Inhibition of human glutathione reductase by 10-arylisoalloxazines: crystalline, kinetic, and electrochemical studies.

A series of newly synthesized N(10)-arylisoalloxazines--some of which are known to be antimalarial agents--were studied as inhibitors of human glutathione reductase (GR;NADPH + GSSG + H(+) <==> NADP(+) + 2GSH). The flavoenzyme was inhibited with IC(50) values between </- 1 and 100 microM in the presence of 100 microM NADPH. The isoalloxazines and N(3)-methylisoalloxazines with a 4'-chlorophenyl or a 3',5'-dichlorophenyl group at N10 were found to be the most promising inhibitors of GR, although even the bulkier 10-naphthyl and -anthryl derivatives were also effective inhibitors. In contrast, at position N3 of the isoalloxazine ring, the size of the substituent was found to strongly influence the inhibitory effect. Introduction of a carboxymethyl group at N3--which markedly increased the solubility of the derivative in aqueous solutions-- caused a rise in the IC(50) values by 1 order of magnitude. 8-Fluoro- and 8-azido-10-arylisoalloxazines were potent inhibitors of GR; consequently position C8 of the benzenoid subnucleus instead of N3 should be considered for introducing substituents. No correlation was observed between the inhibitory strength of several isoalloxazines and their redox potential as measured by cyclovoltammetry. The crystallographic analysis of GR complexed with 10-(4'-chlorophenyl)-3-(carboxymethyl)isoalloxazine and 10-(3',5'-dichlorophenyl)-3-(carboxymethyl)isoalloxazine, respectively, revealed the presence of one inhibitor molecule bound at the 2-fold axis of the homodimeric protein. This location is consistent with fluorescence titration measurements and enzyme kinetic studies in solution which gave no indication for binding at the substrate sites.