Flucloxacillin Protein Binding Leads to the Presentation of Novel MHC Peptides

Abstract

Flucloxacillin is a β-lactam antibiotic associated with a high incidence of idiosyncratic drug-induced liver reactions. Although expression of HLA-B*57:01 increases susceptibility, little is known of the pathological mechanisms involved in the induction of the clinical phenotype. Off-target protein modification is suspected to drive the reaction, either through non-immune mediated pathways, through the modification of peptides that are presented to T cells by the risk allele, or both. In this thesis, the characterization of proteins haptenated by flucloxacillin was performed using proteomic techniques. As protein haptenation, followed by antigen processing and presentation of the drug-derived antigenic determinants may drive the adverse event, the immunopeptidome of HLA-B*57:01 in the presence and absence of flucloxacillin was determined. In order to detect flucloxacillin modified proteins, an antibody specific for flucloxacillin was generated. Characterization of the antibody to determine cross reactivity and selectivity was determined. Anti-flucloxacillin antibody was used to identify flucloxacillin modified proteins in a number of immortalised cell lines and primary human hepatocytes. Western blot, immunocytochemistry and mass spectrometry were used for the detection, localisation and characterization, respectively, of drug modified proteins. C1R-B*57:01, B-lymphoblast cells transfected with HLA-B*57:01, were incubated with flucloxacillin for 48h. HLA peptide complexes were subsequently eluted and processed for mass spectrometric analysis. Finally, bioinformatic pipelines were generated to assist in the characterization of flucloxacillin-modified MHC peptides to allow a more high-throughput approach to immunopeptidomic data analysis. The generation of a high titre flucloxacillin specific antibody was successful, with no cross reactivity with proteins. Isoxazole ring containing β-lactams did cross react, indicating this was the site of recognition. Intracellular protein modification was identified in all the cell lines examined, including primary human hepatocytes. Using the liver cell line HepaRG, localization within the bile canaliculi was observed. The function of the major hepatocellular efflux transporters MRP2 and P-gp was increased in the presence of flucloxacillin in a time-dependant manner. Modification of master regulators of MAP Kinase signalling molecules was detected. Flucloxacillin also modified HLA-B*57:01 protein directly, which could lead to neo-antigens being presented. In depth analysis of the global repertoire of peptides was interrogated. Flucloxacillin was found to alter the C-terminal amino acid on the majority of peptides, where an increase in phenylalanine and a decrease in tryptophan was observed. Peptides unique to flucloxacillin treatment were theoretically weaker binders to HLA-B*57:01, indicating flucloxacillin may assist in their stabilization. Of the peptides eluted from flucloxacillin treated C1R-B*57:01 cells, 7 were fully annotated to show flucloxacillin-lysine covalent binding, with other partially annotated peptides indicating modifications. In this thesis a wide range of off-target protein modification has been determined, including proteins involved in regulatory signalling pathways. The localization of flucloxacillin was identified to occur in the site of clinical disease during flucloxacillin-induced liver injury. It was also demonstrated that drug-modified peptides are presented by HLA-B*57:01 and that global repertoires are altered by flucloxacillin. Further investigation into the immunogenicity of haptenated proteins in the onset of iDILI is required to determine the role of these peptides in drug hypersensitivity.