Identification of flucloxacillin-modified hepatocellular proteins: implications in flucloxacillin-induced liver injury.

Flucloxacillin is a ß-lactam antibiotic associated with a high incidence of drug-induced liver injury. Although expression of HLA-B*57:01 is associated with increased susceptibility, little is known of the pathological mechanisms involved in the induction of the clinical phenotype. Irreversible protein modification is suspected to drive the reaction through the provision of flucloxacillin-modified peptides that are presented to T-cells by the protein encoded by the risk allele. In this study, we have shown that flucloxacillin binds to multiple proteins within human primary hepatocytes, including major hepatocellular proteins (hemoglobin and albumin) and mitochondrial proteins. Inhibition of membrane transporters multidrug resistance-associated protein 2 (MRP2) and P-glycoprotein (P-gp) appeared to reduce the levels of covalent binding. A diverse range of proteins with different functions was found to be targeted by flucloxacillin, including adaptor proteins (14-3-3), proteins with catalytic activities (liver carboxylesterase 1, tRNA-splicing endonuclease subunit Sen2, All-trans-retinol dehydrogenase ADH1B, Glutamate dehydrogenase 1 mitochondrial, Carbamoyl-phosphate synthase [ammonia] mitochondrial), and transporters (hemoglobin, albumin, and UTP-glucose-1-phosphate uridylyltransferase). These flucloxacillin-modified intracellular proteins could provide a potential source of neoantigens for HLA-B*57:01 presentation by hepatocytes. More importantly, covalent binding to critical cellular proteins could be the molecular initiating events that lead to flucloxacillin-induced cholestasis Data are available via ProteomeXchange with identifier PXD038581.

Omics and Multi-Omics Analysis for the Early Identification and Improved Outcome of Patients with Psoriatic Arthritis.

The definitive diagnosis and early treatment of many immune-mediated inflammatory diseases (IMIDs) is hindered by variable and overlapping clinical manifestations. Psoriatic arthritis (PsA), which develops in ~30% of people with psoriasis, is a key example. This mixed-pattern IMID is apparent in entheseal and synovial musculoskeletal structures, but a definitive diagnosis often can only be made by clinical experts or when an extensive progressive disease state is apparent. As with other IMIDs, the detection of multimodal molecular biomarkers offers some hope for the early diagnosis of PsA and the initiation of effective management and treatment strategies. However, specific biomarkers are not yet available for PsA. The assessment of new markers by genomic and epigenomic profiling, or the analysis of blood and synovial fluid/tissue samples using proteomics, metabolomics and lipidomics, provides hope that complex molecular biomarker profiles could be developed to diagnose PsA. Importantly, the integration of these markers with high-throughput histology, imaging and standardized clinical assessment data provides an important opportunity to develop molecular profiles that could improve the diagnosis of PsA, predict its occurrence in cohorts of individuals with psoriasis, differentiate PsA from other IMIDs, and improve therapeutic responses. In this review, we consider the technologies that are currently deployed in the EU IMI2 project HIPPOCRATES to define biomarker profiles specific for PsA and discuss the advantages of combining multi-omics data to improve the outcome of PsA patients.

Basic Science Session 1. Biomarkers for Psoriatic Arthritis Treatment Response and Joint Damage Progression: An Update on 2 Industry-GRAPPA Projects.

The Group for Research and Assessment of Psoriasis and Psoriatic Arthritis (GRAPPA) has identified several priority areas for biomarker development, including biomarkers to predict at baseline which patients may progress to develop joint damage and whether a patient will respond to a specific targeted therapy. Two industry-GRAPPA projects were initiated in 2020 on these biomarker research areas: (1) the Pfizer-GRAPPA project, focused on biomarkers of treatment response to tofacitinib in the Oral Psoriatic Arthritis TriaL program; and (2) the Lilly-GRAPPA project, focused on biomarkers of damage in the ixekizumab SPIRIT-P1 randomized controlled trial. Preliminary results from these 2 projects were presented by the GRAPPA team, with both studies showing promising initial results. Data from these studies will be published when the studies have been completed. Large-scale validation studies are required and are under discussion.

Cell Membrane Transporters Facilitate the Accumulation of Hepatocellular Flucloxacillin Protein Adducts: Implication in Flucloxacillin-Induced Liver Injury.

Flucloxacillin is a ß-lactam antibiotic associated with a high incidence of drug-induced liver reactions. Although expression of HLA-B*57:01 increases susceptibility, little is known about the pathological mechanisms involved in the induction of the clinical phenotype. Irreversible protein modification is suspected to drive the reaction through the presentation of flucloxacillin-modified peptides by the risk allele. In this study, the binding of flucloxacillin to proteins of liver-like cells was characterized. Flucloxacillin was shown to bind to proteins localized in bile canaliculi regions, coinciding with the site of clinical disease. The localization of flucloxacillin was mediated primarily by the membrane transporter multidrug resistance-associated protein 2. Modification of multiple proteins by flucloxacillin in bile canaliculi regions may provide a potential local source of neo-antigens for HLA presentation in the liver.

A high-stringency blueprint of the human proteome.

The Human Proteome Organization (HUPO) launched the Human Proteome Project (HPP) in 2010, creating an international framework for global collaboration, data sharing, quality assurance and enhancing accurate annotation of the genome-encoded proteome. During the subsequent decade, the HPP established collaborations, developed guidelines and metrics, and undertook reanalysis of previously deposited community data, continuously increasing the coverage of the human proteome. On the occasion of the HPP's tenth anniversary, we here report a 90.4% complete high-stringency human proteome blueprint. This knowledge is essential for discerning molecular processes in health and disease, as we demonstrate by highlighting potential roles the human proteome plays in our understanding, diagnosis and treatment of cancers, cardiovascular and infectious diseases.

CDDO-imidazolide Targets Multiple Amino Acid Residues on the Nrf2 Adaptor, Keap1.

Synthetic triterpenoids including CDDO, its methyl ester (CDDO-Me, bardoxolone methyl), and its imidazolide (CDDO-Im) enhance Nrf2-mediated antioxidant and anti-inflammatory activity in many diseases by reacting with thiols on the adaptor protein, Keap1. Unlike monofunctional CDDO-Me, the bifunctional analog, CDDO-Im, has a second reactive site (imidazolide) and can covalently bind to amino acids other than cysteine on target proteins such as glutathione S-transferase pi (GSTP), serum albumin, or Keap1. Here we show for the first time that bifunctional CDDO-Im (in contrast to CDDO-Me), as low as 50 nM, can covalently transacylate arginine and serine residues in GSTP and cross-link them to adjacent cysteine residues. Moreover, we show that CDDO-Im binds covalently to Keap1 by forming permanent Michael adducts with eight different cysteines, and acyl adducts with lysine and several tyrosine residues. Modeling studies suggest that the Tyr 85 adduct stabilizes the Keap1-Cul3 complex, thereby enhancing the potency of CDDO-Im.

HLA DRB1*15:01-DQB1*06:02-Restricted Human CD4+ T Cells Are Selectively Activated With Amoxicillin-Peptide Adducts.

Amoxicillin-clavulanate is the most common cause of idiosyncratic drug-induced liver injury (DILI). Drug-specific CD4+ T cells have been detected in patients with DILI, suggestive of an immune etiology. Furthermore, genetic associations including the human leucocyte antigen (HLA) DRB1*15:01-DQB1*06:02 haplotype influence susceptibility. Amoxicillin forms protein adducts that are postulated to activate T cells, by conjugating with lysine residues. However, a role for such adducts has not been described. This study aimed to (1) investigate whether amoxicillin-modified HLA-DRB1*15:01-DQB1*06:02 binding peptides selectively activate DILI patient T cells and (2) define the nature of the T-cell response with respective to antigen structure. Peptides carrying lysine residues for amoxicillin binding in positions (KP) 2-6 and anchors for the HLA-DRB1*15:01-DQB1*06:02 haplotype were designed. The amoxicillin-modified peptides were characterized by mass spectrometry prior to culturing with patient peripheral blood mononuclear cell. T-cell clones were then tested for specificity with amoxicillin, unmodified- and amoxicillin-modified peptides, and structural variants. Amoxicillin-modified KP-2 and KP-3 peptide-specific CD4+ clones proliferated and secreted interferon gamma (IFN-γ), interleukin (IL)-10, perforin and/or IL-17/IL-22 in a dose-dependent manner and displayed no cross-reactivity with amoxicillin, unmodified peptide or with positional derivatives. The T cells response was HLA class II restricted and the amoxicillin-modified peptides bound selectively to HLA-DRB1*15:01 and/or DQB1*06:02. To conclude, we show that amoxicillin-modified peptides bind to both components of the risk haplotype to stimulate DILI patient T cells and describe the importance of the position of nucleophilic lysine residue in the HLA binding peptide sequence.

Identification of Flucloxacillin-Haptenated HLA-B*57:01 Ligands: Evidence of Antigen Processing and Presentation.

Flucloxacillin is a ß-lactam antibiotic associated with a high incidence of drug-induced liver reactions. Although expression of human leukocyte antigen (HLA)-B*57:01 increases susceptibility, little is known of the pathological mechanisms involved in the induction of the clinical phenotype. Irreversible protein modification is suspected to drive the reaction through the modification of peptides that are presented by the risk allele. In this study, the binding of flucloxacillin to immune cells was characterized and the nature of the peptides presented by HLA-B*57:01 was analyzed using mass spectrometric-based immunopeptidomics methods. Flucloxacillin modification of multiple proteins was observed, providing a potential source of neoantigens for HLA presentation. Of the peptides eluted from flucloxacillin-treated C1R-B*57:01 cells, 6 putative peptides were annotated as flucloxacillin-modified HLA-B*57:01 peptide ligands (data are available via ProteomeXchange with identifier PXD020137). To conclude, we have characterized naturally processed drug-haptenated HLA ligands presented on the surface of antigen presenting cells that may drive drug-specific CD8+ T-cell responses.

Definition of the Chemical and Immunological Signals Involved in Drug-Induced Liver Injury.

Idiosyncratic drug-induced liver injury (iDILI), which is rare and often recognized only late in drug development, poses a major public health concern and impediment to drug development due to its high rate of morbidity and mortality. The mechanisms of DILI are not completely understood; both non-immune- and immune-mediated mechanisms have been proposed. Non-immune-mediated mechanisms including direct damage to hepatocytes, mitochondrial toxicity, interference with transporters, and alteration of bile ducts are well-known to be associated with drugs such as acetaminophen and diclofenac; whereas immune-mediated mechanisms involving activation of both adaptive and innate immune cells and the interactions of these cells with parenchymal cells have been proposed. The chemical signals involved in activation of both innate and adaptive immune responses are discussed with respect to recent scientific advances. In addition, the immunological signals including cytokine and chemokines that are involved in promoting liver injury are also reviewed. Finally, we discuss how liver tolerance and regeneration can have profound impact on the pathogenesis of iDILI. Continuous research in developing in vitro systems incorporating immune cells with liver cells and animal models with impaired liver tolerance will provide an opportunity for improved prediction and prevention of immune-mediated iDILI.

Definition of Haptens Derived from Sulfamethoxazole: In Vitro and in Vivo.

Hypersensitivity reactions occur frequently in patients upon treatment with sulfamethoxazole (SMX). These adverse effects have been attributed to nitroso sulfamethoxazole (SMX-NO), the reactive product formed from auto-oxidation of the metabolite SMX hydroxylamine. The ability of SMX-NO to prime naïve T-cells in vitro and also activate T-cells derived from hypersensitive patients has illustrated that T-cell activation may occur through the binding of SMX-NO to proteins or through the direct modification of MHC-bound peptides. SMX-NO has been shown to modify cysteine residues in glutathione, designer peptides, and proteins in vitro; however, the presence of these adducts have not yet been characterized in vivo. In this study a parallel in vitro and in vivo analysis of SMX-NO adducts was conducted using mass spectrometry. In addition to the known cysteine adducts, multiple SMX-NO-derived haptenic structures were found on lysine and tyrosine residues of human serum albumin (HSA) in vitro. On lysine residues two haptenic structures were identified including an arylazoalkane adduct and a Schiff base adduct. Interestingly, these adducts are labile to heat and susceptible to hydrolysis as shown by the presence of allysine. Furthermore, SMX-modified HSA adducts were detected in patients on long-term SMX therapy illustrated by the presence of an arylazoalkane adduct derived from a proposed carboxylic acid metabolite of SMX-NO. The presence of these adducts could provide an explanation for the immunogenicity of SMX and the strong responses to SMX-NO observed in T-cell culture assays. Also, the degradation of these adducts to allysine could lead to a stress-related innate immune response required for T-cell activation.

Dapsone and Nitroso Dapsone Activation of Naïve T-Cells from Healthy Donors.

Dapsone (DDS) causes hypersensitivity reactions in 0.5-3.6% of patients. Although clinical diagnosis is indicative of a hypersensitivity reaction, studies have not been performed to define whether dapsone or a metabolite activates specific T-cells. Thus, the aims of this study were to explore the immunogenicity DDS and nitroso DDS (DDS-NO) using peripheral blood mononuclear cells from healthy donors and splenocytes from mice and generate human T-cell clones to characterize mechanisms of T-cell activation. DDS-NO was synthesized from DDS-hydroxylamine and shown to bind to the thiol group of glutathione and human and mouse albumin through sulfonamide and N-hydroxyl sulphonamide adducts. Naïve T-cell priming to DDS and DDS-NO was successful in three human donors. DDS-specific CD4+ T-cell clones were stimulated to proliferate in response to drug via a MHC class II restricted direct binding interaction. Cross reactivity with DDS-NO, DDS-analogues, and sulfonamides was not observed. DDS-NO clones were CD4+ and CD8+, MHC class II and I restricted, respectively, and activated via a pathway dependent on covalent binding and antigen processing. DDS and DDS-NO-specific clones secreted a mixture of Th1 and Th2 cytokines, but not granzyme-B. Splenocytes from mice immunized with DDS-NO were stimulated to proliferate in vitro with the nitroso metabolite, but not DDS. In contrast, immunization with DDS did not activate T-cells. These data show that DDS- and DDS-NO-specific T-cell responses are readily detectable.

Mass Spectrometric and Functional Aspects of Drug-Protein Conjugation.

The covalent binding of drugs (metabolites) to proteins to form drug-protein adducts can have an adverse effect on the body. These adducts are thought to be responsible for idiosyncratic drug reactions including severe drug hypersensitivity reactions. Major advances in proteomics technology have allowed for the identification and quantification of target proteins for certain drugs. Human serum albumin (HSA) and Hb have been identified as accessible targets and potential biomarkers for drug-protein adducts formation, for numerous drugs (metabolites) including ß-lactam antibiotics, reactive drug metabolites such as quinone imines (acetaminophen) and acyl glucuronides (diclofenac), and covalent inhibitors (neratinib). For example, MS/MS analysis of plasma samples from patients taking flucloxacillin revealed that flucloxacillin and its 5-hydroxymethyl metabolite formed covalent adducts with lysine residues on albumin via opening of the ß-lactam ring. Other proteins such as P450 and keratin are also potential targets for covalent binding. However, for most drugs, the properties of these target proteins including their location, their quantity, the timing of conjugate generation, and their biological function are not well understood. In this review, currently available proteomic technologies including MS/MS analysis to identify antigens, precise location of modifications, and the immunological consequence of hapten-protein complex are illustrated. Moving forward, identification of the nature of the antigenic determinants that trigger immune responses to drug-protein adducts will increase our ability to predict idiosyncratic toxicity for a given compound.

Amoxicillin and Clavulanate Form Chemically and Immunologically Distinct Multiple Haptenic Structures in Patients.

Amoxicillin-clavulanate (AC) is one of the most common causes of drug induced liver injury (DILI). The association between AC-DILI and HLA alleles and the detection of drug-specific T cells in patients with AC-DILI indicate that the adaptive immune system is involved in the disease pathogenesis. In this study, mass spectrometric methods were employed to characterize the antigen formed by AC in exposed patients and the antigenic determinants that stimulate T cells. Amoxicillin formed penicilloyl adducts with lysine residues on human serum albumin (HSA) in vitro, with K190 and K199 being the most reactive sites. Amoxicillin-modified K190 and K199 have also been detected in all patients, and more extensive modification was observed in patients exposed to higher doses of amoxicillin. In contrast, the binding of clavulanic acid to HSA was more complicated. Multiple adducts were identified at high concentrations in vitro, including those formed by direct binding of clavulanic acid to lysine residues, novel pyrazine adducts derived from binding to the degradation products of clavulanic acid, and a cross-linking adduct. Stable adducts derived from formylacetic acid were detected in all patients exposed to the drug. Importantly, analysis of hapten-protein adducts formed in the cell culture medium revealed that the highly drug-specific T-cell responses were likely driven by the markedly different haptenic structures formed by these two drugs. In this study, the unique haptenic structures on albumin in patients formed by amoxicillin and clavulanic acid have been characterized and shown to function as chemically distinct antigens which can stimulate separate, specific T-cell clones.