Combined morphological and proteome profiling reveals target-independent impairment of cholesterol homeostasis

Morphological Subprofile Analysis for Bioactivity Annotation of Small Molecules

“…Therefore, morphological profiling relies on the profiles of reference compounds for the generation of target or mode-of-action hypothesis. However, we and others noticed that profiles of reference compounds with similar target annotation can differ as the activity may be governed not by the nominal target but rather an off-target (Akbarzadeh et al, 2021, Schneidewind et al, 2021, Cox et al, 2020, Woehrmann et al, 2013, Breinig et al, 2015. Moreover, reference compounds with diverse annotation may form a morphological cluster that is based on the same mode of action rather than on the same target (Schneidewind et al, 2020).…”

“…As a subprofile constitutes only part of the full profile, we used the non-cluster features to gain further bioactivity information. We previously assigned a large number of reference compounds which have very diverse annotated nominal targets, to the lysosomotropism/cholesterol homeostasis cluster (Schneidewind et al, 2021). Potentially, the nominal activity of these compounds may be detectable using CPA but masked by the dominant profile of disturbing cholesterol homeostasis.…”

“…A literature search revealed impairment of microtubules for several compounds and we experimentally validated tubulin targeting for reference compounds that had not been previously linked to tubulin. In addition, 12 % of the reference set (27 % of the active references) belong to a large cluster with deregulation of cholesterol biosynthesis as a common denominator (Schneidewind et al, 2021). This cluster contains compounds with very diverse targets and the activity of approx.…”

“…Ideally, reference compounds sharing the same target shall give rise to similar CPA profiles and profile similarity can then be used to predict a target for a new compound. However, reference compounds often lack complete annotation, may display polypharmacology and address unknown targets which significantly hampers the generation of target hypotheses based solely on profile similarity (Akbarzadeh et al, 2021, Schneidewind et al, 2020, Schneidewind et al, 2021).…”

Cell Morphological Profiling Enables High-Throughput Screening for PROteolysis TArgeting Chimera (PROTAC) Phenotypic Signature

“…There could be several mechanisms leading to toxicity on mitochondria, direct or indirect. Indirectly, it has been described that accumulation of compounds in lysosomes can lead to mitotoxicity, and lysosomotropic compounds have been shown in previous studies to share similar profiles in phenotypic assays including the Cell Painting assay. − In addition to protonation and trapping in lysosomes, cationic amphiphilic drugs can cause phospholipidosis and may accumulate in mitochondria, thus leading to mitotoxicity. We therefore investigated what type of mitotoxicity could have been identified in our Cell Painting study.…”