Lipophilic antifolate trimetrexate is a potent inhibitor of Trypanosoma cruzi: prospect for chemotherapy of Chagas' disease

Abstract

Trypanosoma cruzi, a protozoan parasite, is the causative agent for Chagas' disease, which poses serious public health problem in Latin America. The two drugs available for the treatment of this disease are effective only against recent infections and are toxic. Dihydrofolate reductase (DHFR) has a proven track record as a drug target. The lipophilic antifolate trimetrexate (TMQ), which is an FDA-approved drug for the treatment of Pneumocystis carinii infection in AIDS patients, is a potent inhibitor of T. cruzi DHFR activity, with an inhibitory constant of 6.6 nM. The compound is also highly effective in killing T. cruzi parasites. The 50 and 90% lethal dose values against the trypomastigote are 19 and 36 nM, and the corresponding values for the amastigote form are 26 and 72 nM, respectively. However, as TMQ is also a good inhibitor of human DHFR, further improvement of the selectivity of this drug would be preferable. Identification of a novel antifolate selective against T. cruzi would open up new therapeutic avenues for treatment of Chagas' disease.

FIG. 1.

Inhibition of TcDHFR-TS by TMQ. Enzyme activity was measured at several fixed concentrations of DHF (5 to 30 μM) and various concentrations of TMQ (0 to 26.22 nM). Reaction rate was measured by monitoring the decrease in absorbance at 340 nM due to oxidation of NADPH (accompanying reduction of DHF). Reaction rate is decreased in the presence of increasing concentrations of the inhibitor. The line I = 0 represents the control, to which no inhibitor was added. Bottom panel: sodium dodecyl sulfate-polyacrylamide gel electrophoresis of recombinant TcDHFR-TS at different stages of purification. Protein samples at various stages of purification were boiled with equal volumes of 2× SDS sample denaturing buffer and subjected to electrophoresis on 10% polyacrylamide gel containing 1% SDS. Ten microliters of total denatured sample was loaded in each lane. Lanes: 1, cell extract; 2, post-Q-Sepharose column; 3, dimeric pool from size exclusion chromatography on Superdex 200; 4, post-MTX-Sepharose column; 5, final protein pool from DEAE-Sephacel column step; 6, low-molecular-weight marker (molecular masses in kDa shown at the right).

FIG. 2.

Antiparasitic activity of TMQ. T. cruzi trypomastigotes and amastigotes were treated with various concentrations of TMQ (0 to 1,000 nM) at 37°C. As with time trypomastigotes tend to transform into amastigotes, the number of trypomastigotes was counted only up to 24 h. In the case of amastigotes counting was continued up to 48 h. The number of live parasites is presented as percentages of control (untreated) organisms, trypomastigotes (white bars), and amastigotes (grey bars). The total numbers of parasites counted in the untreated control were 3.5 × 10⁶ and 2.1 × 10⁶ for trypomastigotes and amastigotes, respectively. No live parasites were observed at doses higher than 128 nM.