Identification of Selective Small Molecule Modulators of mRNA Processing Using a Multiplexed QuantiGene High-Throughput Screening Platform

High-throughput screening (HTS) approaches incorporating multiplexed, cell-based assays are increasingly common for identifying novel modulators of complex biological processes. To enable the discovery of small molecule modulators of mRNA processing, we developed a multiplexed, bead-based, high-throughput QuantiGene assay, leveraging the Luminex platform that is capable of simultaneously quantifying transcript levels of multiple independent target genes within a single HTS campaign. To address plate variability and potential screening artifacts, a pragmatic hit-calling pipeline was implemented utilizing both plate- and well-based normalization strategies. This dual-normalization approach reduced false negatives and produced consistent hit confirmation rates. Application of this methodology led to the identification of unique compounds selectively modulating individual target genes. Strikingly, among the three exemplary genes, only 5% of primary actives demonstrated activity across all three target genes, underscoring the assay's capacity for detecting selective mRNA modulators. Chemical motif analysis of confirmed actives recovered known RNA privileged scaffolds as well as novel scaffolds that are uniquely enriched for individual targets screened. Validation screening using an orthogonal, four-point concentration-response real-time PCR (qPCR) assay in a disease-relevant cell line demonstrated high validation rates, supporting the robustness and translational relevance of this multiplexed HTS platform. These findings establish a scalable and reliable strategy for identifying selective small molecule modulators of mRNA processing, with broad applicability in early drug discovery.