Anti-trypanosomal activity and target validation of the M17 leucyl-aminopeptidase inhibitor DDD00057570 in Trypanosoma cruzi
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Background
Chagas disease, caused by the parasite Trypanosoma cruzi , affects six to seven million people, and its treatment still relies on two drugs introduced more than fifty years ago, both of limited efficacy and poorly tolerated. The major acidic M17 leucyl-aminopeptidase of T. cruzi is expressed in all developmental stages of the parasite and has been proposed as a drug target, but it has not been pharmacologically validated.
Methodology/Principal Findings
We assessed DDD00057570, a compound originally identified as an inhibitor of the orthologue enzyme of Leishmania major , against the recombinant T. cruzi enzyme and against the parasite. The compound inhibited the recombinant enzyme with an IC 50 of 2.7 ± 0.9 µM and reduced the proliferation of insect-stage epimastigotes, with a half-maximal effective concentration of 3.7 ± 1.1 µM. Parasites engineered to overexpress the enzyme were twice as resistant to the compound as wild-type parasites, indicating that the enzyme is a primary target in the living parasite. Treatment increased the proportion of epimastigotes with fragmented DNA. In infected mammalian cells, the compound reduced the release of trypomastigotes, the stage responsible for dissemination in the mammalian host, with a half-maximal effective concentration of 1.3 ± 0.6 µM, while its half-maximal cytotoxic concentration on CHO-K 1 mammalian host cells was 41.5 ± 3.9 µM, resulting in a selectivity index of approximately 32.
Conclusions/Significance
These results validate the major M17 leucyl-aminopeptidase as a drug target in T. cruzi through target overexpression, and identify DDD00057570 as a selective lead structure warranting mechanistic, pharmacokinetic and in vivo studies toward a new treatment for Chagas disease.
AUTHOR SUMMARY
Chagas disease is caused by Trypanosoma cruzi , a parasite spread by blood-feeding triatomine insects that affects millions of people, mostly in Latin America. The only two drugs available were discovered more than half a century ago; both often fail and cause side effects severe enough that many patients abandon treatment. New drugs are urgently needed, and finding them starts with knowing which parasite proteins are worth attacking. Herein we studied an enzyme the parasite uses to trim fragments from proteins and release leucine, a building block it needs to proliferate and sustain the infection. We tested a compound originally designed to block the same enzyme in the related parasite causing leishmaniasis. We found that it blocks the recombinant enzyme, slows the proliferation of the parasite, and reduces the number of parasites bursting from infected mammalian cells, the step by which infection spreads through the body. To confirm the enzyme is what the compound attacks, we engineered parasites carrying extra copies of it; these became more resistant, exactly as expected. The compound was far more toxic to the parasite than to mammalian cells. We therefore propose this enzyme as a genuine drug target, and this compound as a starting point.
