Identification of Potential Targets Mediating Polymyxin Toxicity in Human Alveolar Epithelial Cells

Abstract ID 128217 Poster Board 017 Inhaled polymyxins are highly beneficial for treating lung infections caused by multidrug-resistant Gram-negative bacteria. However, the mechanisms underlying polymyxin-induced toxicity in human lung epithelial cells remain poorly understood. This study investigates the molecular toxicology of polymyxin-induced toxicity in A549 lung epithelial cells employing Thermal proteome profiling (TPP). TPP identified potential binding targets for polymyxin B, where in a protein with higher abundance in the soluble fraction indicated stabilization. A549 cell lysates were incubated for 5 min with either 25 μM or 100 μM polymyxin B, with milli-Q water as the solvent control. Following thermal (60oC) challenge and ultracentrifugation, the soluble fraction underwent trypsinization for 16 h. Peptides and proteins were quantified using HPLC-MS. SpectronautTM 16.0 was used to process raw files using directDIA against the human proteome, and proteins identified with at least two unique peptides across all conditions. Significantly enriched proteins at both polymyxin B concentrations were considered potential hits (log₂ fold change (FC) > 1, adjusted p-value < 0.05). Confocal microscopy at 4 and 24 h was utilized for the colocalization study of polymyxin B and top hits in A549 cells. A total of 1,342 proteins were consistently identified across all samples examined. Principal component analysis and cluster heatmap showed distinct separation and clustering based on conditions. Concentration-dependent stabilization occurred for target proteins with both 25 and 100 μM polymyxin B. Fourteen proteins displayed strong thermal stabilization with polymyxin B compared to the control. Notably, ferritin heavy chain 1 (FTH1) from ferroptosis pathway exhibited 1.74- and 3.92-fold enrichment at 25 and 100 μM polymyxin B compared to the control. Polymyxin B also co-localized with FTH1 at 4 and 24 h in A549 cells. Furthermore, TPP revealed proteins from endocytosis (ARF GTPase activating protein 2, ARFGAP2 and RAB11 family interacting protein 5, RAB11FIP5) and the ubiquitin-degradation system (ubiquitin fold modifier, UFD1 and ubiquitin conjugating enzyme E2, UBE2V1) to be significantly stabilized. In conclusion, TPP identified potential cellular protein targets of polymyxin B in human lung epithelial cells which are involved in endocytosis, ferroptosis, and protein degradation through the ubiquitin system. Further validation of these potential targets is underway in our laboratory. This study offers unique insights into mitigating polymyxin-induced pulmonary toxicity for the development of safer new-generation polymyxins.