Electrophilic reactivity of the Busulfan metabolite, EdAG, towards cellular thiols and inhibition of human thioredoxin-1

Busulfan is an alkylating agent used in chemotherapy conditioning regimens prior to hematopoietic stem cell transplantation (HSCT). However, its administration is associated with a great risk of adverse toxicities, which have been historically attributed to busulfan's mechanism of non-specific DNA alkylation. A phase II generated metabolite of busulfan, EdAG (γ-glutamyldehydroalanylglycine), is a dehydroalanine analog of glutathione (GSH) with an electrophilic moiety, suggesting it may bind to proteins and disrupt biological function. However, EdAG's reactions with common cellular thiols such as glutathione (GSH) and L-cysteine are understudied, along with possible inhibition of glutathionylation-dependent enzymes (with active site cysteine residues). We established a physiologically-relevant in vitro model to readily measure thiol loss over time. Using this model, we compared the apparent rates of thiol depletion in the presence of EdAG or arecoline, a toxic constituent of the areca (betel) nut and known GSH depletor. Simulated kinetic modeling revealed that the mean (±SE) alpha (α) second order rate constants describing GSH and L-cysteine depletion in the presence of EdAG were 0.00522 (0.00845) μM⁻¹∙min⁻¹ and 0.0207 (0.00721) μM⁻¹∙min⁻¹, respectively; in the presence of arecoline, the apparent rates of depletion were 0.0619 (0.009) μM⁻¹∙min⁻¹ and 0.2834 (0.0637) μM⁻¹∙min⁻¹ for GSH and L-cysteine, respectively. Under these experimental conditions, we conclude that EdAG was a weaker electrophile than arecoline. Arecoline and EdAG both depleted apparent L-cysteine concentrations to a much greater extent than GSH, approximately 4.58-fold and 3.97-fold change greater, respectively. EdAG modestly inhibited (∼20%) the human thioredoxin-1 (hTrx-1) catalyzed reduction of insulin with a mean IC₅₀ of 93 μM [95% CI: 78.6–110 μM). In summary, EdAG's ability to spontaneously react with endogenous thiols and inhibit hTrx-1 are potentially biochemically relevant in humans. These findings continue to support the growing concept that EdAG, an underrecognized phase II metabolite of busulfan, plays a role in untoward cellular toxicities during busulfan pharmacotherapy.