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

Guhathakurta, Piyali; Próchniewicz, Ewa; Grant, Benjamin D.; Peterson, Kurt C.; Thomas, David D.

doi:10.1074/jbc.ra118.002702

Cited by 21 publications

(50 citation statements)

References 40 publications

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“…Fluphenazine, thioridazine, and novantrone inhibited ATPase of both muscle types and had reduced the rate of polymerization of both actin isoforms. This is consistent with our previous study, where these three compounds had maximum effect on F-actin structure and functional interaction with myosin (11). Thus, compound-specific effects are transmitted from actin to the myofibril level.…”

Section: Specificity Of Compounds For Muscle Typessupporting

confidence: 93%

“…The pK Ca values of skeletal and cardiac myofibrils are 6.06 ± 0.01 and 5.76 ± 0.02. These values are consistent with previous reports (10,11,18,(20)(21)(22). The myofibril ATPase activities of both muscle systems were not affected either by the presence of 1% DMSO (data not shown) or by 500 nM thapsigargin, a SERCA (sarcoendoplasmic reticulum calcium transport ATPase pump) modulator (data not shown).…”

Section: Effects Of Compounds On Skeletal and Cardiac Myofibril Atpasesupporting

confidence: 92%

“…Most pronounced changes were observed in the presence of fluphenazine, novantrone, and thioriadazine. These compounds also have pronounced effects on skeletal actin filament dynamics and on the environment of the C-terminus of actin, as detected by transient phosphorescence anisotropy (TPA) in our previous study (11). Additionally, thioriadazine, phenothiazine, and tegaserod exhibit significantly different isoform-specificity.…”

Section: Specificity Of Compounds For Actin Isoformsmentioning

confidence: 78%

“…The current study shows that actin-binding compounds, previously discovered by high-throughput FRET screening, have specific biochemical and functional effects that are distinct in cardiac and skeletal actin, and in cardiac and skeletal muscle. This sets the stage to apply the original high-throughput FRET screen to larger small-molecule libraries, leading to the discovery of novel actin-binding compounds with specific therapeutic potential for treating disorders of cardiac or skeletal muscle (11,40).…”

Section: Resultsmentioning

confidence: 99%

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

Guhathakurta

Phung

Próchniewicz

et al. 2020

Preprint

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We 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 highthroughput 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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Section: Specificity Of Compounds For Muscle Typessupporting

confidence: 93%

Section: Effects Of Compounds On Skeletal and Cardiac Myofibril Atpasesupporting

confidence: 92%

Section: Specificity Of Compounds For Actin Isoformsmentioning

confidence: 78%

Section: Resultsmentioning

confidence: 99%

See 2 more Smart Citations

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

Guhathakurta

Phung

Próchniewicz

et al. 2020

Preprint

Self Cite

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“…Since we have no clue on the potential determinant of this interaction, an extensive mutagenesis scanning would have to be undertaken. Moreover, to assess the impact of these mutations on actin binding and channels’ trafficking would require sophisticated imaging techniques such as FRET/BRET (Guhathakurta et al, ). Nevertheless, our data support the idea that Ca V β2a, via its SH3 domain, might constitute a regulatory molecule that controls the plasma membrane expression of ion channels by modulating actin remodeling.…”

Section: Discussionmentioning

confidence: 99%

The Ca²⁺ channel subunit Ca_Vβ2a‐subunit down‐regulates voltage‐activated ion current densities by disrupting actin‐dependent traffic in chromaffin cells

Guerra

González‐Jamett

Báez‐Matus

et al. 2019

Journal of Neurochemistry

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β‐Subunits of the Ca2+ channel have been conventionally regarded as auxiliary subunits that regulate the expression and activity of the pore‐forming α1 subunit. However, they comprise protein–protein interaction domains, such as a SRC homology 3 domain (SH3) domain, which make them potential signaling molecules. Here we evaluated the role of the β2a subunit of the Ca2+ channels (CaVβ2a) and its SH3 domain (β2a‐SH3) in late stages of channel trafficking in bovine adrenal chromaffin cells. Cultured bovine adrenal chromaffin cells were injected with CaVβ2a or β2a‐SH3 under different conditions, in order to acutely interfere with endogenous associations of these proteins. As assayed by whole‐cell patch clamp recordings, Ca2+ currents were reduced by CaVβ2a in the presence of exogenous α1‐interaction domain. β2a‐SH3, but not its dimerization‐deficient mutant, also reduced Ca2+ currents. Na+ currents were also diminished following β2a‐SH3 injection. Furthermore, β2a‐SH3 was still able to reduce Ca2+ currents when dynamin‐2 function was disrupted, but not when SNARE‐dependent exocytosis or actin polymerization was inhibited. Together with the additional finding that both CaVβ2a and β2a‐SH3 diminished the incorporation of new actin monomers to cortical actin filaments, β2a‐SH3 emerges as a signaling module that might down‐regulate forward trafficking of ion channels by modulating actin dynamics.

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The Application of High‐Throughput Approaches in Identifying Novel Therapeutic Targets and Agents to Treat Diabetes

Lim

2022

Advanced Biology

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An early example of HTS in drug development came from the identification of new antibiotics using 96-well microtiter plates, whereby fermentations of an established actinomycetes library were incubated with a panel of microorganisms, and after optimization, a screening capacity of 10 000 fermentation broths per week was achieved. [1] In the following decades, the rapid development of molecular biology techniques and advances in automated equipment have changed how new drugs are identified, and a variety of HTS-identified drugs have been approved to treat different diseases. [2] For a comprehensive list of HTS-identified drugs, please refer to the excellent review by Macarron et al. [2] High-throughput applications can also be combined with other unbiased "Omics" technologies used to study genomics, epigenomics, transcriptomics, proteomics, and metabolomics. [3] Diabetes is an incurable disease characterized by the inability of the body to maintain normoglycemia due to decreased sensitivity of various tissues to insulin and insufficient amounts of circulating insulin, a hormone produced by pancreatic β-cells. [4] Diabetes is associated with chronic complications, like macrovascular diseases (e.g., cardiovascular diseases) and microvascular diseases (e.g., damages in nerve, kidney, eye, and foot). [5,6] More than 537 million adults are currently living with diabetes, and this number is expected to increase to 693 million by 2045. In 2021, 6.7 million deaths worldwide were attributed to diabetes, [7] which made it the ninth leading cause of death. [8] Globally, the prevalence of diabetes also brings a heavy burden to healthcare systems, with an estimated expenditure of over 966 billion U.S. dollars per year. [7] Diabetes is generally categorized into two groups: Type I diabetes mellitus (T1DM) and Type II diabetes mellitus (T2DM), but there are still some cases that have different etiologies, such as gestational diabetes mellitus and monogenic diabetes. [9] Since the discovery of insulin in 1921, [10] significant progress has been made in diabetes pharmacotherapy and therapeutic technology; [11][12][13] however, since a permanent cure is unavailable, the discovery and development of anti-diabetic drugs is still at the forefront of diabetes research. With the rapid advancement in high-throughput omics technologies, combined with accumulated genome-wide association study (GWAS) data, a better understanding of diabetes pathophysiology may one day lead to the discovery of novel therapeutic targets. [14,15] This review will During the past decades, unprecedented progress in technologies has revolutionized traditional research methodologies. Among these, advances in highthroughput drug screening approaches have permitted the rapid identification of potential therapeutic agents from drug libraries that contain thousands or millions of molecules. Moreover, high-throughput-based therapeutic target discovery strategies can comprehensively interrogate relationships between biomolecules (e.g., gene, RNA, and protein) and diseas...

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

Cited by 21 publications

References 40 publications

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

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

The Ca²⁺ channel subunit Ca_Vβ2a‐subunit down‐regulates voltage‐activated ion current densities by disrupting actin‐dependent traffic in chromaffin cells

The Application of High‐Throughput Approaches in Identifying Novel Therapeutic Targets and Agents to Treat Diabetes

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

Cited by 21 publications

References 40 publications

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

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

The Ca2+ channel subunit CaVβ2a‐subunit down‐regulates voltage‐activated ion current densities by disrupting actin‐dependent traffic in chromaffin cells

The Application of High‐Throughput Approaches in Identifying Novel Therapeutic Targets and Agents to Treat Diabetes

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Product

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The Ca²⁺ channel subunit Ca_Vβ2a‐subunit down‐regulates voltage‐activated ion current densities by disrupting actin‐dependent traffic in chromaffin cells