Specific chemotherapy of Chagas disease: relevance, current limitations and new approaches.

Trypanosoma cruzi is the causative agent of Chagas' disease. The major protease, cruzain, is a target for the development of new chemotherapy. We report the first successful treatment of an animal model of Chagas' disease with inhibitors designed to inactivate cruzain. Treatment with fluoromethyl ketone-derivatized pseudopeptides rescued mice from lethal infection. The optimal pseudopeptide scaffold was phenylalanine-homophenylalanine. To achieve cure of infection, this pseudopeptide scaffold was incorporated in a less toxic vinyl sulfone derivative. N-methyl piperazine-Phe-homoPhe-vinyl sulfone phenyl also rescued mice from a lethal infection. Six of the treated mice survived over nine months, three without further treatment. Three mice that had entered the chronic stage of infection were retreated with a 20-d regimen. At the conclusion of the experiments, five of the six mice had repeated negative hemacultures, indicative of parasitological cure. Studies of the effect of inhibitors on the intracellular amastigote form suggest that the life cycle is interrupted because of inhibitor arrest of normal autoproteolytic cruzain processing at the level of the Golgi complex. Parasites recovered from the hearts of treated mice showed the same abnormalities as those treated in vitro. No abnormalities were noted in the Golgi complex of host cells. This study provides proof of concept that cysteine protease inhibitors can be given at therapeutic doses to animals to selectively arrest a parasitic infection.

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Cysteine Protease Inhibitors Cure an Experimental Trypanosoma cruzi Infection

Nifurtimox and benznidazole are the front-line drugs used to treat Chagas disease, the most important parasitic infection in the Americas. These agents function as prodrugs and must be activated within the parasite to have trypanocidal effects. Despite >40 years of research, the mechanism(s) of action and resistance have remained elusive. Here, we report that in trypanosomes, both drugs are activated by a NADH-dependent, mitochondrially localized, bacterial-like, type I nitroreductase (NTR), and that down-regulation of this explains how resistance may emerge. Loss of a single copy of this gene in Trypanosoma cruzi, either through in vitro drug selection or by targeted gene deletion, is sufficient to cause significant cross-resistance to a wide range of nitroheterocyclic drugs. In Trypanosoma brucei, loss of a single NTR allele confers similar cross-resistance without affecting growth rate or the ability to establish an infection. This potential for drug resistance by a simple mechanism has important implications, because nifurtimox is currently undergoing phase III clinical trials against African trypanosomiasis.

https://www.pnas.org/content/pnas/105/13/5022.full.pdf

A mechanism for cross-resistance to nifurtimox and benznidazole in trypanosomes.

Current knowledge of the biochemistry of Trypanosoma cruzi has led to the development of new drugs and the understanding of their mode of action. Some trypanocidal drugs such as nifurtimox and benznidazole act through free radical generation during their metabolism. T. cruzi is very susceptible to the cell damage induced by these metabolites because enzymes scavenging free radicals are absent or have very low activities in the parasite. Another potential target is the biosynthetic pathway of glutathione and trypanothione, the low molecular weight thiol found exclusively in trypanosomatids. These thiols scavenge free radicals and participate in the conjugation and detoxication of numerous drugs. Inhibition of this key pathway could render the parasite much more susceptible to the toxic action of drugs such as nifurtimox and benznidazole without affecting the host significantly. Other drugs such as allopurinol and purine analogs inhibit purine transport in T. cruzi, which cannot synthesize purines de novo. Nitroimidazole derivatives such as itraconazole inhibit sterol metabolism. The parasite's respiratory chain is another potential therapeutic target because of its many differences with the host enzyme complexes. The pharmacological modulation of the host's immune response against T. cruzi infection as a possible chemotherapeutic target is discussed. A large set of chemicals of plant origin and a few animal metabolites active against T. cruzi are enumerated and their likely modes of action are briefly discussed.

Mode of action of natural and synthetic drugs against Trypanosoma cruzi and their interaction with the mammalian host.

The treatment of human African trypanosomiasis.

The susceptibility and natural resistance to two nitroheterocyclic derivatives used clinically in Chagas disease (nifurtimox and benznidazole) were investigated in 47 Trypanosoma cruzi strains isolated from human patients, domestic vectors and sylvatic reservoirs or vectors. A large gradient of drug efficacy from 0% to 100% was detected. Drug susceptibility apparently related to geographical distribution of some T. cruzi strains was also observed. Drug resistance was identified among T. cruzi populations isolated from sylvatic vectors from an area where autochthonous human Chagas disease does not exist. Thus, natural drug-resistance of sylvatic strains might be a way of introducing this character into a T. cruzi domestic cycle. Most of the 47 studied strains were either sensitive or resistant to both compounds, an intriguing finding considering that nifurtimox and benznidazole apparently have different mechanisms of action against T. cruzi.

L.S. Filardi

Papers

Susceptibility and natural resistance of Trypanosoma cruzi strains to drugs used clinically in Chagas disease.