The new anthelmintic tribendimidine is an L-type (levamisole and pyrantel) nicotinic acetylcholine receptor agonist - PubMed (original) (raw)
The new anthelmintic tribendimidine is an L-type (levamisole and pyrantel) nicotinic acetylcholine receptor agonist
Yan Hu et al. PLoS Negl Trop Dis. 2009.
Abstract
Background: Intestinal parasitic nematodes such as hookworms, Ascaris lumbricoides, and Trichuris trichiura are amongst most prevalent tropical parasites in the world today. Although these parasites cause a tremendous disease burden, we have very few anthelmintic drugs with which to treat them. In the past three decades only one new anthelmintic, tribendimidine, has been developed and taken into human clinical trials. Studies show that tribendimidine is safe and has good clinical activity against Ascaris and hookworms. However, little is known about its mechanism of action and potential resistance pathway(s). Such information is important for preventing, detecting, and managing resistance, for safety considerations, and for knowing how to combine tribendimidine with other anthelmintics.
Methodology/principal findings: To investigate how tribendimidine works and how resistance to it might develop, we turned to the genetically tractable nematode, Caenorhabditis elegans. When exposed to tribendimidine, C. elegans hermaphrodites undergo a near immediate loss of motility; longer exposure results in extensive body damage, developmental arrest, reductions in fecundity, and/or death. We performed a forward genetic screen for tribendimidine-resistant mutants and obtained ten resistant alleles that fall into four complementation groups. Intoxication assays, complementation tests, genetic mapping experiments, and sequencing of nucleic acids indicate tribendimidine-resistant mutants are resistant also to levamisole and pyrantel and alter the same genes that mutate to levamisole resistance. Furthermore, we demonstrate that eleven C. elegans mutants isolated based on their ability to resist levamisole are also resistant to tribendimidine.
Conclusions/significance: Our results demonstrate that the mechanism of action of tribendimidine against nematodes is the same as levamisole and pyrantel, namely, tribendimidine is an L-subtype nAChR agonist. Thus, tribendimidine may not be a viable anthelmintic where resistance to levamisole or pyrantel already exists but could productively be used where resistance to benzimidazoles exists or could be combined with this class of anthelmintics.
Conflict of interest statement
The authors have declared that no competing interests exist.
Figures
Figure 1. Intoxication of C. elegans by tribendimidine.
A. L4 worms exposed to no drug (upper) or 100 µg/mL tribendimidine (lower) for 24 h at 25° and photographed at 30× magnification. Tribendimidine causes most C. elegans animals to coil. Scale bar applies to both panels. B and C. 600× magnification of animals under various conditions. B. Wild-type control animal without drug showing healthy intestine (between black arrowheads). White arrowheads (here and in other panels) point to cuticular regions within which the pharyngeal isthmus is contained. C. Animals on 100 µg/mL tribendimidine. Top row: wild-type animals on tribendimidine. Significant damage to the intestine (between black arrowheads) is evident, as well as degradation of the body around the pharyngeal isthmus of the left-most animal. Bottom row: tribendimidine resistant animals on tribendimidine. Note, all have healthy intestines and no degradation of body cavity is evident. Scale bar in B applies to all images in B and C. wt = wild type. Alleles used are trb-1(ye492), trb-2(ye493), trb-3(ye494), and trb-4(ye495).
Figure 2. Dose response of wild-type C. elegans to tribendimidine.
A. Response of wild-type (N2) C. elegans to tribendimidine as measured by the effect of various doses of the drug on the ability of larvae to develop to adulthood. B. Response of C. elegans to tribendimidine measured by the effect of various doses of the drug on viability. LC50 value is given in Table 2. For both A and B, each data point represents on average 180 nematodes (n = 3 repeats; 3 replicate wells per repeat). Error bars represent standard error of the mean for the three independent experiments. For converting to a mM dose, 100 µg/mL tribendimidine is equivalent to 0.22 mM.
Figure 3. trb mutants resist tribendimidine-induced mortality.
The response of wild-type N2 and four trb mutant animals to various doses of tribendimidine as measured by viability after 6 days at 25°. Each data point represents on average 180 worms (n = 3 repeats; 3 replicate wells per repeat). Error bars represent standard error of the mean for the three independent experiments. Allele designations are as in Figure 1. The LC50 value for wild-type is reported in Table 2.
Figure 4. trb mutants resist tribendimidine-induced sterility.
Brood sizes of N2 wild-type (wt) and trb mutant hermaphrodites after 64 h on either no drug or three different doses of tribendimidine. The experiment was done at 25° and repeated a total of three times with five wells/experiment/genotype/dose (thus each bar represents brood sizes from 15 worms). Whereas the brood size of wild-type animals decreases upon exposure to tribendimidine (P<0.01 for 0 vs. 50 µg/mL; P<0.001 for 0 vs. 100 and 200 µg/mL), the brood sizes of all four _trb_ mutants are unaffected even by the highest dose of tribendimidine tested (P>0.05 for all pair-wise comparisons of all doses for any given mutant). The relative brood sizes of wild-type animals at 50, 100, and 200 µg/mL are respectively 31%, 12%, and 5% of the brood size without toxin. The brood size of trb-4 animals in the absence of drug is clearly lower than that of the other genotypes. Further data and discussion of brood sizes of wild-type vs. trb animals in the absence of drug is given in Figure S2. Error bars represent standard deviations. Allele designations are as in Figure 1.
Figure 5. trb mutants are qualitatively resistant to levamisole, and pyrantel.
L4 hermaphrodites of the genotype indicated above the panels were seeded onto plates with no anthelmintic (control), 0.22 mM tribendimidine (100 µg/mL), 1 mM levamisole, or 2 mM pyrantel. Animals were incubated at 25° for 24 h. Photos were taken under the dissecting scope at 30× magnification (scale bar in first panel = 0.5 mm). Wild-type (wt) N2 animals are immotile and cluster on plates with any of these drugs. trb-1, trb-2, and trb-3 animals are motile on all three drugs and do not cluster, indicating their resistance to all three. trb-4 animals are motile on these drugs but are Unc even in the absence of drugs and therefore cannot move normally. Some of these animals cluster and some do not on the drugs, but they are clearly resistant. Allele designations are as in Figure 1.
Figure 6. trb mutants are quantitatively resistant to levamisole.
Mortality of wild-type (wt) N2 and trb-1, -2, -3, and -4 mutant animals exposed to varying doses of levamisole for 6 days at 25°. Each data point represents on average 180 worms (n = 3 repeats; 3 replicate wells per repeat). Error bars represent standard error of the mean for the three independent experiments. Allele designations are as in Figure 1. The LC50 value for each genotype is reported in Table 2. For converting to a mM dose, 100 µg/mL levamisole is equivalent to 0.42 mM.
Figure 7. Mutations in trb-1(ye492) are associated with exon/intron boundaries of the unc-63 gene.
Above, nucleotide sequence of the unc-63 gene at the exon 9/intron 9 and intron 9/exon 10 boundaries. Below, three altered nucleotides in trb-1(ye492) as indicated by down arrows. Assuming the mutant intron is not spliced, then inclusion of intron 9 would result in a translated protein with two missense mutations at amino acids 458 and 459 followed by a premature stop codon (the full length protein is normally 502 aa).
Figure 8. Levamisole-resistant mutants are resistant to tribendimidine.
Eleven mutants isolated based on their ability to resist levamisole were subjected to dose-dependent tribendimidine mortality assays. Each data point represents viability for three independent trials for each mutant, with three wells per trial (average of 180 worms per data point). Error bars represent standard error of the mean.
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