A new class of natural anthelmintics targeting lipid metabolism

Abstract

Parasitic helminths are a major global health threat, infecting nearly one-fifth of the human population and causing significant losses in livestock and crops. Resistance to the few anthelmintic drugs is increasing. Here, we report a set of avocado fatty alcohols/acetates (AFAs) that exhibit nematocidal activity against four veterinary parasitic nematode species: Brugia pahangi, Teladorsagia circumcincta and Heligmosomoides polygyrus, as well as a multidrug resistant strain (UGA) of Haemonchus contortus. AFA shows significant efficacy in H. polygyrus infected mice. In C. elegans, AFA exposure affects all developmental stages, causing paralysis, impaired mitochondrial respiration, increased reactive oxygen species production and mitochondrial damage. In embryos, AFAs penetrate the eggshell and induce rapid developmental arrest. Genetic and biochemical tests reveal that AFAs inhibit POD-2, encoding an acetyl CoA carboxylase, the rate-limiting enzyme in lipid biosynthesis. These results uncover a new anthelmintic class affecting lipid metabolism.

Fig. 1. A screen using C. elegans and P. pacificus identifies novel anthelmintic compounds.

a Treatment with AFA hits induced slow growth, sterility, larval lethality, larval arrest, adult lethality or paralysis or embryonic lethality (n = 3 biological replicates (BRs) with 2 technical replicates (TRs) each) (scale bar = 1 mm). b Chemical structure of avocado-derived compounds. All except the furans show anthelmintic activity against C. elegans and P. pacificus. c Incorporation of avocadene acetate in C. elegans embryos and larvae as detected by the ¹H NMR spectrum of the pure reference compound compared with the corresponding spectra of CHCl₃ fraction extracts from embryos and L4 larvae, with and without treatment with 20 μM or 10 μM avocadene acetate, respectively.

Fig. 2. AFAs impair development and survival in C. elegans and P. pacificus.

a-c Dose-response curves of the 8 AFA compounds. a Percentage of animals developing past the L1 stage after 5 days of treatment beginning at the L1 stage. b Percentage of adults surviving after 48 hours of treatment. c Percentage of eggs hatched after 48 hours of treatment. d Calculated LD₅₀ values (concentration at which development or survival was reduced by 50%) of AFA compounds and ivermectin in each assay. e Percentage of eggs hatched in N2 and PS312 after 48 hours of treatment with existing anthelmintics and avocadyne acetate at 5 and 10 µM. Lower and upper bounds of the box correspond to the first and third quartiles (25th and 75th percentile), whiskers extend to the largest or smallest value no further than 1.5 times the inter-quartile range (IQR). f Dose-response curves of avocadyne acetate on the percentage survival of N2 dauers and daf-2 dauers upon 48 hours of exposure to the compound. Each data point represents mean ± SEM; for (a-c,f) n = 3 BRs with 4 TRs each with >50 worms/concentration; for (e) n = 3 BRs with 3 TRs each with >100 individual eggs/concentration. In all experiments, DMSO was used as a negative control. Source data are provided as a Source Data file.

Fig. 3. AFAs are active in vivo.

Eggs per gram of faeces and adult worm counts are reduced after avocatin A (100 mg/kg) treatment in mice infected with H. polygyrus. C57BL/6 mice were infected with 200 H. polygyrus larvae and were given Avocatin A or control vehicle on alternate days from day 9 to 19; egg counts and adult worm numbers were enumerated at day 21 post infection. Data represent pooled results from two independent experiments, each with 5 mice per group, and analyzed by unpaired two-tailed t-test (p = 0.0076 for eggs and p = 0.0188 for worms). Where applicable, data represents mean ± SEM. Source data are provided as a Source Data file.

Fig. 4. Avocadene acetate causes paralysis and ROS production in C. elegans.

a Stereoscope live imaging demonstrating the paralysis phenotype upon avocadene acetate treatment (n = 3 BRs) (scale bar = 1 mm). b Dose response curve of motility of young adult worms treated with varying concentrations of avocadene acetate for 5 minutes. c Dose response curve of pharyngeal pump frequency of young adult worms treated with avocadene acetate. Datapoints in (b and c) represent mean ± SEM; n = 3 BRs with 5 TRs each for (b) and n = 3 BRs with > 20 individual worms per concentration for (c). d Representative images of body wall muscle mitochondria in C. elegans strain SJ4103, illustrating the five morphological classes of progressive mitochondrial deterioration: Class 1, tubular network (similar to wild type); Classes 2-4, altered morphology with blebbing, fragmentation, and enlarged, rounded appearance (Class 2, abundant mitochondria; Class 3, fewer mitochondria; Class 4, very few mitochondria); Class 5, extremely few or no mitochondria detected (scale bar = 10 µm). L1 animals were treated for 48-hours with 0.5% DMSO (control) or 5 µM avocadyne acetate. e Distribution of mitochondrial defects in control and treated animals, (n = 12). Around 50 muscles from each group from two independent experiments were analyzed (p = 0.005, two sided Chi-square test with df = 4). Controls for all treatments were DMSO. f Kinetic oxygen consumption rate (OCR) measuring basal respiration, FCCP-driven OCR (maximal respiration) and sodium azide-driven OCR (non-mitochondrial respiration). Each time point represents the average OCR ± SEM of five individual wells, expressed as pmol/min/animal. g Intracellular ROS in 10 µM avocadene acetate treated animals (L1s treated for 48 hours) normalized to controls, using CM-H2DCFDA method. Three independent experiments were performed (unpaired two-tailed t-test, p < 0.0001). h Representative images of Mitosox fluorescence in the posterior pharyngeal region (left) with DIC overlay (right) in control and 10 µM avocadene acetate treated worms (L1s for 42 hours) (scale bar = 20 µm). i Mitosox fluorescence quantification using ImageJ software (n = 20; unpaired two-tailed t-test, p = 0.01). j Pre-treatment with 10 mM N-acetyl cysteine (NAC) partially rescued lethality induced by 15 µM avocadene acetate on L4 stage animals (n = 3 BRs, one-way ANOVA, p < 0.0001). Source data are provided as a Source Data file.

Fig. 5. AFAs target fatty lipid metabolism.

a LD₅₀ values of avocadene acetate in pod-2(ye60) and N2 control (L4 stage), in presence or absence of 50 µM malonyl-CoA supplementation. Data represent three biological replicate (n = 3) experiments with 4 technical replicates (n = 50 animals) each. b ¹H NMR stacked plot highlighting the decrease of the acetyl methyl peak of acetyl-CoA (AcCoA) at 2.2 ppm, after addition of a C. elegans protein fraction containing POD-2 to a standard reaction mixture (“Reference”: 50 µM AcCoA in D₂O + 50 µM ATP + 30 mM (NH₄)HCO₃). c Time course of the t₀-normalized AcCoA concentrations assessed from NMR spectra after POD-2 addition. The reported curves refer to the normalized AcCoA concentration under 4 different composition conditions, namely Reference solution (red), the Reference with 20 µM (purple) or 50 µM (green) avocadene acetate (Avo Ac), and the Reference devoid of ATP (violet). Each curve reports the signal intensity data from a single sample (n = 1). The error bars represent the maximal experimental uncertainty affecting the NMR signal areas. d NMR-based consumption of AcCoA relative to the Reference solution, measured 180 minutes after POD-2 addition. The color code and the error bars are the same as in (c). Each bar represents a single sample (n = 1) with the complement to unity of the 180-minutes concentration data-point reported in (c), after normalization over the corresponding value of the Reference solution. e Structural modeling of avocadene acetate and avocadyne acetate complexed with POD-2. (i) Model homodimer of POD-2 CT domain (residues 1438-2165) with biotin and CoA in the active site (boxed). Dimer and ligand bind sites were modeled using solved yeast CT dimer complexes (PDB ID: 1w2x, 5csl). (ii, iii) Top 3 conformations, as ranked by affinity, of (ii) avocadene acetate and (iii) avocadyne acetate complexed with POD-2. (iv) Overlap of biotin and CoA with the top avocadene acetate conformation at the CT active site illustrates how avocado-derived compounds block POD-2 activity. f DIC images showing that ACC inhibitors CP-640186 and sethoxydim impair embryonic development (n = 10), (scale bar = 10 µm). Source data are provided as a Source Data file.