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.