Fig 1.
The novel nematicide wact-86 interacts with aldicarb to kill C. elegans.
(A) The chemical structure of wact-86. (B) Combination dose-response matrix for wact-86 and aldicarb. Worm abundance, relative to the DMSO control, is represented by a colour-coded scale ranging from 0 (no viable worms) to ≥1 (at least as many viable worms as DMSO control). See Methods for how the relative worm abundance value was calculated.
Fig 2.
Wact-86 resistant mutants harbour missense mutations in the carboxylesterase gene ges-1.
(A) wact-86 dose-response assays for wild-type worms and the three wact-86 resistant mutants. For each resistant strain the ges-1 allele and the GES-1 amino acid substitution are indicated (see S2 File for the whole genome sequencing data obtained for the resistant mutants). (B) Sequence alignment of the C. elegans GES-1 protein with the orthologous carboxylesterases from fly, fish, mouse, and human. For clarity, only the segment that is mutated in the wact-86 resistant strains is shown. Conserved residues are highlighted in black. The two GES-1 residues that are mutated in the wact-86 resistant strains are highlighted in grey. The asterisk denotes the conserved histidine that is part of the enzyme’s catalytic triad.
Fig 3.
Wact-86-resistant mutants have increased GES-1-dependent hydrolysis of wact-86.
(A) HPLC analysis of wact-86 metabolism for the indicated genotypes. HPLC-DAD chromatograms for the lysates of live worms incubated in DMSO alone, the lysates of live and dead worms incubated in wact-86, and 5 nmol of wact-86 stock compound are shown. The magenta asterisks indicate the DMSO peak, the red asterisks indicate the peaks of endogenous worm metabolites, the white arrow indicates the wact-86 parent structure peak, and the yellow arrow indicates the presumptive wact-86 metabolite peak. The peak that elutes at ~4.3 minutes in all of the chromatograms is a background instrument peak. (B and C) Mass spectral data for the DMSO control and metabolite fractions collected from the lysates of worms with the indicated genotypes, as well as tandem MS/MS fragmentation spectra for the 283.1 mass found exclusively in the metabolite fractions (see S1 Table for accurate mass data). (D and E) Quantification of wact-86 metabolite (D) and parent structure (E) accumulation in worms incubated in 30 μM wact-86. The genotypes are indicated. ok2716 and tm4694 are deletion alleles of ges-1. For control purposes, quantification was also performed for worms incubated in DMSO alone, as well as for dead worms incubated in wact-86, where appropriate. Area under the curve (AUC) was calculated at a wavelength of 290 nm and a retention time of 3.7 minutes for the metabolite. For the wact-86 parent structure, AUC was calculated at a wavelength of 316 nm and a retention time of 4.5 minutes. One, two, and three asterisks indicate student’s t-test p < 0.05, p < 0.01, and p < 0.001, respectively, compared to wild-type. Error bars represent the s.e.m.
Fig 4.
Aldicarb potentiates wact-86 activity by inhibiting its GES-1-dependent hydrolysis.
(A) wact-86 dose-response assays, plus and minus 20 μM aldicarb, for wild-type worms and two strains harbouring ges-1 deletion alleles. (B) Quantification of wact-86 metabolite accumulation in wild-type worms incubated in wact-86 alone or in combination with aldicarb. (C) Quantification of wact-86 accumulation in wild-type worms incubated in wact-86 alone or in combination with aldicarb. For B and C, the area under the curve (AUC) values for the wact-86 parent and metabolite were calculated as in Fig 3. One and three asterisks indicate student’s t-test p < 0.05 and p < 0.001, respectively, compared to the aldicarb-untreated condition. Error bars represent the s.e.m.
Table 1.
The HPLC solvent and flow rate gradients used herein.
Solvent A is 4.9:95:0.1 (ACN:H2O:Acetic Acid); Solvent B is 95:4.9:0.1 (ACN: H2O:Acetic Acid).