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Development and application of an animal-free test strategy based on in silico and in vitro methods for the efficient detection of substance effects using morphological and molecular fingerprints in HT / HC screening (MORPHEUS: MORPHology-based Endocrine DisrUptor Screening)

04/2022-03/2025

Funding programme / funding institution: Bundesministerium für Bildung und Forschung (BMBF) - Deutschland

Grant number: 16LW0137K

Project homepage: http://www.bf3r.de

Project description:

Overall goals of the project

In toxicology, the carcinogenic effects of substances, for example by disrupting the endogenous hormone system (so-called endocrine disruptors), or substances that endanger human health through specific organ toxicity, are primarily examined in very complex animal experiments that involve a large number of test animals. In the MORPHEUS project, the E-Morph Assay (recently published by VP1, Kornhuber et al. 2021), an in vitro method for the detection of estrogenic substances in a human breast cancer cell line, will be further developed and its applicability for the detection of carcinogenicity and organ toxicity significantly expanded. By means of Cell Painting and "unbiased phenotypic profiling", substance/group-specific morphological changes in sub-/cellular structures will be identified, which, as "morphological fingerprints", allow a reliable differentiation of endocrine and (organ)toxic activity of test substances by phenotypic high-throughput/high-content (HT/HC) screening. In addition, the morphological fingerprints (morphological changes in the cells) will be combined with "molecular fingerprints" (structural properties of the substances) in an iterative process in order to develop in silico prediction models that enable the effective identification of substances with endocrine or (organ) toxic effects from extensive chemical libraries. A testing strategy of in vitro and in silico methods developed as part of the MORPHEUS project is intended to contribute to reducing animal testing in the future and is intended to be used both in toxicological safety testing and in biomedical and pharmaceutical research.

Scientific and technical work objectives of the project

In the first year ("from images to numbers"), two methods established by VP1 and VP2 are combined (E-Morph Assay, Cell Painting) and corresponding technologies (HT/HC screening and image analysis pipelines) are exchanged, implemented and adapted. The implementation of the E-Morph Assay, which has been adapted for Cell Painting and HT/HC screening, is laid down in detail in an SOP (Standard Operating Procedure) and the successful transfer of the combined assay is ensured by subsequent pre-validation with suitable reference substances. During this period, VP3 is simultaneously developing in silico methods based on morphological input data for the planned identification of morphological fingerprint patterns (year 2, "from numbers to patterns") and the prediction of substance effects (year 3, "from patterns to predictions”). To identify specific morphological and molecular fingerprints, two different substance libraries (“Bioactive Library”, “BfR Library”) are analyzed with the combined Cell Painting E-Morph Assay by VP1 and VP2 as part of a “pilot screening”. The results obtained allow not only the identification of relevant fingerprint patterns (VP3) but also a more detailed assessment of the transferability of the E-Morph Assay as a decisive planning basis for future validation studies. The identified fingerprints are then used for the development of in silico prediction models for active substances (VP3). In an iterative process, the predictions are checked and refined as part of a "proof-of-concept screening" by in vitro "phenotypic profiling" with the combined Cell Painting E-Morph Assay and thus continuously further developed for future use as part of a "Follow-up Screening”.

Project partners

• Institut für Physiologie (Institut für Physiologie) (Charité - Universitätsmedizin Berlin)
• Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e. V. (FMP) - Deutschland

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