching titration (tsFQT)[3] This approach is time-consuming, requires relatively high protein concentrations, is low throughput, and has other methodological limitations, which make it unsuitable for drug discovery applications. DcpS-binding ligands have been studied using other methods, including high-performance liquid c hromatography[33], microscale thermophoresis34, tsFQT35, and radioactive assays[15]

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