Abstract 5660: Optimizing and integrating high-throughput cell-based assays to enable SAR studies of metarrestin to target cancer metastasis
Authors: Asia S. Almarales, Yi-Han Lin, Harrison Daly, Samarjit Patnaik, Abhijeet Kapoor, Jing Ran, Soumita De, Udo Rudloff, Juan Marugan, Mark Henderson
Journal: Cancer Research (2025)

Abstract The perinucleolar compartment (PNC), a marker of genome organization / a membrane-less organelle (condensate) adjacent but structurally distinct from the nucleolus, associates with metastatic behavior in cancer cells and has been used as biomarker in high-throughput screening assays to identify small molecules that target metastasis. We discovered metarrestin, an investigational agent that disassembles the PNC in multiple cancer cell lines as a novel treatment for cancer metastasis. Metarrestin is currently in phase I clinical trials. Metarrestin treatment effects phase-separation, rapidly disrupts nucleolar function and inhibits ribosomal RNA synthesis. Pre-clinical structure activity relationship (SAR) studies, to further improve metarrestin’s pharmacological properties, are ongoing. These require robust cell-based assays to evaluate PNC disassembly, rRNA synthesis and growth phenotypes. Our prior studies relied heavily on the Incucyte live-cell imaging system to characterize the cytostatic effect of metarrestin derivatives by tracking the growth of treated cells over days. However, this experimental setup requires long machine times and is limited by the capacity of a single Incucyte to accommodate six 384-well format plates. To address these throughput limitations, we optimized an orthogonal viability assay using Cell Titer Glo which measures luminescence and detects ATP in metarrestin treated cells. Using 1536-well plates, we examined the assay performance using cell numbers ranging from 125-1, 000/well, and incubation time between 48-96 hr, in two cell lines PANC1, a pancreatic cancer cell line and PC3M, a prostate cancer cell line. Unlike the measurement of cytotoxic effect, which is typically performed at higher cell densities and shorter incubation times, the cytostatic effect was best captured after 96 hr incubation with cells seeded at 250/well in a 1536-well plate. For detection of rRNA inhibition and PNC disassembly in cells, we utilized a rRNA selective fluorescent probe (Probe 1) and a GFP-based reporter of the PNC, respectively, followed by confocal microscopy using an Opera Phenix. Using this suite of cell-based assays, we evaluated the compound of ∼160 metarrestin derivatives and performed comparative analyses of compound activities in measurements of PNC disassembly, rRNA synthesis inhibition, and cytostatic growth effect in PANC1 and PC3M cells. Citation Format: Asia S. Almarales, 1 Yi-Han Lin, 1 Harrison Daly, 1 Samarjit Patnaik, 1 Abhijeet Kapoor, 1 Jing Ran, 1 Soumita De, 2 Udo Rudloff, 2 Juan Marugan, 1 Mark Henderson1. Optimizing and integrating high-throughput cell-based assays to enable SAR studies of metarrestin to target cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5660.