The identified hits can be grouped into mitochondrial and non-mitochondrial localization, which both could contribute to the observed side effects and were thus investigated subsequently. Non-mitochondrial proteins comprise PTGR2, SCARB1, NUDT1 and ADAL1, which were either cloned and recombinantly expressed or obtained from commercial sources. The activity of PTGR2, a prostaglandin reductase involved in adipogenesis and lipid metabolism, was not significantly affected by Ciprofloxacin (tested up to 250 µM, data not shown) and for SCARB1 that is involved in reverse cholesterol transport weak binding was detected for Cipro P1, but no binding affinity could be determined for the parent compound Ciprofloxacin via microscale thermophoresis (MST, Figure ) due to aggregation/precipitation at high compound concentrations excluding both as specific targets. The enzyme NUDT1 (also called MTH1) hydrolyses oxidized purine nucleoside triphosphates (2-oxo-ATP, 2-oxo-dATP, 8-oxo-GTP, 8-oxo-dGTP) to their respective monophosphates sanitizing the NTP pool of ROS-damaged DNA and RNA precursors and thereby, protecting cells from missense base-paring (Figure ). NUDT1 is non-essential in healthy cells, however crucial under high oxidative stress, e.g. in cancer cells. In fact, various NUDT1 inhibitors have been developed eradicating cancer by "cancer phenotypic lethality", including quinoline and quinolinedione compounds sharing structural similarities with FQs. As also FQs increase ROS, a potential NUDT1 activity modulation by FQs is likely to contribute to their human side-effects. Indeed, incubation with Ciprofloxacin affected NUDT1 in vitro activity in a dose-dependent manner with significant partial inhibition at a high but physiologically relevant concentration of 100 µM (Figure ). Inhibition of NUDT1 by Levofloxacin was only significant at the highest concentration tested. The known specific inhibitor (S)-crizotinib served as positive control (Figure ). Additionally, gel-based labeling of recombinant NUDT1 spiked into human lysate as background by Cipro P2 and Levo P2 was abolished for temperature-denatured NUDT1 and competed by parent Ciprofloxacin (Figure , S20C), further corroborating NUDT1 as an off-target of FQs. Of note, the inhibition of NUDT1 is in line with elevated 8-oxo-dG levels, which have been observed in cells treated with Ciprofloxacin and could thus contribute to its long-lasting side-effects. Adenosine deaminase-like protein ADAL1, the most stabilized protein in TPP, operates downstream of NUDT1 by converting N 6 -methyl dAMP/AMP to dIMP/IMP in the purine salvage pathway (Figure ). Activity assays in presence of Ciprofloxacin showed partial enzyme inhibition only at very high concentrations ≥ 300 µM (Figure ). Although this is likely not relevant for the side effects of FQs, it could explain the stabilization observed with TPP. For Levofloxacin even at high concentrations no inhibitory effect was detected (Figure ).