Fluorescent peptide displacement as a general assay for screening small molecule libraries against RNA

Cai, Zhipeng; Newson, Colby N.; Hargrove, Amanda E.

doi:10.1039/c8ob02467g

Cited by 35 publications

(29 citation statements)

References 80 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…More recently, Hargrove and co-workers demonstrated the use of FTatRhd to screen simultaneously small molecular ligands against multiple RNA targets of interest while assessing the associated binding affinities and selectivities using the same assay. 33 In this work, similar sized RNA targets (HIV-1-TAR, HIV-2-TAR, A-site, RRE-IIB) possessing different secondary structure were shown to bind to FTatRhd with low nanomolar affinities. This improved system required only low concentrations of RNA (nM) to be effective.…”

Section: Ida-based Sensors For Nucleic Acidsmentioning

confidence: 89%

Indicator displacement assays (IDAs): the past, present and future

et al. 2021

View full text Add to dashboard Cite

show abstract

Section: Ida-based Sensors For Nucleic Acidsmentioning

confidence: 89%

Indicator displacement assays (IDAs): the past, present and future

et al. 2021

View full text Add to dashboard Cite

show abstract

“…Binding constant of the fluorescently labeled peptide for EV71 SLII RNA, was measured using previously reported procedure 20…”

Section: Binding Assay For Indicator Peptide To Ev71 Slii Rnamentioning

confidence: 99%

“…Titrations of amiloride derivatives for binding to both the wild type and mutant RNAs was performed as previously described 3,20 . Briefly, small molecules were diluted in the assay buffer to achieve final assay concentrations between 0-334 µM.…”

Section: Full Binding Titrations Of Amiloride Derivatives With Ev71 Sliimentioning

confidence: 99%

Small Molecule Targeting IRES Domain Inhibits Enterovirus 71 Replication via an Allosteric Mechanism that Stabilizes a Ternary Complex

Davila‐Calderon

Chiu

Sugarman

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

We herein report an RNA-targeting antiviral small molecule that reduces replication of the human enterovirus 71 (EV71) via stabilization of an inhibitory small molecule-RNA-protein ternary complex. The EV71 virus poses serious threats to human health, particularly in regions of Southeast Asia, and no FDA approved drugs or vaccines are available. We first screened an RNA-biased small molecule library using a peptide-displacement assay to identify ligands for the stem loop II structure of the EV71 internal ribosomal entry site, which was previously shown to impact viral translation and replication. One ligand, DMA-135, decreased viral translation and replication in cell-based studies in a dosedependent manner with no significant toxicity. Structural, biophysical, and biochemical characterization support an allosteric mechanism in which DMA-135 induces a conformational change in the RNA structure that stabilizes a ternary complex with the AUF1 protein that then represses translation. This mechanism was further supported by pull-down experiments in cell culture. These detailed studies establish enterovirus RNA structures as promising drug targets while revealing an approach and mechanism of action that should be broadly applicable to functional RNA targeting.

show abstract

“…Next, we tested the ability of the compounds to displace peptides mimics of the cognate proteins that bind TAR and RREIIB using fluorescence-based assays. These assays have previously been used in screening for an evaluating TAR and RRE binders (Matsumoto et al 2000;Hamasaki and Ueno 2001;Patwardhan et al 2017Patwardhan et al , 2019Zeiger et al 2014). The TAR assay measures fluorescence resonance energy transfer (FRET) of a dual-labeled arginine rich motif (ARM) Tat-mimic peptide (Matsumoto et al 2000;Ganser et al 2018), and the RRE assay measures fluorescence anisotropy of an ARM Rev-mimic peptide (Luedtke and Tor 2003;Chu et al 2019).…”

Section: Assaying Binding Using Rna-peptide Displacement Assaysmentioning

confidence: 99%

Revealing the high propensity of RNAs to non-specifically bind drug-like small molecules

Kelly

Chu²,

Shi

et al. 2020

Preprint

View full text Add to dashboard Cite

Identifying small molecules that selectively bind a single RNA target while discriminating against all other cellular RNAs is an important challenge in RNA-targeted drug discovery. Much effort has been directed toward identifying drug-like small molecules that minimize electrostatic and stacking interactions that lead to non-specific binding of aminoglycosides and intercalators to a variety of RNAs. Many such compounds have been reported to bind RNAs and inhibit their cellular activities, however the ability of such compounds to discriminate against RNA stem-loops commonly found in the transcriptome has not been thoroughly assessed in all cases. Here, we examined the propensities of three drug-like compounds, previously shown to bind and inhibit the cellular activity of three distinct RNAs, to non-specifically bind two HIV-1 stem-loop RNAs: the transactivation response element (TAR) and stem IIB in the rev response element (RREIIB). All three compounds bound to TAR and RREIIB in vitro, and two inhibited TAR-dependent transactivation and RRE-dependent viral export in cell-based assays while also exhibiting substantial off-target interactions consistent with non-specific cellular activity. A survey of X-ray and NMR structures of RNA-small molecule complexes revealed that drug-like molecules form hydrogen bonds with functional groups commonly accessible in canonical stem-loop RNA motifs, much like aminoglycosides, and in contrast to ligands that specifically bind riboswitches. Our results support extending the group of non-selective RNA-binders beyond aminoglycosides and intercalators to encompass drug-like compounds with capacity for non-specific hydrogen-bonding and reinforce the importance of assaying for off-target interactions and RNA selectivity in vitro and in cells when assessing novel RNA-binders.

show abstract

Fluorescent peptide displacement as a general assay for screening small molecule libraries against RNA

Abstract: A fluorescent peptide displacement assay combined with statistical analysis is used for screening small molecules against different RNA targets and profiling their affinity/selectivity patterns.

Cited by 35 publications

References 80 publications

Indicator displacement assays (IDAs): the past, present and future

Indicator displacement assays (IDAs): the past, present and future

Small Molecule Targeting IRES Domain Inhibits Enterovirus 71 Replication via an Allosteric Mechanism that Stabilizes a Ternary Complex

Revealing the high propensity of RNAs to non-specifically bind drug-like small molecules

Contact Info

Product

Resources

About