Structural studies on the inactivation of γ-glutamylcysteine synthetase by the disulphide analogues of radioprotective cysteamine derivatives. Effects of aminoalkyl and hydroxyalkyl chain length and ββ-bis-dimethylation

Abstract

Disulphide compounds have been shown to inactivate gamma-glutamylcysteine synthetase, the rate-limiting enzyme for GSH synthesis. Such compounds bind to a cysteine residue at or near the glutamate-binding site of the enzyme. This phenomenon is thought to be responsible for the synergistic toxicity of the thiophosphate radio- and chemo-protective agent WR2721 and the oxygen-radical generator 6-hydroxydopamine (2,4,6-trihydroxyphenethylamine). 6-Hydroxy-dopamine enhances conversion of WR2721 into its disulphide metabolite NN′-bis-(3-aminopropyl)cystamine, which, in turn, paralyses the synthetase. In an effort to identify radio- and chemo-protective thiols and thiol derivatives that do not have this toxicity, we have begun to define the structure-activity relationship that governs inactivation of the enzyme by analogues of WR2721 disulphide. NN′-Bis(aminoalkyl)cystamines and bis(hydroxyalkyl)cystamines with an alkyl chain length of C5 or greater are not inactivators of the synthetase. That this is not due solely to the size of these compounds is shown by the potent inactivation of the enzyme by SAPH3 disulphide, an extremely bulky cystamine analogue. beta beta-Bis-dimethylation of the cystamine portion of the molecule also obviates inactivation. This is almost certainly due to steric interference with disulphide interchange. These findings may facilitate the safe adjunctive use of the thiol counterparts of such compounds with oxygen-radical-generating chemotherapeutic agents, and may shed light on the structure of the region of the synthetase adjacent to the glutamate-binding site.