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While general mechanisms for the fluoroquinolone\u2010associated disability (FQAD) have been identified, the underlying molecular targets of toxicity remain elusive. In this study, focusing on the most commonly prescribed FQs Ciprofloxacin and Levofloxacin, whole proteome analyses revealed prominent mitochondrial dysfunction in human cells, specifically of the complexes I and IV of the electron transport chain (ETC). Furthermore, global untargeted chemo\u2010proteomic methodologies such as photo\u2010affinity profiling with FQ\u2010derived probes, as well as derivatization\u2010free thermal proteome profiling, were applied to elucidate human protein off\u2010targets of FQs in living cells. Accordingly, the interactions of FQs with mitochondrial AIFM1 and IDH2 have been identified and biochemically validated for their contribution to mitochondrial dysfunction. Of note, the FQ induced ETC dysfunction\n                    <jats:italic>via<\/jats:italic>\n                    AIFM1 activates the reverse carboxylation pathway of IDH2 for rescue, however, its simultaneous inhibition further enhances mitochondrial toxicity. This off\u2010target discovery study provides unique insights into FQ toxicity enabling the utilization of identified molecular principles for the design of a safer FQ generation.\n                  <\/jats:p>","DOI":"10.1002\/anie.202421424","type":"journal-article","created":{"date-parts":[[2025,2,18]],"date-time":"2025-02-18T11:33:07Z","timestamp":1739878387000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":21,"title":["Chemical Proteomics Reveals Human Off\u2010Targets of Fluoroquinolone Induced Mitochondrial Toxicity"],"prefix":"10.1002","volume":"64","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2967-0828","authenticated-orcid":false,"given":"Till","family":"Reinhardt","sequence":"first","affiliation":[{"name":"Center for Functional Protein Assemblies Department of Bioscience TUM School of Natural Sciences Technische Universit\u00e4t M\u00fcnchen  Ernst-Otto-Fischer-Stra\u00dfe 8 85748 Garching, 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