Three high throughput compatible cell-based assays for identifying small molecule JAG1 upregulators for Alagille syndrome.

Haploinsufficiency disorders arise when loss of function mutations in one allele of a gene reduce gene dosage below the level required for normal physiology. Pharmacologic upregulation of the remaining functional allele represents a promising therapeutic strategy but requires screening assays capable of detecting modest changes in endogenous gene expression. Here we developed and compared three high throughput cell-based assays for identifying small molecule upregulators of JAG1, the gene most frequently mutated in Alagille syndrome (ALGS). The assays measure JAG1 expression at different molecular levels: RNA fluorescence in situ hybridization (RNA FISH) for JAG1 mRNA, immunofluorescence (IF) for endogenous JAG1 protein, and a HiBiT luminescence assay using CRISPR engineered LX-2 hepatic stellate cells expressing HiBiT tagged JAG1. Each assay was optimized in 384-well format and benchmarked using a panel of 32 histone deacetylase inhibitors (HDACi), compounds known to broadly increase gene expression. All three assays detected JAG1 upregulation and identified overlapping sets of active compounds. The homogeneous HiBiT assay showed the most favorable high throughput screening statistics (S/B = 2.7 and Z' > 0.5) and the lowest well to well variability, whereas the RNA FISH and IF assays provided higher signal to basal ratios and single cell resolution. Entinostat, Mocetinostat, and Chidamide were consistently identified as the most potent JAG1 upregulators across all assays. These complementary assays provide a flexible platform for identifying small molecule modulators of gene dosage and may be broadly applicable to drug discovery efforts targeting haploinsufficiency diseases.