The busulfan metabolite EdAG irreversibly glutathionylates glutaredoxins

Abstract

The DNA alkylating agent busulfan is used to ‘precondition’ patients with leukemia, lymphomas and other hematological disorders prior to hematopoietic stem cell transplants. Busulfan is metabolized via conjugation with glutathione (GSH) followed by intramolecular rearrangement to the GSH analog γ–glutamyl-dehydroalanyl-glycine (EdAG). EdAG contains the electrophilic dehydroalanine, which is expected to react with protein nucleophiles, particularly proteins with GSH binding sites such as glutaredoxins (Grx’s). Incubation of EdAG with human Grx-1 or Grx-2 results in facile adduction of cys-23 and cys-77, respectively, as determined by ESI-MS/MS. The resulting modified proteins are catalytically inactive. In contrast, the glutathione transferase A1-1 includes a GSH binding site with a potentially reactive tyrosinate (Tyr-9) but it does not react with EdAG. Similarly, Cys-112 of GSTA1-1, which lies outside the active site and is known to form disulfides with GSH, does not react with EdAG. The results provide the first demonstration of the reactivity of any busulfan metabolites with intact proteins, and they suggest that GSH-binding sites containing thiolates are most susceptible. The adduction of Grx’s by EdAG suggests the possible alteration of proteins that are normally regulated via Grx-dependent reversible glutathionylation or deglutathionylation. Dysregulation of Grx-dependent processes could contribute to cellular toxicity of busulfan.

Epigallocatechin-3-gallate enhances key enzymatic activities of hepatic thioredoxin and glutathione systems in selenium-optimal mice but activates hepatic Nrf2 responses in selenium-deficient mice

“…Trx activity was determined using the method of Holmgren and Björnstedt with rat TrxR1 as a Trx reductase, the activity was presented in terms of µmol of NADPH oxidized/min/mg protein [27]. GR activity was measured by the method of Carlberg and Mannervik with oxidized glutathione as a substrate [28], and Grx activity was determined according to the method of Scian and Atkins with 2-hydroxyethyl disulfide as a model substrate [29]. Both GR and Grx activities were presented in terms of nmol of NADPH oxidized/min/mg protein.…”

Clarifying busulfan metabolism and drug interactions to support new therapeutic drug monitoring strategies: a comprehensive review

“…The work of Younis et al . [24] found that EdAG binds to GST A1, the major GST isoform responsible for Bu conjugation; per contra, Scian et al [23] demonstrated that EdAG does not bind to the GSH binding site on GST A1, but does irreversibly bind to a cysteine residue in the GSH binding pocket of glutaredoxin, resulting in a catalytically inactive protein [23]. …”

Supporting data for characterization of the busulfan metabolite EdAG and the Glutaredoxins that it adducts

“…EdAG has been shown to irreversibly glutathionylate and inhibit Grx's which play a critical role in the glutathionylation and deglutathionylation of many proteins. Grx's are important for many cellular regulatory processes [1] . Many other redoxins that contain active site cys residues in GSH binding sites, or other proteins with nucleophilic cys residues, may be targets for EdAG as well.…”

“… This article describes data related to a research article titled “The Busulfan Metabolite EdAG Irreversibly Glutathionylates Glutaredoxins” [1] . EdAG is an electrophilic GSH analog formed in vivo from busulfan, which is used in hematopoietic stem cell transplants.…”