Inhibitors of Glutathione Reductase as Potential Antimalarial Drugs Kinetic Cooperativity and Effect of Dimethyl Sulphoxide on Inhibition Kinetics

Abstract

We have developed inhibitors of glutathione reductase that improve on the inhibition of literature lead compounds by up to three orders of magnitude. Thus, analogues of Safranine 0 and menadione were found to be strong, reversible inhibitors of yeast glutathione reductase. Safranine 0 exhibited partial, uncompetitive inhibition with Ki and a values of 0.5mM and 0.15, respectively. Thionine 0 was a partial (hyperbolic) uncompetitive inhibitor with Ki and 1y values of 0.4 pM and 0.15, respectively. LY83583 and 2-anilino-l.4-naphthoquinone also showed (hyperbolic) partial, uncompetitive inhibition with micromolar Ki values. For Nile Blue A a model for two-site binding with (parabolic) uncompetitive inhibition fitted the data with a Ki value of 1 1 pM and a kinetic cooperativity between the sites of 0.12, increased to 0.46 by preincubation 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 conformational change in the homodimeric enzyme, the first such indication of kinetic cooperativity in the native enzyme to our knowledge. Further evidence of conformational changes for this enzyme came from studies of the effects of dimethyl sulphoxide which indicated that this co-solvent, which at low concentrations has no apparent effect on initial velocities under normal assay conditions, induced a slow conformational change in the enzyme. Thionine 0, Nile Blue A and LY83583 were redox-cycling substrates producing superoxide ion, detectable by means of cytochrome c reduction, but leading to no loss of glutathione reductase activity, under aerobic or anaerobic conditions. The water-soluble Safranine analogues Methylene Blue, Methylene Green, Nile Blue A and Thionine 0 (5 mg/kg i.p. x 5) were effective antimalarial agents in vivo against P . berghei, but their effect was small and a higher dose (50mg/kg i.p. x 1) was toxic in mice. Comparison was made with human glutathione reductase and its literaturereported interactions with several tricyclic inhibitors as studied by X-ray diffraction. It is possible that the conformational changes detected in the present study from alterations *Corresponding author. Fax: 0161 275 2396. E-mail: Ken.Douglas@man.ac.uk. 327 328 R.M. LUOND el al.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.

Coupling of a Competitive and an Irreversible Ligand Generates Mixed Type Inhibitors ofTrypanosomacruziTrypanothione Reductase

“…In the assays started with substrate, EI can be formed before GSSG is added, whereas in assays started with enzyme or NADPH, no preformed EI complex is present. Another explanation is that the DMSO present as solvent in the assay is responsible for a slow conformational change as reported earlier for yeast GR …”

Superoxide constricts rat pulmonary arteries via Rho-kinase-mediated Ca2+ sensitization

“…We also eliminated the possibility that LY83583 was causing constriction via inhibition of endothelial NO production [3,13] or inhibition of glutathione reductase [48] or by an endogenous constrictor prostanoid produced as a result of cyclooxygenase stimulation [16]. As shown in Supplementary Fig.…”

“…LY83583-mediated constriction in IPA was independent of inhibition of guanylate cyclase or glutathione reductase by LY83583 [46,48], either directly or via superoxide, because constriction to LY83583 persisted in the presence of the structurally unrelated inhibitors ODQ and BCNU, respectively, which did not themselves cause constriction. The LY83583-mediated constriction in IPA also did not depend on an intact endothelium and was therefore independent of NO production or endogenous prostanoid release, despite evidence suggesting that the harmful vasoconstrictor and proliferative effects of superoxide in the vasculature are due to NO scavenging and subsequent uncoupling of e-NOS by peroxynitrite [1,3,11].…”

A Prodrug Form of a Plasmodium falciparum Glutathione Reductase Inhibitor Conjugated with a 4-Anilinoquinoline

“…We focused on GR inhibitors as CQ sensitizers because these compounds are promising antimalarial agents on their own merits. Different series of GR inhibitors including nitrosoureas, quinones, − 3,7-diamino-2,8-dimethyl-5-phenylphenazinium chloride (safranin), 6-hydroxy-3-oxo-3 H -xanthene-9-propionic acid, 10-arylisoalloxazines, , the dye methylene blue, and ajoene have been described. GSH depletion in P. falciparum trophozoite-infected red blood cells by GR inhibitors such as 1,3-bis(2-chloroethyl)-1-nitrosourea, 10-arylisoalloxazines, methylene blue, and ajoene confers a drastic antiplasmodial effect. ,− Noteworthy is that three base exchanges found in the GR cDNAs from the chloroquine-sensitive strain 3D7 and the chloroquine-resistant strain K1 resulted in a GR with a 5-fold catalytic efficiency to regenerate GSH …”