scholarly journals Efficient High-Throughput Screening by Endoplasmic Reticulum Ca2+ Measurement to Identify Inhibitors of Ryanodine Receptor Ca2+-Release Channels

2018 ◽  
Vol 94 (1) ◽  
pp. 722-730 ◽  
Author(s):  
Takashi Murayama ◽  
Nagomi Kurebayashi ◽  
Mari Ishigami-Yuasa ◽  
Shuichi Mori ◽  
Yukina Suzuki ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Ryanodine Receptor ◽  
High Throughput ◽  
High Throughput Screening

scholarly journals Cardiac ryanodine receptor N-terminal region biosensors identify novel inhibitors via FRET-based high-throughput screening

2021 ◽  
pp. 101412
Author(s):  
Jingyan Zhang ◽  
Daniel P. Singh ◽  
Christopher Y. Ko ◽  
Roman Nikolaienko ◽  
Siobhan M. Wong King Yuen ◽  
...  
Keyword(s):  
Ryanodine Receptor ◽  
High Throughput ◽  
High Throughput Screening ◽  
Terminal Region ◽  
Cardiac Ryanodine Receptor

scholarly journals FRET-based High-throughput Screening Biosensor using the Cardiac Ryanodine Receptor (RyR2) N-terminal “ABC” Domain

2020 ◽  
Vol 118 (3) ◽  
pp. 173a-174a
Author(s):  
Jingyan Zhang ◽  
Robyn T. Rebbeck ◽  
David D. Thomas ◽  
Filip V. Petegem ◽  
Razvan L. Cornea
Keyword(s):  
Ryanodine Receptor ◽  
High Throughput ◽  
High Throughput Screening ◽  
Cardiac Ryanodine Receptor

scholarly journals Identifying Ryanodine Receptor Modulators: from High-Throughput Screening to Single Channel Recording

2020 ◽  
Vol 118 (3) ◽  
pp. 272a
Author(s):  
Manuel Paina ◽  
Jim Goodchild ◽  
Lucy Firth ◽  
Katharina Montag ◽  
Maria Grazia Garibaldi ◽  
...  
Keyword(s):  
Ryanodine Receptor ◽  
High Throughput ◽  
High Throughput Screening ◽  
Single Channel ◽  
Single Channel Recording ◽  
Receptor Modulators

scholarly journals Novel Inhibitors of Leaky Skeletal Ryanodine Receptor Calcium Channels Discovered via FRET-Based High-throughput Screening

2018 ◽  
Vol 114 (3) ◽  
pp. 117a
Author(s):  
Claire E. Haskin ◽  
Robyn T. Rebbeck ◽  
Gregory D. Gillispie ◽  
David D. Thomas ◽  
Razvan L. Cornea
Keyword(s):  
Calcium Channels ◽  
Ryanodine Receptor ◽  
High Throughput ◽  
High Throughput Screening

scholarly journals High-Throughput Screens to Discover Small-Molecule Modulators of Ryanodine Receptor Calcium Release Channels

2016 ◽  
Vol 22 (2) ◽  
pp. 176-186 ◽  
Author(s):  
Robyn T. Rebbeck ◽  
Maram M. Essawy ◽  
Florentin R. Nitu ◽  
Benjamin D. Grant ◽  
Gregory D. Gillispie ◽  
...  
Keyword(s):  
Ryanodine Receptor ◽  
High Throughput ◽  
High Throughput Screening ◽  
Calcium Release ◽  
Striated Muscle ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Pathological State ◽  
Pharmacological Agents ◽  
Intracellular Ca

Using time-resolved fluorescence resonance energy transfer (FRET), we have developed and validated the first high-throughput screening (HTS) method to discover compounds that modulate an intracellular Ca2+ channel, the ryanodine receptor (RyR), for therapeutic applications. Intracellular Ca2+ regulation is critical for striated muscle function, and RyR is a central player. At resting [Ca2+], an increased propensity of channel opening due to RyR dysregulation is associated with severe cardiac and skeletal myopathies, diabetes, and neurological disorders. This leaky state of the RyR is an attractive target for pharmacological agents to treat such pathologies. Our FRET-based HTS detects RyR binding of accessory proteins calmodulin (CaM) or FKBP12.6. Under conditions that mimic a pathological state, we carried out a screen of the 727-compound NIH Clinical Collection, which yielded six compounds that reproducibly changed FRET by >3 SD. Dose–response of FRET and [3H]ryanodine binding readouts reveal that five hits reproducibly alter RyR1 structure and activity. One compound increased FRET and inhibited RyR1, which was only significant at nM [Ca2+], and accentuated without CaM present. These properties characterize a compound that could mitigate RyR1 leak. An excellent Z′ factor and the tight correlation between structural and functional readouts validate this first HTS method to identify RyR modulators.

scholarly journals Efficient High-Throughput Screening for Type 1 Ryanodine Receptor Inhibitors using ER Ca2+Measurements

2019 ◽  
Vol 116 (3) ◽  
pp. 153a
Author(s):  
Takashi Murayama ◽  
Nagomi Kurebayashi ◽  
Mari Yuasa-Ishigami ◽  
Shuichi Mori ◽  
Haruo Ogawa ◽  
...  
Keyword(s):  
Ryanodine Receptor ◽  
High Throughput ◽  
High Throughput Screening

scholarly journals Cell-Based High-Throughput Luciferase Reporter Gene Assays for Identifying and Profiling Chemical Modulators of Endoplasmic Reticulum Signaling Protein, IRE1

2015 ◽  
Vol 20 (10) ◽  
pp. 1232-1245 ◽  
Author(s):  
Juan Rong ◽  
Ian Pass ◽  
Paul W. Diaz ◽  
Tram A. Ngo ◽  
Michelle Sauer ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Reporter Gene ◽  
High Throughput ◽  
High Throughput Screening ◽  
Fine Tuning ◽  
Luciferase Reporter ◽  
Signaling Cascade ◽  
Specific Chemical ◽  
Reporter Gene Assays

Endoplasmic reticulum (ER) stress activates three distinct signal transducers on the ER membrane. Inositol-requiring protein 1 (IRE1), the most conserved signal transducer, plays a key role in ER stress-mediated signaling. During ER stress, IRE1 initiates two discrete signaling cascades: the “adaptive” signaling cascade mediated by the XBP1 pathway and the “alarm” signaling cascade mediated by stress-activated protein kinase pathways. Fine-tuning of the balance between the adaptive and alarm signals contributes significantly to cellular fate under ER stress. Thus, we propose that the design of high-throughput screening (HTS) assays to selectively monitor IRE1 mediated-signaling would be desirable for drug discovery. To this end, we report the generation of stable human neural cell lines and development of cell-based HTS luciferase (Luc) reporter gene assays for the identification of pathway-specific chemical modulators of IRE1. We implemented a cell-based Luc assay using a chimeric CHOP-Gal4 transcription factor in 384-well format for monitoring IRE1 kinase-mediated p38MAPK activation and an unfolded response pathway element (URPE)–Luc cell-based assay in 1536-well format for monitoring IRE1’s RNase-mediated activation of XBP1. Chemical library screening was successfully conducted with both the CHOP/Gal4-Luc cells and UPRE-Luc engineered cells. The studies demonstrate the feasibility of using these HTS assays for discovery of pathway-selective modulators of IRE1.

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