INHIBITORS OF GLUTATHIONE-REDUCTASE AS POTENTIAL ANTIMALARIAL-DRUGS -KINETIC COOPERATIVITY AND EFFECT OF DIMETHYL-SULFOXIDE ON INHIBITION-KINETICS

We have developed inhibitors of glutathione reductase that improve on the inhibition of literature lead compounds by up to three orders of m agnitude. Thus, analogues of Safranine O and menadione were found to b e strong, reversible inhibitors of yeast glutathione reductase. Safran ine O exhibited partial, uncompetitive inhibition with K-i and alpha v alues of 0.5 mM and 0.15, respectively. Thionine O was a partial (hype rbolic) uncompetitive inhibitor with K-i and alpha values of 0.4 mu M and 0.15, respectively. LY83583 and 2-anilino-1,4-naphthoquinone also showed (hyperbolic) partial, uncompetitive inhibition with micromolar K-i values. For Nile Blue A a model for two-site binding with (parabol ic) uncompetitive inhibition fitted the data with a IC, value of 11 mu M and a kinetic cooperativity between the sites of 0.12, increased to 0.46 by pre incubation of the enzyme and Nile Blue A in the presence of glutathione disulphide. Analysis of the effects of preincubation on the kinetics and cooperativity indicated the possibility of a slow co nformational change in the homodimeric enzyme. the first such indicati on of kinetic cooperativity in the native enzyme to our knowledge. Fur ther evidence of conformational changes for this enzyme came from stud ies of the effects of dimethyl sulphoxide which indicated that this co -solvent, which at low concentrations has no apparent effect on initia l velocities under normal assay conditions, induced a slow conformatio nal change in the enzyme. Thionine O, Nile Blue A and LY83583 were red ox-cycling substrates producing superoxide ion, detectable by means of cytochrome c reduction, but leading to no loss of glutathione reducta se activity, under aerobic or anaerobic conditions. The water-soluble Safranine analogues Methylene Blue, Methylene Green, Nile Blue A and T hionine O (5 mg/kg i.p. x 5) were effective antimalarial agents in viv o against P. berghei, but their effect was small and a higher dose (50 mg/kg i.p. x 1) was toxic in mice. Comparison was made with human glu tathione reductase and its literature-reported interactions with sever al tricyclic inhibitors as studied by X-ray diffraction. It is possibl e that the conformational changes detected in the present study from a lterations in detailed kinetic inhibition mechanisms may shed light on information transfer through the glutathione reductase molecule from the dimer interface ligand pocket to the active-site.