High-throughput screen, using time-resolved FRET, yields actin-binding compounds that modulate actin–myosin structure and function

Actin's interactions with myosin and other actin-binding proteins are essential for cellular viability in numerous cell types, including muscle. In a previous high-throughput time-resolved FRET (TR-FRET) screen, we identified a class of compounds that bind to actin and affect actomyosin structure and function. For clinical utility, it is highly desirable to identify compounds that affect skeletal and cardiac muscle differently. Because actin is more highly conserved than myosin and most other muscle proteins, most such efforts have not targeted actin. Nevertheless, in the current study, we tested the specificity of the previously discovered actin-binding compounds for effects on skeletal and cardiac α-actins as well as on skeletal and cardiac myofibrils. We found that a majority of these compounds affected the transition of monomeric G-actin to filamentous F-actin, and that several of these effects were different for skeletal and cardiac actin isoforms. We also found that several of these compounds affected ATPase activity differently in skeletal and cardiac myofibrils. We conclude that these structural and biochemical assays can be used to identify actin-binding compounds that differentially affect skeletal and cardiac muscles. The results of this study set the stage for screening of large chemical libraries for discovery of novel compounds that act therapeutically and specifically on cardiac or skeletal muscle.

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Actin-binding compounds, discovered by FRET-based high-throughput screening, differentially affect skeletal and cardiac muscle

10.1101/2020.05.19.104257 ◽

2020 ◽

Cited By ~ 1

Author(s):

Piyali Guhathakurta ◽

Lien A. Phung ◽

Ewa Prochniewicz ◽

Sarah Lichtenberger ◽

Anna Wilson ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Striated Muscle ◽

Muscle Protein ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Structure And Function ◽

Actin Binding ◽

Myosin Isoforms ◽

And Function

AbstractWe have used spectroscopic and functional assays to evaluate the effects of a group of actin-binding compounds on striated muscle protein structure and function. Actin is present in every human cell, and its interaction with multiple myosin isoforms and multiple actin-binding proteins is essential for cellular viability. A previous high-throughput time-resolved fluorescence resonance energy transfer (TR-FRET) assay from our group identified a class of compounds that bind to actin and affect actomyosin structure and function. In the current study, we tested their effects on the two isoforms of striated muscle α-actins, skeletal and cardiac. We found that a majority of these compounds affected the transition of monomeric G-actin to filamentous F-actin, and that these effects were different for the two actin isoforms, suggesting a different mode of action. To determine the effects of these compounds on sarcomeric function, we further tested their activity on skeletal and cardiac myofibrils. We found that several compounds affected ATPase activity of skeletal and cardiac myofibrils differently, suggesting different mechanisms of action of these compounds for the two muscle types. We conclude that these structural and biochemical assays can be used to identify actin-binding compounds that differentially affect skeletal and cardiac muscles. The results of this study set the stage for screening of large chemical libraries for discovery of novel compounds that act therapeutically and specifically on cardiac or skeletal muscle.

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Structure and Function of Myosin Light Chain Kinase of Smooth Muscle: N-Terminal Actin-Binding Domain.

The Kitakanto Medical Journal ◽

10.2974/kmj.47.57 ◽

1997 ◽

Vol 47 (2) ◽

pp. 57-65

Author(s):

Li-Hong Ye

Keyword(s):

Smooth Muscle ◽

Light Chain ◽

Myosin Light Chain Kinase ◽

Myosin Light Chain ◽

Structure And Function ◽

Binding Domain ◽

Actin Binding ◽

Actin Binding Domain ◽

And Function

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Linking RNA Sequence, Structure, and Function on Massively Parallel High-Throughput Sequencers

Cold Spring Harbor Perspectives in Biology ◽

10.1101/cshperspect.a032300 ◽

2018 ◽

Vol 11 (10) ◽

pp. a032300 ◽

Cited By ~ 5

Author(s):

Sarah K. Denny ◽

William J. Greenleaf

Keyword(s):

High Throughput ◽

Structure And Function ◽

Massively Parallel ◽

Sequence Structure ◽

Rna Sequence ◽

And Function

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High-throughput screen using fluorescence lifetime detects compounds that modulate myosin-binding protein C interactions with actin

10.1101/2021.03.24.436879 ◽

2021 ◽

Author(s):

Thomas A. Bunch ◽

Piyali Guhathakurta ◽

Victoria C. Lepak ◽

Andrew R. Thompson ◽

Rhye-Samuel Kanassatega ◽

...

Keyword(s):

Heart Failure ◽

High Throughput ◽

Fluorescence Lifetime ◽

Protein C ◽

Binding Protein ◽

Actin Binding ◽

High Throughput Screen ◽

Myosin Binding Protein ◽

Myosin Binding Protein C ◽

Myosin Binding

ABSTRACTCardiac myosin-binding protein C (cMyBP-C) interacts with actin and myosin to modulate cardiac contractility. These interactions are regulated by cMyBP-C phosphorylation. Heart failure patients often have decreased cMyBP-C phosphorylation and phosphorylation in model systems appears to be cardioprotective for heart failure. Therefore, cMyBP-C is a potential target for heart failure drugs that mimic phosphorylation and/or perturb its interactions with actin/myosin.We have used a novel fluorescence lifetime-based assay to identify small-molecule inhibitors of actin-cMyBP-C binding. Actin was labeled with a fluorescent dye (Alexa Fluor 568, AF568) near its cMyBP-C binding sites. When combined with cMyBP-C N-terminal fragment, C0-C2, the fluorescence lifetime of AF568-actin decreases. Using this reduction in lifetime as a readout of actin binding, a high-throughput screen of a 1280-compound library identified 3 reproducible Hit compounds that reduced C0-C2 binding to actin in the micromolar range. Binding of phosphorylated C0-C2 was also blocked by these compounds. That they specifically block binding was confirmed by a novel actin-C0-C2 time-resolved FRET (TR-FRET) binding assay. Isothermal titration calorimetry (ITC) and transient phosphorescence anisotropy (TPA) confirmed that the Hit compounds bind to cMyBP-C but not to actin. TPA results were also consistent with these compounds inhibiting C0-C2 binding to actin. We conclude that the actin-cMyBP-C lifetime assay permits detection of pharmacologically active compounds that affect cMyBP-C’s actin binding function. TPA, TR-FRET, and ITC can then be used to understand the mechanism by which the compounds alter cMyBP-C interactions with actin.

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High-Throughput Screen for Inhibitors of 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase by Surrogate Ligand Competition

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057103008003011 ◽

2003 ◽

Vol 8 (3) ◽

pp. 332-339 ◽

Cited By ~ 11

Author(s):

Elizabeth B. Gottlin ◽

R. Edward Benson ◽

Scott Conary ◽

Brett Antonio ◽

Kellie Duke ◽

...

Keyword(s):

High Throughput ◽

Binding Sites ◽

Biosynthetic Pathway ◽

Enzyme Assay ◽

High Throughput Screen ◽

Time Resolved Fluorescence ◽

Time Resolved ◽

Biochemical Assays ◽

Key Enzyme ◽

Nadph Oxidation

1-Deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr) is a key enzyme in a biosynthetic pathway for isoprenoids that is unique to eubacteria and plants. Dxr catalyzes the rearrangement and NADPH-dependent reduction of 1-deoxy-D-xylulose 5-phosphate to 2-C-methyl-D-erythritol 4-phosphate. The authors have purified Escherichia coli Dxr and devised a high-throughput screen (HTS) for compounds that bind to this enzyme at a functional site. Evidence is presented that the surrogate ligand directly binds or allosterically affects both the D-1-deoxyxylulose 5-phosphate (DXP) and NADPH binding sites. Compounds that bind at either or both sites that compete for binding with the surrogate ligand register as hits. The time-resolved fluorescence-based assay represents an improvement over the Dxr enzyme assay that relies on relatively insensitive measurements of NADPH oxidation. Screening 32,000 compounds from a diverse historical library, the authors obtained 89 potent inhibitors in the surrogate ligand competition assay. The results presented here suggest that peptide surrogate ligands may be useful in formatting HTS for proteins with difficult biochemical assays or targets of unknown function. ( Journal of Biomolecular Screening 2003:332-339)

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Implementation of High-Throughput Sequencing (HTS) in Aptamer Selection Technology

International Journal of Molecular Sciences ◽

10.3390/ijms21228774 ◽

2020 ◽

Vol 21 (22) ◽

pp. 8774

Author(s):

Natalia Komarova ◽

Daria Barkova ◽

Alexander Kuznetsov

Keyword(s):

Nucleic Acid ◽

High Throughput ◽

High Throughput Sequencing ◽

Combinatorial Libraries ◽

Structure And Function ◽

Huge Number ◽

Systematic Evolution ◽

And Function ◽

Exponential Enrichment

Aptamers are nucleic acid ligands that bind specifically to a target of interest. Aptamers have gained in popularity due to their high potential for different applications in analysis, diagnostics, and therapeutics. The procedure called systematic evolution of ligands by exponential enrichment (SELEX) is used for aptamer isolation from large nucleic acid combinatorial libraries. The huge number of unique sequences implemented in the in vitro evolution in the SELEX process imposes the necessity of performing extensive sequencing of the selected nucleic acid pools. High-throughput sequencing (HTS) meets this demand of SELEX. Analysis of the data obtained from sequencing of the libraries produced during and after aptamer isolation provides an informative basis for precise aptamer identification and for examining the structure and function of nucleic acid ligands. This review discusses the technical aspects and the potential of the integration of HTS with SELEX.

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A Comparison between Vanadyl, Vanadate, and Decavanadate Effects in Actin Structure and Function: Combination of Several Spectroscopic Studies

Spectroscopy An International Journal ◽

10.1155/2012/532904 ◽

2012 ◽

Vol 27 ◽

pp. 355-359 ◽

Cited By ~ 3

Author(s):

S. Ramos ◽

J. J. G. Moura ◽

M. Aureliano

Keyword(s):

Actin Polymerization ◽

Hydrophobic Surface ◽

Spectroscopic Studies ◽

Structure And Function ◽

Actin Binding ◽

Cysteine Oxidation ◽

Natural Ligand ◽

Function Combination ◽

Actin Structure ◽

And Function

The studies about the interaction of actin with vanadium are seldom. In the present paper the effects of vanadyl, vanadate, and decavanadate in the actin structure and function were compared. Decavanadate clearly interacts with actin, as shown byV51-NMR spectroscopy. Decavanadate interaction with actin induces protein cysteine oxidation and vanadyl formation, being both prevented by the natural ligand of the protein, ATP. Monomeric actin (G-actin) titration with vanadyl, as analysed by EPR spectroscopy, indicates a 1 : 1 binding stoichiometry and akdof 7.5 μM. Both decavanadate and vanadyl inhibited G-actin polymerization into actin filaments (F-actin), with aIC50of 68 and 300 μM, respectively, as analysed by light-scattering assays. However, only vanadyl induces G-actin intrinsic fluorescence quenching, which suggests the presence of vanadyl high-affinity actin-binding sites. Decavanadate increases (2.6-fold) actin hydrophobic surface, evaluated using the ANSA probe, whereas vanadyl decreases it (15%). Finally, both vanadium species increasedε-ATP exchange rate (k=6.5×10−3and4.47×10−3 s−1for decavanadate and vanadyl, resp.). Putting it all together, it is suggested that actin, which is involved in many cellular processes, might be a potential target not only for decavanadate but above all for vanadyl.

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