A predictive nuclear translocation assay for spliced x-box-binding protein 1 identifies compounds with known organ
Christa Hahmann1, Amiee Weiser, Derek Duckett
1Department of Molecular Therapeutics and Translational Research Institute, The Scripps Research Institute, Jupiter, Florida, USA.
Abstract:
Compound toxicity is still the main cause of attrition, emphasizing the need for novel predictive assays to identify toxic compounds early during drug development. Endoplasmic reticulum (ER) stress has recently been discovered as a molecular event that links cellular dysfunction to drug-induced organ toxicity in humans. Among higher organisms the inositol-requiring transmembrane kinase/endoribonuclease pathway plays a major role in mediating the ER stress response. Inositol-requiring transmembrane kinase/endoribonuclease achieves this through its endoribonuclease activity causing a frameshift in the translation of the X-box-binding protein 1 (XBP1) to produce spliced XBP1 (XBP1s), which translocates into the nucleus, where it initiates transcription of ER stress response genes. Based on this biology, we have designed a novel β-galactosidase-based XBP1s-enzyme fragment complementation assay, which enables identification of compound-induced ER stress in human U2OS cells. The XBP1s-enzyme fragment complementation assay was established in a 384-well format and validated using a library of 1280 pharmacologically active compounds. Importantly, the library of pharmacologically active compounds screen identified both well-established ER stress inducers and several compounds that are known organ toxicants but not previously reported to induce ER stress. Implementation of this assay to assess compound-induced ER stress will facilitate decision making for compound selection and we believe that it will significantly increase the ability to reduce toxicity of preclinical drug candidates.
Insights
A new assay detects drug-induced endoplasmic reticulum (ER) stress by monitoring XBP1 splicing. This tool identifies toxic compounds early, aiding drug development and reducing preclinical candidate toxicity.
Area of Science:
- Drug Discovery and Toxicology
- Molecular Biology
- Cellular Stress Response
Background:
- Compound toxicity is a major cause of drug development attrition.
- Endoplasmic reticulum (ER) stress links cellular dysfunction to drug-induced organ toxicity.
- The inositol-requiring transmembrane kinase/endoribonuclease pathway mediates ER stress via XBP1 splicing.
Purpose of the Study:
- To develop a novel assay for identifying compound-induced ER stress.
- To facilitate early identification of toxic compounds in drug development.
Main Methods:
- Designed a β-galactosidase-based XBP1s-enzyme fragment complementation assay.
- Established the assay in human U2OS cells using a 384-well format.
- Validated the assay using a library of 1280 pharmacologically active compounds.
Main Results:
- Successfully identified compound-induced ER stress using the novel assay.
- Detected known ER stress inducers and previously unreported ER stressors among toxicants.
- The assay demonstrated effectiveness in a large-scale compound screen.
Conclusions:
- The XBP1s-enzyme fragment complementation assay enables early identification of ER stress.
- Implementation of this assay can improve decision-making in compound selection.
- This tool has the potential to significantly reduce toxicity in preclinical drug candidates.
