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17 p.-8 fig.-1 tab., Allergic reactions to antibiotics are a major concern in the clinic. ß-lactam antibiotics are the class most frequently reported to cause hypersensitivity reactions. One of the mechanisms involved in this outcome is the modification of proteins by covalent binding of the drug (haptenation). Hence, interest in identifying the corresponding serum and cellular protein targets arises. 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 between amoxicillin haptenation and acetylation of α-enolase was studied in 1D- and 2D-gels that showed decreased haptenation and displacement of the haptenation signal to lower pI spots after chemical acetylation of the protein, respectively. In addition, the peptide containing lysine 239 was identified by mass spectrometry as the amoxicillin target sequence on α-enolase, thus suggesting a selective haptenation under our conditions. The putative 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., This work was supported by grants from the Ministerio de Ciencia e Innovación cofunded by ERDF (SAF2015-68590-R and RTI2018-097624-B-I00 to DPS and PCI2019-111825-2 Proyectos de I+D+I “Programación Conjunta Internacional” EuroNanoMed 2019 and PID2019-104293GB-I00 to EI), the Instituto de Salud Carlos III ERDF (RETIC ARADyAL RD16/0006/0021 to DPS, RETIC ARADyAL RD16/0006/0001 to MS and MIM, CPII20/00028 to MIM and RETIC ARADyAL RD16/0006/0012 to EPI, and CPII20/00028 to MIM), Junta de Andalucía and Universidad de Málaga (UMA18-FEDERJA-007 to EPI), Consejería de Transformación Económica, Industria, Conocimiento y Universidades of Junta de Andalucía (PY20_00384 to EPI). The funders had no role in study design, analysis of the data or decission to publish. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

16 p.-7 fig.-2 tab.-1 graph. abst., Serum and cellular proteins are targets for the formation of adducts with the β-lactam antibiotic amoxicillin. This process could be important for the development of adverse, and in particular, allergic reactions to this antibiotic. In studies exploring protein haptenation by amoxicillin, we observed that reducing agents influenced the extent of amoxicillin-protein adducts formation. Consequently, we show that several thiol-containing compounds, including dithiothreitol, N-acetyl-L-cysteine, and glutathione, perform a nucleophilic attack on the amoxicillin molecule that is followed by an internal rearrangement leading to amoxicillin diketopiperazine, a known amoxicillin metabolite with residual activity. Increased diketopiperazine conversion is also observed with human serum albumin but not with L-cysteine, which mainly forms the amoxicilloyl amide. The effect of thiols is catalytic and can render complete amoxicillin conversion. Interestingly, this process is dependent on the presence of an amino group in the antibiotic lateral chain, as in amoxicillin and ampicillin. Furthermore, it does not occur for other β-lactam antibiotics, including cefaclor or benzylpenicillin. Biological consequences of thiol-mediated amoxicillin transformation are exemplified by a reduced bacteriostatic action and a lower capacity of thiol-treated amoxicillin to form protein adducts. Finally, modulation of the intracellular redox status through inhibition of glutathione synthesis influenced the extent of amoxicillin adduct formation with cellular proteins. These results open novel perspectives for the understanding of amoxicillin metabolism and actions, including the formation of adducts involved in allergic reactions., This work was supported by grant SAF2015-68590-R from MINECO/FEDER, RTI2018-097624-B-I00 and RETIC Aradyal from ISCIII/FEDER RD16/0006/0021 to DP-S; RD16/0006/0001 to MT, RD16/0006/0024 to MB; grants CP15/00103 and PI17/ 01237 from ISCIII/ERDF and PI-0179-2014 from Andalusian Regional Ministry Health to MM. AA holds a “Sara Borrell” research contract (CD17/0146) supported by ISCIII from MINECO [confounded by the European Social Fund (ESF)]. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI)