Detection of a Mitochondrial Stress Phenotype using the Cell Painting Assay

Abstract

Mitochondria are cellular powerhouses and crucial for cell function. However, these organelles are vulnerable to internal and external perturbagens that may impair mitochondrial function and eventually lead to cell death. In particular, small molecules may impact mitochondrial function and cardio- or hepatotoxicity caused by numerous drugs links mitochondrial toxicity to these adverse effects. Therefore, the influence of small molecules on mitochondrial homeostasis is at best assessed early on in the characterization of biologically active small molecules and drug discovery. We demonstrate that unbiased morphological profiling by means of the Cell Painting assay (CPA) can detect mitochondrial stress coupled to the induction of integrated stress response. This activity is common for compounds addressing different targets, is not shared by direct inhibitors of the electron transport chain and enables prediction of mitochondrial stress induction for small molecules that are profiled using CPA.

Identification of Readily Available Pseudo-Natural Products

“…The extracted morphological features are normalized to DMSO controls using a modified Z-score and the final generated profiles are investigated for their similarity to a set of pre-defined 13 clusters using a subprofile analysis. 32,33 Details of the assay are described in the Supporting…”

“…We previously introduced a cluster subprofile analysis which allows the classification of active compounds into currently 13 bioactivity clusters based on activity in the CPA. 32,33 Application of this cluster analysis to the Enamine-PNPs which had displayed bioactivity in CPA at the two concentrations revealed that among the identified clusters lysosomotropism / cholesterol homeostasis, tubulin modulation and DNA synthesis inhibition are the most prominent activities (fig. 3E).…”