A Mechanism for Cross-Resistance to Nifurtimox and Benznidazole in Trypanosomes
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A mechanism for cross-resistance to nifurtimox and benznidazole in trypanosomes Shane R. Wilkinson*†, Martin C. Taylor‡, David Horn‡, John M. Kelly‡, and Ian Cheeseman‡ *School of Biological and Chemical Sciences, Queen Mary University of London, London E1 4NS, United Kingdom; and ‡Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, United Kingdom Edited by P. Borst, The Netherlands Cancer Institute, Amsterdam, The Netherlands, and approved February 12, 2008 (received for review November 21, 2007) Nifurtimox and benznidazole are the front-line drugs used to treat sensitive FAD- or FMN-containing enzymes that mediate a one- Chagas disease, the most important parasitic infection in the Amer- electron reduction of the nitro-group generating an unstable nitro- icas. These agents function as prodrugs and must be activated within radical. In the presence of oxygen, this radical undergoes futile the parasite to have trypanocidal effects. Despite >40 years of cycling to produce superoxide, with the subsequent regeneration of research, the mechanism(s) of action and resistance have remained the parent nitro-compound (12, 13). In trypanosomes, type II elusive. Here, we report that in trypanosomes, both drugs are acti- activity has been proposed as the main activation mechanism (14, vated by a NADH-dependent, mitochondrially localized, bacterial-like, 15). However, the only direct link between drug-induced reactive type I nitroreductase (NTR), and that down-regulation of this explains oxygen species formation and trypanocidal activity stems from how resistance may emerge. Loss of a single copy of this gene in functional studies on the iron-dependent superoxide dismutase Trypanosoma cruzi, either through in vitro drug selection or by tar- SODB1. Parasites lacking this gene are more sensitive to nifurtimox geted gene deletion, is sufficient to cause significant cross-resistance and benznidazole (16). Analysis of other components of the oxi- to a wide range of nitroheterocyclic drugs. In Trypanosoma brucei, dative defense system indicates they do not play a major role in loss of a single NTR allele confers similar cross-resistance without protecting trypanosomes against nitroheterocyclic drugs (17–22). affecting growth rate or the ability to establish an infection. This To resolve how nitroheterocyclic drugs are activated by trypano- potential for drug resistance by a simple mechanism has important somes, we used two approaches: the first involved in vitro selection implications, because nifurtimox is currently undergoing phase III to generate nifurtimox-resistant parasites, and the second entailed clinical trials against African trypanosomiasis. functional analysis of a recently identified trypanosomal type I NTR. Here, we demonstrate that this type I NTR has the capacity activation ͉ nitroheterocyclic drugs ͉ Trypanosoma brucei ͉ Trypanosoma cruzi to metabolize a wide range of nitroheterocyclic drugs, and that a reduction in this activity in both T. cruzi and T. brucei confers resistance to these trypanocidal agents. he protozoan parasites Trypanosoma cruzi, Trypanosoma brucei, Tand Leishmania are the causative agents of Chagas disease, Results African sleeping sickness, and leishmaniasis, respectively. Over 20 Selection of Nifurtimox-Resistant T. cruzi. To investigate the mech- million people are infected by these pathogens, and Ͼ500 million anism of nifurtimox resistance, we cultured T. cruzi in the presence are at risk. Because there is no immediate prospect of vaccines, of 10 M nifurtimox for 8 months (Materials and Methods). This chemotherapy is of importance. Nitroheterocyclic drugs such as drug concentration inhibits parasite growth by Ͼ99%. Several nifurtimox and benznidazole have been used for Ͼ40 years against drug-resistant parasite populations were generated independently, Chagas disease. However, their use is problematic. They can have and these were cloned by limited dilution (designated NifR). We side effects, and some strains are refractory to treatment, the basis first investigated the growth properties of three NifR clones (Fig. for which has yet to be elucidated (1). In addition, medication is 1A). In the absence of nifurtimox, their doubling time was compa- expensive with, for example, nifurtimox regimes requiring 10 mg rable to the parental line. When NifR parasites were grown in per kg of body weight in three or four doses per day over a 60- to medium containing nifurtimox (10 ⌴), the doubling time in- 120-day period. Because of these problems, the recommended creased 2-fold; by comparison, there was no growth in the parental course of treatment is often not completed, resulting in consider- line beyond 1 week. We next determined the extent of nifurtimox able scope for the development of resistance. Plans to extend the resistance by establishing the IC50. All clones were 4-fold more use of nifurtimox in combinational therapies with eflornithine to resistant to nifurtimox than controls (Fig. 1B). When the studies African sleeping sickness are being evaluated (2). This, in conjunc- were expanded to include other nitroheterocyclic drugs, cross- tion with reports that several new nitroheterocycles have trypano- resistance was observed to benznidazole, megazol, and nitrofura- cidal activities with no/low toxicity, has led to a renewed interest in zone (Fig. 1B). The phenotype was specific to this class of com- the use of these compounds as antiparasitic agents (3, 4). pound, because NifR clones had the same sensitivity to the Nitroheterocyclic compounds are characterized by a nitrogroup nonnitro-compound rimantadine as the parental line. linked to an aromatic ring (5). They include the broad-spectrum In vitro drug selection has been shown to give rise to parasites nitrofuran and nitroimidazole antibiotics, which are effective with an altered karyotype (11, 23). To investigate whether this had against a variety of bacterial and parasitic infections (5, 6). These occurred here, we analyzed the chromosomal profiles from parental agents function as prodrugs and must undergo enzyme-mediated and NifR parasites and found that an 800-kbp band present in activation within the pathogen to have cytotoxic effects, reactions, which are catalyzed by nitroreductases (NTRs). Based on oxygen sensitivity, NTRs are divided into two groups (7). Type I NTRs are Author contributions: S.R.W. designed research; S.R.W., M.C.T., D.H., J.M.K., and I.C. oxygen-insensitive, contain FMN as a cofactor, and function via a performed research; S.R.W. analyzed data; and S.R.W. and J.M.K. wrote the paper. series of two-electron reductions of the conserved nitro-group, The authors declare no conflict of interest. leading to moieties that promote DNA damage (8, 9). This class of This article is a PNAS Direct Submission. NTR is characteristically bacterial. The only trypanosomal enzyme †To whom correspondence should be addressed. E-mail: [email protected]. shown to mediate this type of activity is prostaglandin F2␣ synthase This article contains supporting information online at www.pnas.org/cgi/content/full/ (also known as ‘‘old yellow enzyme’’) (10, 11), although only under 0711014105/DCSupplemental. anaerobic conditions. Type II NTRs are ubiquitous oxygen- © 2008 by The National Academy of Sciences of the USA 5022–5027 ͉ PNAS ͉ April 1, 2008 ͉ vol. 105 ͉ no. 13 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0711014105 Downloaded by guest on September 30, 2021 A C D Fig. 1. Nifurtimox-resistant T. cruzi obtained by in vitro culture are cross-resistant to other nitrohetero- cyclic drugs and have lost a copy of a TcNTR-containing chromosome. (A) Cumulative cell density of parental (X10/6) and laboratory-generated nifurtimox-resistant (NifR) lines in medium containing 10 M nifurtimox. Two other NifR clones analyzed in parallel displayed similar growth properties. (B) The drug concentrations that inhibited T. cruzi growth by 50% were estab- B nifurtimox benznidazole nitrofurazone megazol rimantadine ( M) M) ( M) ( M) ( M) lished. The data are means from three experiments Ϯ 14 90 20 4 30 standard deviation (SD). Differences observed in sus- 80 Ͻ 12 3.5 25 ceptibility were statistically significant (P 0.01), as 70 10 15 3 assessed by Student’s t test. Rimantadine was used as a 60 2.5 20 8 50 drug control. (C) Ethidium bromide-stained CHEFE gel 10 2 15 6 40 containing genomic DNA from T. cruzi. The asterisk (*) 30 1.5 10 4 identifies an 800-kbp chromosome present in X10/6 20 5 1 5 R 2 10 0.5 (lane 1), but missing in Nif clones (lane 2). M corre- 0 0 0 0 0 sponds to yeast DNA markers. (D) Autoradiograph of T. cruzi chromosomal DNA separated by CHEFE and hy- TiT. cruzi R cell lines parentltal Nif bridized with TcNTR gene probe (lane order as in B). parental cells was missing from NifR clones (Fig. 1C). This corre- insertionally inactivated one TcNTR gene in the T. cruzi genome lated with a reduction in nifurtimox reducing activity in NifR (Fig. S2). DNA fragments corresponding to the 5Ј and 3Ј regions of parasites compared with that of controls (2.79 Ϯ 0.20 vs. 6.73 Ϯ 1.18 TcNTR were amplified and cloned sequentially on either side of a nmol NADH oxidized minϪ 1⅐mgϪ1 protein). Because nifurtimox cassette containing a puromycin resistance marker. The construct and benznidazole are prodrugs, we postulated that the observed was then used to transform T. cruzi epimastigotes and clones resistance might result from a reduction in the copy number of the selected using puromycin. Southern hybridization of genomic DNA gene encoding the physiological activator, and that it could be from heterozygous parasites (TcNTRϩ/Ϫ) showed that one TcNTR present on this chromosome. Intriguingly, in the parental T. cruzi allele could be disrupted (Fig. S2), with no obvious effect on BIOCHEMISTRY line, a putative type I NTR gene is located on chromosome parasite growth (Fig. 3A). To evaluate whether a reduction in the homologues of 1,000 and 800 kbp (Fig. 1D).