Identification and characterization of PERK activators by phenotypic screening and their effects on NRF2 activation

PloS One
Wensheng XieHu Li

Abstract

Endoplasmic reticulum stress plays a critical role to restore the homeostasis of protein production in eukaryotic cells. This vital process is hence involved in many types of diseases including COPD. PERK, one branch in the ER stress signaling pathways, has been reported to activate NRF2 signaling pathway, a known protective response to COPD. Based on this scientific rationale, we aimed to identify PERK activators as a mechanism to achieve NRF2 activation. In this report, we describe a phenotypic screening assay to identify PERK activators. This assay measures phosphorylation of GFP-tagged eIF2α upon PERK activation via a cell-based LanthaScreen technology. To obtain a robust assay with sufficient signal to background and low variation, multiple parameters were optimized including GFP-tagged eIF2α BacMam concentration, cell density and serum concentration. The assay was validated by a tool compound, Thapsigargin, which induces phosphorylation of eIF2α. In our assay, this compound showed maximal signal window of approximately 2.5-fold with a pEC50 of 8.0, consistent with literature reports. To identify novel PERK activators through phosphorylation of eIF2α, a focused set of 8,400 compounds was screened in this assay at 10 µM. A ...Continue Reading

References

Oct 1, 2003·Molecular and Cellular Biology·Sara B CullinanJ Alan Diehl
Feb 12, 2005·Antioxidants & Redox Signaling·Makoto Kobayashi, Masayuki Yamamoto
Nov 18, 2005·The International Journal of Biochemistry & Cell Biology·Sara B Cullinan, J Alan Diehl
Sep 14, 2006·Annual Review of Pharmacology and Toxicology·Thomas W KenslerShyam Biswal
Oct 10, 2006·Biochemical and Biophysical Research Communications·Takao Iwawaki, Ryoko Akai
Oct 24, 2007·Methods in Molecular Biology·James A FornwaldRobert S Ames
Dec 2, 2008·Nature Reviews. Drug Discovery·Inki KimJohn C Reed
Dec 24, 2008·Proceedings of the National Academy of Sciences of the United States of America·Thomas E SussanShyam Biswal
Jan 22, 2011·Genes to Cells : Devoted to Molecular & Cellular Mechanisms·Keiko TaguchiMasayuki Yamamoto
Feb 15, 2011·Journal of Molecular Medicine : Official Organ of the Gesellschaft Deutscher Naturforscher Und Ärzte·Taehong MinNeeraj Vij
Jun 16, 2012·Current Molecular Medicine·C M P Ribeiro, W K O'Neal
Jul 21, 2012·Molecular Neurobiology·Ricardo J S VianaCecília M P Rodrigues
Aug 1, 2012·Pulmonary Pharmacology & Therapeutics·Steven G Kelsen
Aug 1, 2012·Experimental Physiology·Anita Somborac-BacuraAntoon J M van Oosterhout
Sep 25, 2012·Biochemical Pharmacology·Axel H Schönthal
Nov 22, 2012·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·Harshavardhan KencheAnna Blumental-Perry
May 24, 2013·Cancer Chemotherapy and Pharmacology·Huaping LiuTheodore J Lampidis

❮ Previous
Next ❯

Citations

Mar 30, 2017·Pulmonary Pharmacology & Therapeutics·Li LinQiuyue Wang
May 11, 2016·Human Molecular Genetics·Gali MaorMia Horowitz
Sep 27, 2018·Cancers·Eoghan P McGrathAfshin Samali
Oct 23, 2019·Neural Regeneration Research·Wensheng Lin, Sarrabeth Stone
Sep 21, 2016·Expert Opinion on Therapeutic Patents·Jeffrey M Axten
Nov 1, 2016·IUBMB Life·Hector VegaMarek Michalak
Sep 6, 2020·The Journal of Biological Chemistry·Julia M D Grandjean, R Luke Wiseman

❮ Previous
Next ❯

Methods Mentioned

BETA
protein folding
LanthaScreen
transfection
ELISA
Assay
FRET
PCR

Software Mentioned

Graphpad Prism
LanthaScreen

Related Concepts

Related Feeds

Blood And Marrow Transplantation

The use of hematopoietic stem cell transplantation or blood and marrow transplantation (bmt) is on the increase worldwide. BMT is used to replace damaged or destroyed bone marrow with healthy bone marrow stem cells. Here is the latest research on bone and marrow transplantation.

B-Cell Lymphoma

B-cell lymphomas include lymphomas that affect B cells. This subtype of cancer accounts for over 80% of non-Hodgkin lymphomas in the US. Here is the latest research.

Apoptosis

Apoptosis is a specific process that leads to programmed cell death through the activation of an evolutionary conserved intracellular pathway leading to pathognomic cellular changes distinct from cellular necrosis