Cell-Based High-Throughput Luciferase Reporter Gene Assays for Identifying and Profiling Chemical Modulators of Endoplasmic Reticulum Signaling Protein, IRE1.
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.
Cell Death and Survival Networks Program, Sanford-Burnham-Prebys Medical Discovery Institute, La Jolla, CA, USA Conrad Prebys Center for Chemical Genomics, Sanford-Burnham-Prebys Medical Discovery Institute, La Jolla, CA, USA.
Juan
J
Rong
Conrad Prebys Center for Chemical Genomics, Sanford-Burnham-Prebys Medical Discovery Institute, La Jolla, CA, USA.
Ian
I
Pass
Cell Death and Survival Networks Program, Sanford-Burnham-Prebys Medical Discovery Institute, La Jolla, CA, USA.
Paul W
PW
Diaz
Conrad Prebys Center for Chemical Genomics, Sanford-Burnham-Prebys Medical Discovery Institute, La Jolla, CA, USA.
Tram A
TA
Ngo
Conrad Prebys Center for Chemical Genomics, Sanford-Burnham-Prebys Medical Discovery Institute, La Jolla, CA, USA.
Michelle
M
Sauer
Conrad Prebys Center for Chemical Genomics, Sanford-Burnham-Prebys Medical Discovery Institute, La Jolla, CA, USA.
Gavin
G
Magnuson
Conrad Prebys Center for Chemical Genomics, Sanford-Burnham-Prebys Medical Discovery Institute, La Jolla, CA, USA.
Fu-Yue
FY
Zeng
Conrad Prebys Center for Chemical Genomics, Sanford-Burnham-Prebys Medical Discovery Institute, La Jolla, CA, USA.
Christian A
CA
Hassig
Conrad Prebys Center for Chemical Genomics, Sanford-Burnham-Prebys Medical Discovery Institute, La Jolla, CA, USA.
Michael R
MR
Jackson
Cell Death and Survival Networks Program, Sanford-Burnham-Prebys Medical Discovery Institute, La Jolla, CA, USA Conrad Prebys Center for Chemical Genomics, Sanford-Burnham-Prebys Medical Discovery Institute, La Jolla, CA, USA.
Nicholas D P
ND
Cosford
Cell Death and Survival Networks Program, Sanford-Burnham-Prebys Medical Discovery Institute, La Jolla, CA, USA smatsuzawa@SBPdicovery.org reedoffice@SBPdiscovery.org.
Shu-Ichi
S
Matsuzawa
Cell Death and Survival Networks Program, Sanford-Burnham-Prebys Medical Discovery Institute, La Jolla, CA, USA Conrad Prebys Center for Chemical Genomics, Sanford-Burnham-Prebys Medical Discovery Institute, La Jolla, CA, USA.
John C
JC
Reed
D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000378:metabolism; D059865:Endoplasmic Reticulum Stress; D004722:Endoribonucleases / Q000037:antagonists & inhibitors* / Q000502:physiology; D004789:Enzyme Activation; D017930:Genes, Reporter; D006367:HeLa Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D020935:MAP Kinase Signaling System; D009474:Neurons; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000502:physiology; D000071701:Regulatory Factor X Transcription Factors; D054852:Small Molecule Libraries; D019284:Thapsigargin / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D000071717:X-Box Binding Protein 1
D004268:DNA-Binding Proteins; D000071701:Regulatory Factor X Transcription Factors; D054852:Small Molecule Libraries; D014157:Transcription Factors; D000071717:X-Box Binding Protein 1; C000605543:XBP1 protein, human; D019284:Thapsigargin; D008156:Luciferases; C528229:ERN1 protein, human; D017346:Protein Serine-Threonine Kinases; D004722:Endoribonucleases
ER stress; IRE1; XBP1; high-throughput screen; p38MAPK