Figure 1.

High throughput screening for small molecule inhibitors of mitochondrial apoptosis. (A) A schematic description of the intrinsic (mitochondrial) pathway of apoptosis. Cytokines induce intracellular stress and ultimately apoptosis. A high‐content image‐based phenotypic assay was designed to identify small molecules that protect beta cells from cytokine‐induced activation of mitochondrial apoptosis by monitoring cleaved caspase‐9 levels. (B) Representative screening set‐up from a subset of Life Chemicals Library (30 000 compounds), which identified the hit ATV399. Rat insulin‐producing INS1E cells were pretreated with compounds for 24 h. Cytokines (2.5 ng·mL⁻¹ IL‐1β and 100 ng·mL⁻¹ IFNγ) were then supplemented in the presence of high glucose (33 mM) to induce intracellular stress and activate cleaved caspase‐9. Compounds reducing cleaved caspase‐9 levels by >50% in the presence of cytokines were considered as hits. Z' > 0.7 was calculated based on DMSO versus cytokine‐treated wells, indicating that the assay was robust and suitable for high throughput screening. ATV399 was identified as one of the most potent hit compounds. (C) Schematic workflow of the high‐throughput screen. (D) Chemical structures of the selected hit compounds. Everolimus, IKK‐16, baricitinib and flumazenil were identified as hit compounds with known mechanisms of action.