Levofloxacin-Induced Insulin Aggregation: Biophysical and Computational Insights into Amyloidogenic Potential

Protein misfolding, a hallmark of conformational diseases known as proteinopathies, arises when hydrophobic regions of proteins are exposed, leading to aggregation, loss of functionality and potential cytotoxicity. With the increasing prevalence of multidrug-resistant bacteria, repurposing antibiotics has gained attention for therapeutic applications beyond their conventional use. In this study, we investigated the amyloidogenic and anti-amyloidogenic potential of levofloxacin (LFX) using human insulin (HI) as a model, given its propensity to aggregate at injection sites in diabetes patients. Using various biophysical techniques, we observed a concentration-dependent increase in light scattering (LS) intensity and a corresponding rise in Thioflavin T (ThT) fluorescence intensity in the presence of LFX, indicating enhanced amyloid formation. Time-dependent ThT measurements revealed a shortened lag phase and a higher apparent rate constant for HI fibrillation under LFX exposure. Circular dichroism (CD) spectra showed a negative peak at 218 nm, signifying a transition to [Formula: see text]-sheet-rich structures, consistent with amyloid formation. Complementary in silico analyses corroborated these findings, with DSSP-based secondary structure analysis and Ramachandran plots confirming significant [Formula: see text]-sheet enrichment induced by LFX. These results suggest that LFX promotes HI aggregation, raising important implications for diabetic patients where LFX may exacerbate aggregation-related complications. This research underscores the critical need for evaluating potential interactions between antibiotics and proteins prone to misfolding, particularly in vulnerable populations. Beyond highlighting the risks of unintended drug-induced protein aggregation, this work also emphasizes the broader public health importance of raising awareness about drug interactions to minimize adverse effects, reduce healthcare costs and improve patient outcomes. By providing insights into the amyloidogenic potential of LFX, this study contributes to the understanding of antibiotic–protein interactions, paving the way for safer therapeutic applications and better management strategies in clinical practice.