Screening Trimethoprim Primary Metabolites for Covalent Binding to Albumin

Abstract

Modification of endogenous proteins by drugs and drug metabolites are thought to be a cause of idiosyncratic adverse drug reactions (IADRs). Trimethoprim (TMP) is a commonly prescribed antibiotic that has been implicated in IADRs; however, there is no known mechanism by which this drug or its metabolites modify proteins. This study describes the results of screening trimethoprim and its primary metabolites for the ability to covalently modify human serum albumin (HSA). The first step of the screen was in vitro reactions of the compounds with HSA followed by western blotting with antisera specific to drug-modified proteins. Compounds with positive signal in the western blot were then screened using an untargeted peptide profiling method to discover modified peptides. This strategy identified two sites in HSA that are modified by incubation with a TMP metabolite, α-hydroxy trimethoprim (Cα-OH-TMP).

Keywords: Trimethoprim; adverse drug reaction; metabolism; protein adduct.

Fig. 1

Primary trimethoprim metabolites a, TMP b, Cα-OH-TMP c, 4-DM-TMP d, 3-DM-TMP, e, 1-NO-TMP f, 3-NO-TMP

Fig. 2

Western blot analysis a, TMP and TMP metabolites incubated with HSA b, dose- dependent HSA treatment with Cα-OH-TMP

Fig. 3

Representative extracted ion chromatograms (EICs) and area boxplots from XCMS data a, EIC for Peptide 1 (681.1) b, EIC for Peptide 2 (816.37) c, boxplot for Peptide 1 (681.1) d, boxplot for Peptide 2 (816.37). Boxes depict the 25–75^th percentiles with the whiskers depicting the minimum and maximum values

Fig. 4

Fragmentation spectra a, spectrum of Peptide 1 (m/z 681.1) showing b and y ions b, zoomed in spectrum of Peptide 1 (m/z 681.1) showing 289.13 reporter c, zoomed out spectrum of Peptide 2 (m/z 816.37) showing 289.13 reporter d, spectrum of Peptide 2 (m/z 816.37) showing b and y ions

Fig. 5

Cα-NAC-TMP structure showing fragmentation to form 289.13 fragment

Fig. 6

Measurements of peptides during a time course treatment of HSA with Cα-OH-TMP a, time course EIC for Peptide 1 (681.1) b, time course EIC for Peptide 2 (816.37) c, Mass Lynx area count time course for Peptide 1 (681.1) and Peptide 2 (816.37)