Date Published: October 31, 2007
Publisher: Public Library of Science
Author(s): Vanesa Olivares-Illana, Adela Rodríguez-Romero, Ingeborg Becker, Miriam Berzunza, Juventino García, Ruy Pérez-Montfort, Nallely Cabrera, Francisco López-Calahorra, Marieta Tuena de Gómez-Puyou, Armando Gómez-Puyou, John Dalton
Abstract: BackgroundChagas disease affects around 18 million people in the American continent. Unfortunately, there is no satisfactory treatment for the disease. The drugs currently used are not specific and exert serious toxic effects. Thus, there is an urgent need for drugs that are effective. Looking for molecules to eliminate the parasite, we have targeted a central enzyme of the glycolytic pathway: triosephosphate isomerase (TIM). The homodimeric enzyme is catalytically active only as a dimer. Because there are significant differences in the interface of the enzymes from the parasite and humans, we searched for small molecules that specifically disrupt contact between the two subunits of the enzyme from Trypanosoma cruzi but not those of TIM from Homo sapiens (HTIM), and tested if they kill the parasite.Methodology/Principal FindingsDithiodianiline (DTDA) at nanomolar concentrations completely inactivates recombinant TIM of T. cruzi (TcTIM). It also inactivated HTIM, but at concentrations around 400 times higher. DTDA was also tested on four TcTIM mutants with each of its four cysteines replaced with either valine or alanine. The sensitivity of the mutants to DTDA was markedly similar to that of the wild type. The crystal structure of the TcTIM soaked in DTDA at 2.15 Å resolution, and the data on the mutants showed that inactivation resulted from alterations of the dimer interface. DTDA also prevented the growth of Escherichia coli cells transformed with TcTIM, had no effect on normal E. coli, and also killed T. cruzi epimastigotes in culture.Conclusions/SignificanceBy targeting on the dimer interface of oligomeric enzymes from parasites, it is possible to discover small molecules that selectively thwart the life of the parasite. Also, the conformational changes that DTDA induces in the dimer interface of the trypanosomal enzyme are unique and identify a region of the interface that could be targeted for drug discovery.
Partial Text: Triosephosphate isomerase (TIM) is a ubiquitous enzyme that catalyzes the interconversion between glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. In most of the species the enzyme is formed by two identical monomers of approximately 250 amino acids. TIM belongs to the family of α-β barrels proteins, in which 8 central β strands are surrounded by 8 α helices; the strands and helices are joined by loops. It is one of the most thoroughly studied enzymes. Its kinetics are well established [1,2], the crystal structure of the enzyme from 15 different species is available, and significant advances have been made on the dynamics of the enzyme when it is in the resting state and during active catalysis [3–5].
TcTIM  and TIMs from T. brucei, L. mexicana, and Homo sapiens were expressed in Escherichia coli and purified as described in the indicated references. After purification, the enzymes dissolved in 100 mM triethanolamine, 10 mM EDTA and 1 mM dithiothreitol (pH 8) were precipitated with ammonium sulfate (75% saturation) and stored at 4°C. Before use, the enzymes were extensively dialyzed against 100 mM triethanolamine/10 mM EDTA (pH 7.4). Protein was determined from their absorbance at 280 nm . The ε M−1 cm−1 was 36440 for TcTIM, and 33460 for TbTIM and LmTIM.
As noted, we found that a contaminant that formed during the synthesis of 3-(-2-benzothiazolylthio)-1 propanethioaniline was a powerful inhibitor of the activity of TcTIM. The contaminant was identified as 2,2′-dithiodianiline (DTDA). The compound was subsequently synthesized, and the product characterized by mass spectrometry and NMR analysis. When the effect of DTDA was assessed in TIMs from various sources (Fig. 2), it was found that 260 nM induced 50% inhibition of the activity of TcTIM, and that 10 µM did not affect the activity of TbTIM, LmTIM and TIM from H. sapiens (HTIM). In connection to the inhibiting effect of DTDA on TcTIM, it is pointed out that the inhibition of activity was accompanied by aggregation of the enzyme, indicating that the compound induced drastic structural alterations. It is also noted, that at concentrations higher than those used in the experiment of Table 1, DTDA affected the activity of HTIM. Fifty percent inhibition of HTIM activity was achieved with 98 µM DTDA; thus, the selectivity for TcTIM in reference to HTIM is nearly 400-times. It is also noteworthy that TbTIM and LmTIM were completely insensitive to DTDA concentrations as high as 200 µM.
DTDA is a powerful inhibitor of the activity of TIM from T. cruzi. It is also effective in human TIM, but at concentrations that are nearly 400 times higher. Remarkably, the compound fails to affect the activity of TIM from T. brucei, and L. mexicana, albeit these enzymes are markedly similar to TcTIM in amino acid sequence and three-dimensional structure. An additional salient property of DTDA is that it is able to cross biological membranes as evidenced by the data with E. coli. These experiments also showed that DTDA does not affect the growth of intact E. coli, whereas in cells that depend on the function of TcTIM, low micromolar concentrations induce a strong inhibition of cell growth. These findings thus indicate that cell membranes are permeable to DTDA and that it affects adversely the life of cells that depend on the function of TcTIM. In consonance with these data, it was found that at concentrations of 4–8 µM, the compound induces a significant inhibition of the growth of T. cruzi epimastigotes, and that 10 µM and 15 µM causes death of parasites in cell cultures. Although the overall data suggest that the detrimental effect of DTDA on intact T. cruzi parasites is due to inhibition of the activity of their TIM, the results do not prove unambiguously that death of the parasites is due exclusively to the inhibition of that enzyme.