High-Throughput FRET Assay Yields Allosteric SERCA Activators

Cornea, Rǎzvan L.; Gruber, Simon; Lockamy, Elizabeth L.; Muretta, Joseph M.; Jin, Dayong; Chen, Jiqiu; Dahl, Russell; Bartfai, Tamás; Zsebo, Krisztina M.; Gillispie, Gregory D.; Thomas, David D.

doi:10.1177/1087057112456878

Cited by 80 publications

(104 citation statements)

References 37 publications

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“…Here we assessed the metabolic effects of a small molecule SERCA2 activator, CDN1163, that acts directly on SERCA enzyme probably via allosteric mechanism to activate SERCA2's Ca 2ϩ -ATPase activity and improve Ca 2ϩ homeostasis (16). We demonstrate that SERCA2b activation via CDN1163 attenuates ER stress, ameliorates mitochondrial efficiency, and improves glucose and lipid metabolism.…”

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confidence: 99%

Small Molecular Allosteric Activator of the Sarco/Endoplasmic Reticulum Ca2+-ATPase (SERCA) Attenuates Diabetes and Metabolic Disorders

Kang

Dahl

Hsieh

et al. 2016

Journal of Biological Chemistry

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158

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Dysregulation of endoplasmic reticulum (ER) Ca2؉ homeostasis triggers ER stress leading to the development of insulin resistance in obesity and diabetes. Impaired function of the sarco/endoplasmic reticulum Ca 2؉ -ATPase (SERCA) has emerged as a major contributor to ER stress. We pharmacologically activated SERCA2b in a genetic model of insulin resistance and type 2 diabetes (ob/ob mice) with a novel allosteric activator, CDN1163, which markedly lowered fasting blood glucose, improved glucose tolerance, and ameliorated hepatosteatosis but did not alter glucose levels or body weight in lean controls. Importantly, CDN1163-treated ob/ob mice maintained euglycemia comparable with that of lean mice for >6 weeks after cessation of CDN1163 administration. CDN1163-treated ob/ob mice showed a significant reduction in adipose tissue weight with no change in lean mass, assessed by magnetic resonance imaging. They also showed an increase in energy expenditure using indirect calorimetry, which was accompanied by increased expression of uncoupling protein 1 (UCP1) and UCP3 in brown adipose tissue. CDN1163 treatment significantly reduced the hepatic expression of genes involved in gluconeogenesis and lipogenesis, attenuated ER stress response and ER stress-induced apoptosis, and improved mitochondrial biogenesis, possibly through SERCA2-mediated activation of AMP-activated protein kinase pathway. The findings suggest that SERCA2b activation may hold promise as an effective therapy for type-2 diabetes and metabolic dysfunction.Obesity and insulin resistance are major causes of type 2 diabetes (T2D), 2 which represents an enormous health burden to societies worldwide. T2D is now one of the most prevalent diseases globally and is the fourth leading cause of death in many developed countries (1). Endoplasmic reticulum stress (ER stress) has emerged as an important cause of the metabolic syndrome and T2D. ER stress and the unfolded protein response have now been described in organs playing key roles in metabolic homeostasis such as liver, pancreatic ␤-cells, adipose tissue, and hypothalamus in both obese and/or diabetic humans and rodents (2-5) and have recently emerged as key pathophysiological pathways triggering insulin resistance and T2D (4). Amelioration of ER stress through chemical chaperones has been demonstrated to be a promising pharmacological strategy for treatment of T2D (6 -10). The ER is the main storage site of intracellular Ca 2ϩ , and alterations in Ca 2ϩ homeostasis have been demonstrated to trigger ER stress and activation of the unfolded protein response (11, 12). The sarco/endoplasmic reticulum Ca 2ϩ -ATPase (SERCA) pumps Ca 2ϩ from the cytoplasm into the ER. Recent studies demonstrate that SERCA dysfunction leads to elevation of cytoplasmic calcium and triggers ER stress. SERCA2 activity and expression is diminished in islets (13), liver (2, 14), and heart (15) in animal models of obesity/ diabetes, highlighting a potential pathological role for SERCA2 dysfunction and disturbed ER Ca 2ϩ homeostasis in the dev...

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confidence: 99%

Small Molecular Allosteric Activator of the Sarco/Endoplasmic Reticulum Ca2+-ATPase (SERCA) Attenuates Diabetes and Metabolic Disorders

Kang

Dahl

Hsieh

et al. 2016

Journal of Biological Chemistry

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158

150

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“…We hypothesize that this TR-FRET approach can be used as a tool to screen for compounds that rescue defects in actomyosin structure and function. Small-molecule modulators of actomyosin structural dynamics represent potential leads for future drug development, and this search is greatly facilitated by recent developments in high-throughput FRET-based screening methods, which measure the effects of compounds on the distance between donor and acceptor probes on interacting proteins (1,(12)(13)(14)(15).…”

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confidence: 99%

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

Guhathakurta

Próchniewicz

Grant

et al. 2018

Journal of Biological Chemistry

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We have used a novel time-resolved FRET (TR-FRET) assay to detect small-molecule modulators of actin-myosin structure and function. Actin-myosin interactions play crucial roles in the generation of cellular force and movement. Numerous mutations and post-translational modifications of actin or myosin disrupt muscle function and cause life-threatening syndromes. Here, we used a FRET biosensor to identify modulators that bind to the actin-myosin interface and alter the structural dynamics of this complex. We attached a fluorescent donor to actin at Cys-374 and a nonfluorescent acceptor to a peptide containing the 12 N-terminal amino acids of the long isoform of skeletal muscle myosin's essential light chain. The binding site on actin of this acceptor-labeled peptide (ANT) overlaps with that of myosin, as indicated by () a similar distance observed in the actin-ANT complex as in the actin-myosin complex and () a significant decrease in actin-ANT FRET upon binding myosin. A high-throughput FRET screen of a small-molecule library (NCC, 727 compounds), using a unique fluorescence lifetime readout with unprecedented speed and precision, showed that FRET is significantly affected by 10 compounds in the micromolar range. Most of these "hits" alter actin-activated myosin ATPase and affect the microsecond dynamics of actin detected by transient phosphorescence anisotropy. We conclude that the actin-ANT TR-FRET assay enables detection of pharmacologically active compounds that affect actin structural dynamics and actomyosin function. This assay establishes feasibility for the discovery of allosteric modulators of the actin-myosin interaction, with the ultimate goal of developing therapies for muscle disorders.

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“…45,46 Further, a high-throughput fluorescence resonance energy transfer assay has been used to find allosteric SERCA2a activators capable of decreasing SERCA2a/PLN-derived fluorescence resonance energy transfer. 47 In this study, several compounds previously reported to correct aberrant Ca 2+ regulation in HF were shown to activate SERCA2a activity, thus validating the mechanism of action for these drugs.…”

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confidence: 75%

SERCA2a: its role in the development of heart failure and as a potential therapeutic target

Fragoso-Medina¹,

Zarain‐Herzberg²

2014

RRCC

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Abstract:The complexity of heart physiology has delayed the implementation of efficient, feasible, and safe therapies to fight against heart diseases for many years. As knowledge of the precise mechanisms governing cardiac hypertrophy and heart failure development increases, the availability of new therapeutic alternatives also grows. Since the cardiomyocyte physiology deeply depends on the correct calcium handling, many efforts to describe accurately the excitation-contraction coupling process in the heart and the proteins involved have been made. Among the proteins participating in calcium handling, sarco/endoplasmic reticulum Ca 2+ adenosine triphosphatase-2a (SERCA2a), whose expression and function is decreased in heart failure, stands out because of its critical role regulating Ca 2+ concentration in the cardiomyocyte. The importance of SERCA2a has been reflected in numerous studies aimed to describe its expression and function. Recently, gene therapy to deliver SERCA2a has shown promising results in human clinical trials. This paper reviews the current literature knowledge exploring diverse approaches to rescue SERCA2a expression in heart failure. It also discusses some data suggesting other possible therapies that could improve SERCA2a expression and function in cardiac diseases.

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High-Throughput FRET Assay Yields Allosteric SERCA Activators

Cited by 80 publications

References 37 publications

Small Molecular Allosteric Activator of the Sarco/Endoplasmic Reticulum Ca2+-ATPase (SERCA) Attenuates Diabetes and Metabolic Disorders

Small Molecular Allosteric Activator of the Sarco/Endoplasmic Reticulum Ca2+-ATPase (SERCA) Attenuates Diabetes and Metabolic Disorders

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

SERCA2a: its role in the development of heart failure and as a potential therapeutic target

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