In light of our whole proteome analysis data showing significant downregulation of both the electron transport chain, especially complexes I and IV, and the destabilized mito-ribosomal subunits in the TPP experiment, we next focused our target validation efforts on mitochondrial protein hits. We started with ClpP, the proteolytic subunit of the mitochondrial caseinolytic protease P involved in the maintenance, maturation and quality control of various mitochondrial proteins, including the ETC and the mito-ribosome. We recombinantly expressed and purified ClpP and tested its activity against the compounds in ClpP peptidase and ClpXP protease assays. Unexpectedly, while literature-known positive controls for enzyme inhibition (TG42) and overactivation (ONC201) resulted in clear effects, Ciprofloxacin evoked no significant concentration-dependent activation or inhibition (Figure ). In line with this observation, a full proteome analysis of HEK-239 wild-type and ClpP knockout cells in presence of Ciprofloxacin revealed almost identical changes within up-and downregulated proteins (Ciprofloxacin vs. vehicle control) suggesting that a potential interaction between Ciprofloxacin and ClpP does not play a major role for proteome dysregulation and thereby for toxicity (Figure ).