Abstract
Clavulanic acid (CLV) and amoxicillin, frequently administered in combination, can be independently involved in allergic reactions. Protein haptenation with β-lactams is considered necessary to activate the immune system. The aim of this study was to assess the suitability of biotinylated analogues of CLV as probes to study protein haptenation by this β-lactam. Two synthetic approaches afforded the labeling of CLV through esterification of its carboxylic group with a biotin moiety, via either direct binding (CLV-B) or tetraethylenglycol linker (CLV-TEG-B). The second analogue offered advantages as solubility in aqueous solution and potential lower steric hindrance for both intended interactions, with the protein and with avidin. NMR reactivity studies showed that both CLV and CLV-TEG-B reacts through β-lactam ring opening by aliphatic amino nitrogen, however with different stability of resulting conjugates. Unlike CLV conjugates, that promoted the decomposition of clavulanate fragment, the conjugates obtained with the CLV-TEG-B remained linked, as a whole structure including biotin, to nucleophile and showed a better stability. This was a desired key feature to allow CLV-TEG-B conjugated protein detection at great sensitivity. We have used biotin detection and mass spectrometry (MS) to detect the haptenation of human serum albumin (HSA) and human serum proteins. MS of conjugates showed that HSA could be modified by CLV-TEG-B. Remarkably, HSA preincubation with CLV excess only reduced moderately the incorporation of CLV-TEG-B, which could be attributed to different protein interferences. The CLV-TEG-B fragment with opened β-lactam was detected bound to the 404–430HSA peptide of the treated protein. Incubation of human serum with CLV-TEG-B resulted in the haptenation of several proteins that were identified by 2D-electrophoresis and peptide mass fingerprinting as HSA, haptoglobin, and heavy and light chains of immunoglobulins. Taken together, our results show that tagged-CLV keeps some of the CLV features. Moreover, although we observe a different behavior in the conjugate stability and in the site of protein modification, the similar reactivity indicates that it could constitute a valuable tool to identify protein targets for haptenation by CLV with high sensitivity to get insights into the activation of the immune system by CLV and mechanisms involved in β-lactams allergy.
Highlights
Diagnosis of a suspected reaction after AX-CLV intake is a challenge, as it involves determining which of the two drugs is the responsible one (Torres et al, 2016)
We have recently reported the identification of a CLV determinant: N-protein, 3oxopropanamide, which was addressed through a synthetic approach of its analogues and their ability to activate basophils in a higher proportion of patients compared with the native CLV (Barbero et al, 2019)
Regarding CLV, only a couple of recent studies have reported human serum albumin (HSA) haptenation, identifying stable N-protein, 3-oxopropanamide determinant on lysine residues in in vitro conjugation at physiological pH (Barbero et al, 2019) and in patients treated with the drug (Meng et al, 2016), whereas direct binding of CLV to lysine residues, subsequent degradation products, pyrazine conjugates and cross-linking conjugates were identified at high concentrations in vitro (Meng et al, 2016)
Summary
Diagnosis of a suspected reaction after AX-CLV intake is a challenge, as it involves determining which of the two drugs is the responsible one (Torres et al, 2016). In vitro tests such as basophil activation tests or histamine release tests have been used for the evaluation of patients (Torres et al, 2010; Pineda et al, 2015; Salas et al, 2018; Barbero et al, 2019) These functional assays use the CLV molecule to evaluate whether the drug induces cellular activation, but they show suboptimal sensitivity (Ariza et al, 2016b; Doña et al, 2017). Unlike AX, no antibody targeting CLV is available, which has hampered the immunological detection of protein-CLV conjugates To overcome this issue, in this work we envisaged an alternative strategy consisting in the design of appropriate biotinylated derivatives of CLV as highly sensitive and straightforward tools to study haptenation and developing methods to identify CLV target proteins. The biotinylated derivative was demonstrated to be a straightforward tool to identify serum proteins target of modification
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