Given the role of
trypanothione in the redox defenses of pathogenic trypanosomal and leishmanial parasites, in contrast to
glutathione for their mammalian hosts, selective inhibitors of
trypanothione reductase are potential drug leads against
trypanosomiasis and
leishmaniasis. In the present study, the rational drug design approach was used to discover tricyclic
neuroleptic molecular frameworks as lead structures for the development of inhibitors, selective for
trypanothione reductase over host
glutathione reductase. From a homology-modeled structure for
trypanothione reductase, replaced in the later stages of the study by the X-ray coordinates for the
enzyme from Crithidia fasciculata, a series of inhibitors based on
phenothiazine was designed. These were shown to be reversible inhibitors of
trypanothione reductase from Trypanosoma cruzi, linearly competitive with
trypanothione as substrate and noncompetitive with
NADPH, consistent with ping-pong bi bi kinetics. Analogues, synthesized to define structure-activity relationships for the active site, included N-acylpromazines, 2-substituted
phenothiazines, and trisubstituted promazines. Analysis of Ki and I50 data, on the basis of calculated log P and molar refractivity values, provided evidence of a specially favored fit of small 2-substituents (especially 2-chloro and 2-trifluoromethyl), with a remote hydrophobic patch on the
enzyme accessible for larger, hydrophobic 2-substituents. There was also evidence of an additional hydrophobic enzymic region available to suitable N-substituents of the
promazine nucleus. Ki data also indicated that the
phenothiazine nucleus can adopt more than one inhibitory orientation in its binding site. Selected compounds were tested for in vitro activity against Trypanosoma brucei, T. cruzi, and Leishmania donovani, with selective activities in the micromolar range being determined for a number of them.