AlphaScreen-based characterization of the bifunctional kinase/RNase IRE1alpha: a novel and atypical drug target.
Assay technologies that were originally developed for high-throughput screening (HTS) have recently proven useful in drug discovery for activities located upstream (target identification and validation) and downstream (ADMET) of HTS. Here the authors investigated and characterized the biological properties of a novel target, IRE1alpha, a bifunctional kinase/RNase stress sensor of the endoplasmic reticulum (ER). They have developed a novel assay platform using the HTS technology AlphaScreen to monitor the dimerization/oligomerization and phosphorylation properties of the cytosolic domain of IRE1alpha. They show in vitro that dimerization/oligomerization of the cytosolic domain of IRE1 correlated with the autophosphorylation ability of this domain and its endoribonuclease activity toward XBP1 mRNA. Using orthogonal in vitro and cell-based approaches, the authors show that the results obtained using AlphaScreen were biologically relevant. Preliminary characterization of assay robustness indicates that both AlphaScreen assays should be useful in HTS for the identification of IRE1 activity modulators.
1Inserm, U889, Avenir, Bordeaux, France.
Marion
M
Bouchecareilh
Marie-Elaine
ME
Caruso
Philippe
P
Roby
Stéphane
S
Parent
Nathalie
N
Rouleau
Said
S
Taouji
Olivier
O
Pluquet
Roger
R
Bossé
Michel
M
Moenner
Eric
E
Chevet
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004722:Endoribonucleases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006367:HeLa Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D010766:Phosphorylation; D055503:Protein Multimerization; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D015203:Reproducibility of Results
C528229:ERN1 protein, human; D017346:Protein Serine-Threonine Kinases; D004722:Endoribonucleases