Summary assay for the identification of small molecule activators of the apoptotic arm of the Unfolded Protein response
Tremendous advances in our understanding of pathologic mechanisms have recently revealed that defective protein processing within the secretory pathway is an integral component of many genetic and environmental diseases. Diverse disease states ranging from diabetes, Alzheimer's disease, and Parkinson's disease, to hemophilia and lysosomal storage diseases have all been characterized by folding more ..
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego)
Network: NIH Molecular Libraries Production Centers Network (MLPCN)
Grant Number: 1 R03 MH089782-01
Assay Provider: Dr. Randal J Kaufman, University of Michigan
Tremendous advances in our understanding of pathologic mechanisms have recently revealed that defective protein processing within the secretory pathway is an integral component of many genetic and environmental diseases. Diverse disease states ranging from diabetes, Alzheimer's disease, and Parkinson's disease, to hemophilia and lysosomal storage diseases have all been characterized by folding defects or impaired transport from the endoplasmic reticulum (ER). Very recently it has come to light that deregulation of protein synthesis may be a key component in the pathogenesis of cancer and metastasis. When misfolded protein accumulates in the ER lumen, the cell activates the Unfolded Protein Response (UPR) to clear the malfolded proteins and restore homeostatic protein processing. When a stress is prolonged or robust the UPR employs a genetic pathway that results in cell death. The proposed studies are to focus attention to identify and characterize drug-like small molecule activators of the apoptotic pathway.
In this project we investigate the hypotheses that specific and robust activation of the PERK-eIF2a-CHOP (apoptotic) arm will exacerbate the UPR and lead to cell death in malignant cells with hyper-active secretory machinery. This aim is predicated on the hypothesis that the adaptive arm of the UPR can be pharmacologically overwhelmed and lead to a productive CHOP-mediated apoptotic response in human cancer cells. Specifically, we aim to identify small molecule, specific activators of the CHOP pathway via high-throughput screening and to further improve potency and pathway specificity via focused SAR refinement studies.
This AID summarizes SBCCG's probe development efforts to identify activators of the PERK-eIF2a-CHOP (apoptotic) arm of the UPR.
From the primary HTS (AID 449763) and subsequent hit confirmation (AID 463112) several hit compounds were identified. Hits were clustered into scaffolds and SAR studies were performed on compounds from several scaffolds reconfirming potency (AID 489024). Selected compounds were then ordered via the MLSMR as solid powder samples and reconfirmed in dose response (AID 540312) and tested in the XBP1 counterscreen (AID 489040) to assess UPR pathway specificity. SAR efforts on promising lead scaffolds lead to the synthesis and/or purchase from commercial sources of numerous related compounds. These samples were also tested in a dose dependent manner in the UPR-CHOP primary (AID 588594, 602434) and UPR-XBP1 counterscreen (AID 588582, 602416) assays. Compounds exhibiting CHOP pathway specificity were tested for cytotoxicity in both the CHOP (AID 588557, 588556) and XBP1 (AID 588570, 588558) cell lines following exposures of 6 and 24 hours. These efforts led to the successful development of a first-in-class, potent (762 nM EC50), not generally cytotoxic, chemical probe, ML291, that selectively activates the apoptotic but not the adaptive arm of the UPR, that demonstrates efficacy in inducing cell death through activation of the apoptotic arm in relevant cells, and moreover activate genes associated with the apoptotic arm of the UPR.
Details of protocols, compound structures, and results from the original assays can be found in PubChem at the respective AIDs listed below.
Please see pertinent AIDs: 449763, 463112, 489024, 489040, 540305, 540312, 588556, 588557, 588558, 588570, 588582, 588594, 602416, 602434
Probe molecules are defined as the positives of this assay and assigned a score of 100. Two substances have been identified as probe molecules, SID123083137 and SID134228465.
Categorized Comment - additional comments and annotations
From MLP Probe Report:
Probe count: 1
MLP Probe ML# for probe 1: ML291
PubChem Substance ID (SID) for probe 1: 123083137
PubChem Compound ID (CID) for probe 1: 52940465
Probe type for probe 1: Activator
IC50/EC50 (nM) for probe 1: 762
Target for probe 1: UPRCHOP
Disease relevance for probe 1: Cancer
Anti-target for probe 1: UPR-XBP1
Fold selectivity for probe 1: >105
NCBI Book chapter link for probe 1: http://www.ncbi.nlm.nih.gov/books/NBK133431/ (ID: 3025078)
Grant number for probe 1: MH089782-01
NCBI Book chapter title for probe 1: Selective small molecule activator of the apoptotic arm of the UPR
Data Table (Concise)