Genome-wide mutagenesis and multi-drug resistance in American trypanosomes induced by the front-line drug benznidazole

Abstract

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and affects 5–8 million people in Latin America. Although the nitroheterocyclic compound benznidazole has been the front-line drug for several decades, treatment failures are common. Benznidazole is a pro-drug and is bio-activated within the parasite by the mitochondrial nitroreductase TcNTR-1, leading to the generation of reactive metabolites that have trypanocidal activity. To better assess drug action and resistance, we sequenced the genomes of T. cruzi Y strain (35.5 Mb) and three benznidazole-resistant clones derived from a single drug-selected population. This revealed the genome-wide accumulation of mutations in the resistant parasites, in addition to variations in DNA copy-number. We observed mutations in DNA repair genes, linked with increased susceptibility to DNA alkylating and inter-strand cross-linking agents. Stop-codon-generating mutations in TcNTR-1 were associated with cross-resistance to other nitroheterocyclic drugs. Unexpectedly, the clones were also highly resistant to the ergosterol biosynthesis inhibitor posaconazole, a drug proposed for use against T. cruzi infections, in combination with benznidazole. Our findings therefore identify the highly mutagenic activity of benznidazole metabolites in T. cruzi, demonstrate that this can result in multi-drug resistance, and indicate that vigilance will be required if benznidazole is used in combination therapy.

Identification of di-substituted ureas that prevent growth of trypanosomes through inhibition of translation initiation

“…1,3-diarylureas have two available cyclic rings that can be readily substituted by various functional groups, allowing the synthesis of several improved compounds 53 . The structure of I-17 could be used to design Trypanosoma kinases activators capable of interfering with eIF2α stress responses, potentially useful in combinatory therapies with current drugs, such as benznidazole and nifurtimox, known to be potent oxidative stress inducers 8 , 54 .…”

Challenges in Chagas Disease Drug Development

“…Reductive drug metabolism generates a series of reactive intermediates that have trypanocidal activity, most notably glyoxal in the case of benznidazole [ 24 ], and an unsaturated open-chain nitrile with nifurtimox [ 25 ] ( Figure 1 A,B). For benznidazole, drug-induced mutagenesis has been identified as a possible mode of action, resulting, for example, in disruption to DNA-repair mechanisms, and chromosome instability [ 26 , 27 , 28 , 29 , 30 ]. Possible inducers of mutagenesis include reactive drug metabolites, enhanced oxidative stress resulting from drug adduct interactions with trypanothione, and the production of 8-oxo-guanine and other oxidised nucleotides.…”

Experimental Strategies to Explore Drug Action and Resistance in Kinetoplastid Parasites

“…In general, whole genome sequencing (WGS) allows the detection of mutations such as single nucleotide polymorphisms (SNPs), as well as insertions and deletions (Indels), which can lead to resistance when occurring in drug targets or transporters [ 26 , 27 , 28 , 29 , 30 , 31 , 32 ]. Additionally, copy number variations (CNV) do occur in kinetoplastids and should therefore be incorporated in the analysis of altered drug tolerance [ 33 , 34 , 35 , 36 , 37 , 38 , 39 ]. CNVs arise as a result of the peculiar mechanism of genetic regulation in kinetoplastids where individual gene promotors are lacking and transcriptional regulation relies on RNA stability, resulting in the development of alternative tools to increase or decrease gene expression [ 40 , 41 , 42 , 43 ].…”