Abstract
Trypanothione reductase is a key enzyme in the trypanothione-based redox metabolism of pathogenic trypanosomes. Because this system is absent in humans, being replaced with glutathione and glutathione reductase, it offers a target for selective inhibition. The rational design of potent inhibitors requires accurate structures of enzyme-inhibitor complexes, but this is lacking for trypanothione reductase. We therefore used quinacrine mustard, an alkylating derivative of the competitive inhibitor quinacrine, to probe the active site of this dimeric flavoprotein. Quinacrine mustard irreversibly inactivates Trypanosoma cruzi trypanothione reductase, but not human glutathione reductase, in a time-dependent manner with a stoichiometry of two inhibitors bound per monomer. The rate of inactivation is dependent upon the oxidation state of trypanothione reductase, with the NADPH-reduced form being inactivated significantly faster than the oxidized form. Inactivation is slowed by clomipramine and a melarsen oxide-trypanothione adduct (both are competitive inhibitors) but accelerated by quinacrine. The structure of the trypanothione reductase-quinacrine mustard adduct was determined to 2.7 A, revealing two molecules of inhibitor bound in the trypanothione-binding site. The acridine moieties interact with each other through pi-stacking effects, and one acridine interacts in a similar fashion with a tryptophan residue. These interactions provide a molecular explanation for the differing effects of clomipramine and quinacrine on inactivation by quinacrine mustard. Synergism with quinacrine occurs as a result of these planar acridines being able to stack together in the active site cleft, thereby gaining an increased number of binding interactions, whereas antagonism occurs with nonplanar molecules, such as clomipramine, where stacking is not possible.
Highlights
The atomic coordinates and structure factors have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ
Reaction of Quinacrine Mustard with Trypanothione Reductase—Both oxidized (E) and NADPH-reduced (EH2) forms of trypanothione reductase (TryR) show time-dependent inactivation by 20 M quinacrine mustard (QM) (Fig. 2). This effect was specific for TryR because exposure of reduced human glutathione reductase to 100 M QM for 30 min did not cause any inhibition
QM-inactivated TryR could be readily visualized under ultraviolet light in SDS-PAGE gels because of the intense fluorescence of the acridine moiety (Fig. 3)
Summary
The atomic coordinates and structure factors (code 1GXF) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/). A promising target for the design of such inhibitors is the thiol metabolism of these protozoa This is dependent upon trypanothione (T[SH]2 or N1,N8-bis(glutathionyl)spermidine), a low molecular mass dithiol that is absent from humans but used by the kinetoplastids to maintain their intracellular redox balance [3]. This thiol acts to counteract environmental stress through a variety of enzymatic and nonenzymatic reactions and has been implicated in acquired resistance to chemotherapeutic agents [4, 5]. One of the best characterized of these inhibitors is quinacrine, an acridine derivative that is an effective and selective inhibitor of TryR [16, 17]
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