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Three high throughput compatible cell-based assays for identifying small molecule JAG1 upregulators for Alagille
Molly R Kulikauskas1, Xiuli Huang1, Ivan Pavlinov1
1National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, USA.
SLAS Discovery : Advancing Life Sciences R & D
|July 24, 2026
Summary
Researchers developed three high-throughput assays to find drugs that increase JAG1 gene expression for Alagille syndrome (ALGS). The HiBiT assay showed the best screening performance, identifying potent JAG1 upregulators like Entinostat.
Area of Science:
- Genetics
- Pharmacology
- Biotechnology
Background:
- Haploinsufficiency disorders result from reduced gene dosage due to mutations in one gene allele.
- Pharmacologic upregulation of the functional allele is a potential therapeutic strategy.
- Developing sensitive screening assays is crucial for identifying such therapeutic compounds.
Purpose of the Study:
- To develop and compare three high-throughput cell-based assays for identifying small molecule upregulators of JAG1.
- To benchmark these assays using histone deacetylase inhibitors (HDACi).
- To identify potent JAG1 upregulators for Alagille syndrome (ALGS) drug discovery.
Main Methods:
- Developed and optimized three high-throughput assays: RNA FISH for JAG1 mRNA, immunofluorescence (IF) for JAG1 protein, and a HiBiT luminescence assay for HiBiT-tagged JAG1.
- Assays were performed in 384-well format and benchmarked with 32 HDAC inhibitors.
- Evaluated assay performance using screening statistics (S/B, Z') and well-to-well variability.
Main Results:
- All three assays detected JAG1 upregulation and identified overlapping sets of active compounds.
- The HiBiT assay demonstrated favorable high-throughput screening statistics (S/B = 2.7, Z' > 0.5) and low variability.
- RNA FISH and IF assays offered higher signal-to-basal ratios and single-cell resolution.
- Entinostat, Mocetinostat, and Chidamide were consistently identified as potent JAG1 upregulators.
Conclusions:
- Complementary assays provide a flexible platform for identifying small molecule modulators of gene dosage.
- The developed assays are applicable to drug discovery for haploinsufficiency diseases.
- The HiBiT assay is particularly promising for high-throughput screening of JAG1 upregulators.
