|Title:||Morphological profiling of small molecules for mode-of-action studies using the Cell Painting Assay|
|Abstract:||The Cell Painting Assay (CPA) is an unbiased morphological profiling approach that generates a holistic view of the bioactivity space. Therefore, it is a powerful tool to deconvolute targets or mode-of-action (MoA) of small molecules, which is especially important for non-protein targets as they are difficult to identify with commonly applied methods. In order to examine the applicability of the CPA to identify non-proteins targets, the morphological fingerprint of the iron chelator Deferoxamine (DFO), as well as references and so far uncharacterized compounds with similar fingerprints, were investigated. Reference compounds, biosimilar to DFO, possess different annotated targets and activities but share a common MoA of cell cycle arrest. This was experimentally confirmed for a representative selection of references. The cluster analysis enabled the identification of novel and so far uncharacterized chelating agents and DNA synthesis modulators. Furthermore, hierarchical clustering, using the CPA fingerprints, revealed a first insight into the different mechanisms of action. To investigate the bioactivity of a small tetrahydroindolo[2,3-a]quinolizine compound class, a combination of morphological profiling using the CPA and proteome profiling was pursued. The results revealed an altered cholesterol homeostasis induced by the compound’s physicochemical properties that led to an accumulation and an increased pH in lysosomes. More than 400 reference compounds and well-characterized drugs with different annotated targets and activities share high biosimilarity to the most active derivative. The majority of the compounds in this cluster also possess physicochemical properties, that are predictive for the accumulation in lysosomes. Modulation of cholesterol homeostasis was experimentally confirmed for a representative selection of references. Therefore, this cluster can be used to identify novel modulators of cholesterol homeostasis but also to associate the regulation of corresponding genes or proteins to an effect induced by the physicochemical properties of the compounds. The findings presented in this thesis emphasize the power of the CPA to evaluate bioactive small molecules and to predict diverse MoA for uncharacterized compounds as well as to uncover and expand so far unknown activity for already characterized small molecules and drugs.|
|Subject Headings (RSWK):||Wirkstoffforschung|
|Appears in Collections:||Chemische Biologie|
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