Cell Painting in activated cells illuminates phenotypic dark space and uncovers novel drug mechanisms of action

Cell Painting in activated cells illuminates phenotypic dark space and uncovers novel drug mechanisms of action | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Cell Painting in activated cells illuminates phenotypic dark space and uncovers novel drug mechanisms of action Matylda Zietek, Akshar Lohith, Derfel Terciano, Beverley Rabbitts, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6734784/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract As drug and natural product libraries expand, assays for assessing mechanisms of action (MoA) are increasingly critical. Performing cytological profiling using the Cell Painting (CP) assay enables image-based profiling of cellular states upon treatment, yet many bioactive compounds remain uncharacterized due to undetectable cellular effects under standard conditions. To address this, we combined drug dosing with cell activation using the protein kinase C (PKC) agonist phorbol myristate acetate (PMA). Profiling A549 lung cancer cells treated with 8,387 compounds at two concentrations (1 and 10 µM) in both resting and PMA-activated states allowed us to detect phenotypic effects for up to 40% of all screened compounds, effectively illuminating new phenotypic “dark space”. Over 1,000 compounds exhibited phenotypes exclusively under PMA activation, establishing its advantage for MoA studies. We introduce novel quality control measures for CP screens and demonstrate that integrating phenotypic signatures enhances MoA discovery. Notably, 2-methoxycinnamaldehyde clustered with glucocorticoid receptor modulators and induced nuclear translocation, emphasizing the power of this approach in uncovering novel drug mechanisms and, therefore, aiding in improving therapeutic strategies. Biological sciences/Drug discovery/Target identification Biological sciences/Drug discovery/Drug screening/High-throughput screening Biological sciences/Drug discovery/Drug screening/Phenotypic screening Biological sciences/Biological techniques/High-throughput screening Biological sciences/Computational biology and bioinformatics/High-throughput screening cell painting phenotypic screening high-content screening high-throughput screening glucocorticoid receptor Full Text Additional Declarations There is NO Competing Interest. Supplementary Files ZietekSuppTables.docx Supplementary Tables ZietekSuppSourceDataTable.xlsx Supplementary Data Set 1 Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6734784","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":463316934,"identity":"dd5762cc-2108-4e89-ac72-bee411215e4c","order_by":0,"name":"Matylda 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