AMOXICILLIN HAPTENATION OF α-ENOLASE

Allergic reactions to antibiotics are a major concern in the clinic, with almost 15% of the patients presenting with adverse reactions against β-lactam antibiotics. One of the mechanisms involved in this outcome is the modification of proteins by covalent binding of the drug (haptenation). Hence, the interest in identifying the corresponding serum and cellular protein targets. Importantly, haptenation susceptibility and extent can be modulated by the context, including factors affecting protein conformation or the occurrence of other posttranslational modifications. We previously identified the glycolytic enzyme α-enolase as a target for haptenation by amoxicillin, both in cells and in the extracellular milieu. Here, we performed an in vitro study to analyze amoxicillin haptenation of α-enolase using gel-based and activity assays. Moreover, the possible interplay or interference of amoxicillin haptenation with acetylation was also studied in 1D- and 2D-gels that showed decreased haptenation and displacement of the haptenation signal to lower pI spots upon chemical acetylation, respectively. Mass spectrometry identified lysine 239 as the amoxicillin target residue on α enolase, thus suggesting a selective haptenation under our conditions. The amoxicillin binding site and the surrounding interactions were investigated using the α-enolase crystal structure and molecular docking. Altogether, the results obtained provide the basis for the design of novel diagnostic tools or approaches in the study of amoxicillin-induced allergic reactions.