Mechanism of action, metabolism, and toxicity of buthionine sulfoximine and its higher homologs, potent inhibitors of glutathione synthesis

Abstract

Buthionine sulfoximine (S-(n-butyl)homocysteine sulfoximine) is a potent and specific inhibitor of γ-glutamylcysteine synthetase; administered to animals or incorporated into tissue culture media it inhibits glutathione biosynthesis and causes depletion of cellular glutathione levels. The present studies establish that γ-glutamylcysteine synthetase catalyzes the phosphorylation of buthionine sulfoximine by MgATP; 1 molar equivalent of the product, buthionine sulfoximine phosphate, is tightly bound to the fully inhibited enzyme. Kinetic studies indicate that the initial binding constant for DL-buthionine-SR-sulfoximine is <100 μM and that the limiting pseudo-first order rate constant for inactivation is 3.7 min-1 (t 1/2 ~ 11s). Inhibition is apparently irreversible while the enzyme is maintained in nondenaturing buffers containing MgATP. D-Buthionine-SR-sulfoximine, DL-buthionine-SR-sulfoxide, and DL-buthionine sulfone are shown to be very weak or ineffective inhibitors of the enzyme. Three higher homologs of buthionine sulfoximine, pentathionine sulfoximine, hexathionine sulfoximine, and heptathionine sulfoximine are potent inhibitors of γ-glutamylcysteine synthetase in vitro and produce glutathione depletion in liver and kidney when administered to mice. In marked contrast to buthionine sulfoximine, hexathionine sulfoximine and hepathionine sulfoximine are toxic; heptathionine sulfoximine caused the most extensive glutathione depletion of the compounds mentioned. When L-[35S]buthionine-SR-sulfoximine is administered to mice or rats, the radiolabel is recovered almost quantitatively in the urine within 24 h. About 60% of the recovered radiolabel is unchanged buthionine sulfoximine, and >90% of the remainder is a metabolite tentatively identified as N(α)-acetyl-L-buthionine-SR-sulfoximine. The very limited metabolism of buthionine sulfoximine is also apparent in the observation that <3% of DL-[1-14C]buthionine-SR-sulfoximine given to mice is recovered as 14CO2.