R. Scott Lokey

We developed a high‐content image‐based screen that utilizes the pro‐inflammatory stimulus lipopolysaccharide (LPS) and murine macrophages (RAW264.7) with the goal of enabling the identification of novel anti‐inflammatory lead compounds. We screened 2,259 bioactive compounds with annotated mechanisms of action (MOA) to identify compounds that block the LPS‐induced phenotype in macrophages. We utilized a set of seven fluorescence microscopy probes to generate images that were used to train and optimize a deep neural network classifier to distinguish between unstimulated and LPS‐stimulated macrophages. The top hits from the deep learning classifier were validated using a linear classifier trained on individual cells and subsequently investigated in a multiplexed cytokine secretion assay. All 12 hits significantly modulated the expression of at least one cytokine upon LPS stimulation. Seven of these were allosteric inhibitors of the mitogen‐activated protein kinase kinase (MEK1/2) and showed similar effects on cytokine expression. This deep learning morphological assay identified compounds that modulate the innate immune response to LPS and may aid in identifying new anti‐inflammatory drug leads.