Multiple Adduction Reactions of Nitroso Sulfamethoxazole with Cysteinyl Residues of Peptides and Proteins: Implications for Hapten Formation

Abstract

Sulfamethoxazole (SMX) induces immunoallergic reactions that are thought to be a result of intracellular protein haptenation by its nitroso metabolite (SMX-NO mass, 267 amu). SMX-NO reacts with protein thiols in vitro, but the conjugates have not been defined chemically. The reactions of SMX-NO with glutathione (GSH), a synthetic peptide (DS3), and two model proteins, human GSH S-transferase pi (GSTP) and serum albumin (HSA), were investigated by mass spectrometry. SMX-NO formed a semimercaptal (N-hydroxysulfenamide) conjugate with GSH that rearranged rapidly (1-5 min) to a sulfinamide. Reaction of SMX-NO with DS3 also yielded a sulfinamide adduct (mass increment, 267 amu) on the cysteine residue. GSTP was exclusively modified at the reactive Cys47 by SMX-NO and exhibited mass increments of 267, 283, and 299 amu, indicative of sulfinamide, N-hydroxysulfinamide, and N-hydroxysulfonamide adducts, respectively. HSA was modified at Cys34, forming only the N-hydroxysulfinamide adduct. HSA modification by SMX-NO under these conditions was confirmed with ELISA and immunoblotting with an antisulfonamide antibody. It is proposed that cysteine-linked N-hydroxysulfinamide and N-hydroxysulfonamide adducts of SMX are formed via the reaction of SMX-NO with cysteinyl sulfoxy acids. Evidence for a multistep assembly of model sulfonamide epitopes on GSH and polypeptides via hydrolyzable intermediates is also presented. In summary, novel, complex, and metastable haptenic structures have been identified on proteins exposed in vitro to the nitroso metabolite of SMX.

Mass Spectrometric Characterization of Circulating and Functional Antigens Derived from Piperacillin in Patients with Cystic Fibrosis

“…We have recently developed and employed MS methods to qualify the site(s) of drug-protein conjugation (20)(21)(22)(23). Drugs and drug metabolites bind in a dose-dependent manner to proteins such as albumin and can display different preferences for the sites of modification.…”

“…For Ag formation, the b-lactam ring is targeted by nucleophilic lysine residues, leading to ring opening and binding of the penicilloyl group (17)(18)(19). We have developed novel mass spectrometric techniques to define unequivocally the chemistry of drug-protein conjugation in patients under physiological conditions (20)(21)(22)(23). In this study, we report on the methods we have developed to detect and fully characterize circulating Ags derived from piperacillin and its metabolite in patients undergoing therapy.…”

Adduct Formation and Context Factors in Drug Hypersensitivity: Insight from Proteomic Studies

“…Other synthetic peptides and proteins containing nucleophilic residues have been used as models to explore the reactivity of drugs. The binding capacity of a nitrosylated form of sulfamethoxazole has been analyzed by in vitro incubations with the synthetic peptide "DS3", observing that this compound reacts mainly with the cysteine residue [63]. The detoxifying protein GSTP1-1, which contains several reactive cysteines, has been frequently used as a model for exploring adduct formation in vitro and in cells [41,64], although the direct involvement of drug-modified GSTP1-1 in hypersensitivity reactions is not established.…”

Update on Advances in Research on Idiosyncratic Drug-Induced Liver Injury

“…[41][42][43] Using advanced technologies, such as mass spectrometry, it is possible to define the chemistry of drug-protein conjugation in patients and the nature of the drug-derived epitopes, which can function as an antigen to stimulate T cells. [44][45][46][47][48] There is another mechanism underlying drug-specific T-cell activation. This concept, referred to as the "pharmacological interaction of drugs with immune receptors (p-i concept), " states that drugs by themselves act as antigens interacting in a reversible fashion with immunological receptors.…”