Compounds and methods for treating against nematode infections and methods for compound discovery for same

Abstract

Provided methods of inhibiting fatty acid synthesis and / or fatty acid oxidation pathways, methods of inhibiting nematode growth, and methods of treating a subject having a nematode-based infections. Also provided are compounds and compositions for uses thereof are also disclosed. Also provided are methods for identifying compounds suitable for inhibiting fatty acid synthesis and / or fatty acid oxidation pathways, methods of inhibiting nematode growth, and / or methods of treating a subject having a nematode-based infections Compounds of the present disclosure may have the following structure: (I) or (II) or (III) or (IV).

Description

Attorney Docket No.: 058636.00708 COMPOUNDS AND METHODS FOR TREATING AGAINST NEMATODE INFECTIONS AND METHODS FOR COMPOUND DISCOVERY FOR SAME CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 509,493, filed June 21, 2023, the disclosure of which is incorporated herein by reference in their entirety. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing, which is submitted in .xml format and is hereby incorporated by reference in its entirety. Said .xml file is named “058636_00708_ST26.xml”, was created on June 21, 2024, and is 10,351 bytes in size. BACKGROUND OF THE DISCLOSURE

[0003] More than 1.5 billion people are infected with helminth parasites, primarily affecting marginalized populations in low- and middle-income countries, according to the World Health Organization. These parasites are transmitted by skin-penetrating larvae or by eggs that are shed in human faeces and contaminate soil in areas where sanitation is poor. Helminth infections also have a devastating impact on livestock production globally and are damaging to all major crops, causing huge economic losses of over $80 billion per year.

[0004] Helminths compete with insects as the most evolutionarily successful animal clade on the planet, having colonized almost all known environments, including several extreme ones. Parasitic helminths use animals or plants as their host, resulting in both direct and indirect negative impacts on public health, agricultural systems, and the ecology and conservation of wild species. Helminth infections are particularly challenging to treat without causing toxicity to mammalian hosts, due to strong similarities in their physiology and a multitude of mechanisms to block uptake, metabolize or expel exogenous poisons. Despite the global challenges they pose, only a few drugs are commonly used to treat helminth infections, and rising drug resistance among human and animal parasitic helminths adds to the complexities of control efforts. New chemical classes with novel mechanisms of action are thus urgently needed to overcome the threat of resistance. One promising direction for the discovery of novel anthelmintic drugs is to identify molecules that target nematode chokepoint enzymes, and cocktails of drugs with different MOAs, which could provide greater protection against the development of resistance.

[0005] Nature has been a valuable source of anthelmintic compounds, exemplified by the discovery of ivermectin as a product of the soil bacterium Streptomyces avermitilis in the 1970s. However, direct high-throughput drug screening (HTS) of parasitic helminths is challenging because their life cycle requires a phase in the host, adding layers of complexity to the screening process. The free-living nematode C. elegans has already been established as a model for anthelmintic drug discovery, due to its ease of laboratory culture, amenability to HTS for rapid whole-organism phenotypic screening, and the availability of extensive forward and reverse genetic methods. These molecular genetic tools, combined with chemical perturbation, provide a powerful toolbox for in vivo mode-of-action (MOA) studies to identify molecular targets. Moreover, parallel screening in C. elegans and the distantly related free-living nematode P. pacificus enhances the discovery of candidates that may also display activity against helminth parasites. BRIEF SUMMARY OF THE DISCLOSURE

[0006] The present disclosure provides methods of inhibiting fatty acid synthesis and / or fatty acid oxidation pathways, methods of inhibiting nematode growth, and methods of treating a subject having a nematode-based infections. Also provided are compounds for uses thereof.

[0007] In an aspect, the present disclosure provides compounds. The compounds may be used to inhibit fatty acid synthesis and / or fatty acid oxidation pathways, inhibit nematode growth, treat a subject having or suspected of having a nematode-based infection, or generally as an anthelmintic.

[0008] The compounds of the present disclosure may have the following structure: orwhere X is a group formed from an anthelmintic compound. Y . R' is OH or O. R is H or -COCH3. R'' is H or an alkoxy. may be the same or different. In various examples, a compound of the present disclosure has the following structure: , ,, or of the present disclosure has the followingstructure: oror .administered as a composition comprising any combination of the compounds disclosed herein. For example, when the combination of compounds is:, A. In various examples, when the combination of compounds is: , B.

[0012] In an aspect, the present disclosure provides compositions comprising one or more compound(s) of the present disclosure. The compositions may further comprise one or more pharmaceutically acceptable carrier(s).

[0013] In an aspect, the present disclosure provides various methods. The methods may be methods of inhibiting fatty acid synthesis and / or fatty acid oxidation pathways, methods of inhibiting nematode growth, and / or methods of treating a subject having a nematode-based infections.

[0014] In an aspect, the present disclosure provides a method for identifying if a compound / drug is an anthelmintic. The method may be performed on known libraries or libraries made via OBOC (one bead one compound) methods.

[0015] In an example, the method comprises contacting a culture comprising one or more nematodes with the compound or drug; incubating the culture for a period of time; imaging at least a portion of the plurality of the nematodes after the incubating; and comparing the images of the at least a portion of the plurality of nematodes from the culture with a control, wherein the control comprises nematodes that have not been contacted with the compound / drug. In various examples, the culture further comprises E. coli. In various examples, if there is a plurality of nematodes, the nematodes may be a plurality of different nematode species or a single species of nematode. In various examples, if there is a plurality of nematodes, the nematodes may be at different developmental stages or roughly around the same developmental stage or the same developmental stage.BRIEF DESCRIPTION OF THE FIGURES

[0016] For a fuller understanding of the nature and objects of the disclosure, reference should be made to the following detailed description taken in conjunction with the accompanying figures.

[0017] FIG.1A. A screen using C. elegans and P. pacificus identifies novel anthelmintic compounds. Treatment with library compound hits induced slow growth, sterility, larval lethality, larval arrest, paralysis or embryonic lethality.

[0018] FIG.1B. A screen using C. elegans and P. pacificus identifies novel anthelmintic compounds. Chemical structure of avocado-derived compounds. All except the furans show anthelmintic activity against C. elegans and P. pacificus.

[0019] FIG.1C. A screen using C. elegans and P. pacificus identifies novel anthelmintic compounds. Incorporation of avocadene acetate in C. elegans embryos and larvae as detected by the1H NMR spectrum of the pure reference compound compared with the corresponding spectra of CHCl3fraction extracts from embryos and L4 larvae, with and without treatment with avocadene acetate.

[0020] FIG.2A. AFAs impair development and survival in C. elegans and P. pacificus. Dose-response curves of the 8 AFA compounds. Percentage of animals developing past the L1 stage after 5 days of treatment beginning at the L1 stage. Each data point represents mean±SEM; N=3 replicates; >50 individual worms / eggs per concentration. In all experiments, DMSO was used as a negative control.

[0021] FIG.2B. AFAs impair development and survival in C. elegans and P. pacificus. Dose-response curves of the 8 AFA compounds. Percentage of adults surviving after 48 hours of treatment. Each data point represents mean±SEM; N=3 replicates; >50 individual worms / eggs per concentration. In all experiments, DMSO was used as a negative control.

[0022] FIG.2C. AFAs impair development and survival in C. elegans and P. pacificus. Dose-response curves of the 8 AFA compounds. Percentage of eggs hatched after 48 hours of treatment. Each data point represents mean±SEM; N=3 replicates; >50 individual worms / eggs per concentration. In all experiments, DMSO was used as a negative control.

[0023] FIG.2D. AFAs impair development and survival in C. elegans and P. pacificus. Calculated LD50values (concentration at which development or survival was reduced by 50%) of AFA compounds and ivermectin in each assay. Each data point represents mean±SEM; N=3 replicates. In all experiments, DMSO was used as a negative control.

[0024] FIG.2E. AFAs impair development and survival in C. elegans and P. pacificus. Percentage of eggs hatched in N2 and PS312 after 48 hours of treatment with existing anthelmintics and avocadyne acetate at 5 and 10 µM. Each data point represents mean±SEM; N=3 replicates; >100 individual worms / eggs per concentration. In all experiments, DMSO was used as a negative control.

[0025] FIG.2F. AFAs impair development and survival in C. elegans and P. pacificus. 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; N=3 replicates; >50 individual worms / eggs per concentration. In all experiments, DMSO was used as a negative control.

[0026] FIG.3A. AFAs are effective against a multi-drug resistant parasitic nematode strain. Rates of larval lethality of parasitic worms after treatment with avocatin A or B. bData represent pooled results from two independent experiments each with 5 mice per group and analyzed by unpaired t-test.

[0027] FIG.3B. AFAs are effective against a multi-drug resistant parasitic nematode strain. Rates of larval lethality of B. pahangi adults, L3 and microfilariae (mf) after treatment with avocatin A. Data represent pooled results from two independent experiments each with 5 mice per group and analyzed by unpaired t-test.

[0028] FIG.3C. AFAs are effective against a multi-drug resistant parasitic nematode strain. Calculated LD50 values of AFA compounds in the anthelmintic-resistant C. elegans mutant strains. Data represent pooled results from two independent experiments each with 5 mice per group and analyzed by unpaired t-test.

[0029] FIG.3D. AFAs are effective against a multi-drug resistant parasitic nematode strain. Eggs per gram of feces and adult worm counts are reduced after avocatin A treatment in mice infected with H. polygyrus. C57BL / 6 mice were infected with 200 H. polygyrus larvae and 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 t-test.

[0030] FIG.4A. Avocadene acetate causes paralysis and ROS production in C. elegans. Stereoscope live imaging demonstrating the paralysis phenotype upon avocadene acetate treatment.

[0031] FIG.4B. Avocadene acetate causes paralysis and ROS production in C. elegans. Dose response curve of motility of young adult worms treated with varyingconcentrations of avocadene acetate for 5 minutes. Datapoints represent mean±SEM; N=3 replicates; >50 individual worms / eggs per concentration.

[0032] FIG.4C. Avocadene acetate causes paralysis and ROS production in C. elegans. Dose response curve of pharyngeal pump frequency of young adult worms treated with avocadene acetate. Datapoints represent mean±SEM; N=3 replicates; >50 individual worms / eggs per concentration.

[0033] FIG.4D. Avocadene acetate causes paralysis and ROS production in C. elegans. 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 average OCR±SEM of five individual wells, expressed as pmol / min / animal.

[0034] FIG.4E. Avocadene acetate causes paralysis and ROS production in C. elegans. Intracellular ROS in avocadyne acetate treated animals normalized to controls, using CM-H2DCFDA method. Three independent experiments were performed.(Unpaired t- test, p-value < .0001)

[0035] FIG.4F. Avocadene acetate causes paralysis and ROS production in C. elegans. Representative images of Mitosox fluorescence in the posterior pharyngeal region (left) with DIC overlay (right) in control and AFA-treated worms (scale bar = 20 µm).

[0036] FIG.4G. Avocadene acetate causes paralysis and ROS production in C. elegans. Mitosox fluorescence quantification using ImageJ software (N=20; unpaired t- test, p-value = 0.01).

[0037] FIG.4H. Avocadene acetate causes paralysis and ROS production in C. elegans. Pre-treatment with 10 mM N-acetyl cysteine (NAC) partially rescued lethality induced by 15 µM avocadene acetate (one way-ANOVA, p-value < .0001).

[0038] FIG.5A. AFAs target fatty lipid metabolism. LD50values of avocadene acetate in pod-2(ye60) and N2 control, in presence or absence of 50 µM malonyl-CoA supplementation. Data represent three biological replicate experiments with four technical replicates (n=50) each.

[0039] FIG.5B. AFAs target fatty lipid metabolism. NMR-based consumption of Acetyl-CoA by POD-2 enzyme (red, Reference) in the presence of 20 µM (purple) or 50 µM (green) avocadene acetate (Avo Ac) or in the absence of ATP (violet). Error bars represent the maximal experimental uncertainty affecting NMR signal areas.

[0040] FIG.5C. AFAs target fatty lipid metabolism. Structural modeling of avocadene acetate and avocadyne acetate complexed with POD-2. (i) Model homodimer ofthe 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) Top 3 conformations of avocadene acetate complexed with POD-2 as ranked by affinity. (iii) Top 3 conformations of 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.

[0041] FIG.5D. AFAs target fatty lipid metabolism. DIC images showing that ACC inhibitors CP-640186 and sethoxydim impair embryonic development; CP-640186 treatment phenocopies the avocadene acetate embryonic phenotype.

[0042] FIG.6A. Dose / response of AFA compounds in HCS panels. Cell counts in control (DMSO) or AFA-treated samples are shown.

[0043] FIG.6B. Dose response curves for replicate assays (blue), showing per-well cell counts (y-axis) vs. compound concentration in µM (x-axis). Median (dashed line) and range (dotted lines, minimum and maximum) of cell counts in DMSO controls and 30% threshold for cytotoxicity (red line) are shown for comparison.

[0044] FIG.7. Persenone A, persin and avocadane acetate impair L1 development. Exemplary images show the inhibition of development in N2 L1 stage worms upon treatment with persenone A, persin and avocadane acetate at different concentrations.

[0045] FIG.8. Anthelmintic-resistant mutants are sensitive to AFA compounds. Avocadene acetate is effective against ivermectin-resistant mutant strain JD608 avr- 14(ad1302); avr-15(ad1051) glc-1(pk54), levamisole-resistant mutant strain CB193 unc- 29(e193), monepantel resistant mutant strain RB2119 acr-23(ok2804) and benzimidazoles resistant mutant strain CB3474 ben-1(e1880).

[0046] FIG.9. Kinetic response of young adult worms treated with varying concentrations of avocadene acetate over a time course of 3 hours.

[0047] FIG.10A. AFA affects the development of all embryonic C. elegans stages. Graphical representation of different embryonic stages and development time course after treatment with avocadene acetate.

[0048] FIG.10B. AFA affects the development of all embryonic C. elegans stages. Timelapse videos showing treatment with 20 µM avocadene acetate (right), and DMSO control (left).

[0049] FIG.11A. Reduced respiration capacities in avocadyne acetate treated animals and pre-treatment with NAC and Vitamin B12 rescue avocadyne acetate phenotype. Graphical comparison of basal OCR, maximal respiration, and spare capacity (maximal OCR– basal OCR) per animal in avocadyne acetate treated and DMSO controls. (Comparison between groups were done using multiple unpaired t-test and statistical significance indicated by stars above the compared samples, *p-value < 0.05, **p-value < 0.001 and ***p-value < 0.0001.

[0050] FIG.11B. Reduced respiration capacities in avocadyne acetate treated animals and pre-treatment with NAC and Vitamin B12 rescue avocadyne acetate phenotype. Representative images to show that pre-treatment with 10mM N-acetyl cysteine partially rescued the worms from avocadyne acetate Ac induced toxicity.

[0051] FIG.11C. Reduced respiration capacities in avocadyne acetate treated animals and pre-treatment with NAC and Vitamin B12 rescue avocadyne acetate phenotype. Representative images show N2 L1 stage worms pre-treated with / without 64nM vitamin B12 until stage L4, followed by the treatment of 6µM, 8µM and 10µM of avocadyne acetate. Survival scores represent the development of the worms upon 5 days of treatment, with 0 being 100% arrested / lethal and 5 being 100% developed / alive.

[0052] FIG.11D. Reduced respiration capacities in avocadyne acetate treated animals and pre-treatment with NAC and Vitamin B12 rescue avocadyne acetate phenotype. Representative histogram show N2 L1 stage worms pre-treated with / without 64nM vitamin B12 until stage L4, followed by the treatment of 6µM, 8µM and 10µM of avocadyne acetate. Survival scores represent the development of the worms upon 5 days of treatment, with 0 being 100% arrested / lethal and 5 being 100% developed / alive. Each bar in the histograms represent mean ± S.E.M.; N=4 replicates; >50 individual worms. Comparison between groups were done using multiple unpaired t-test and statistical significance indicated by stars above the compared samples, *p-value < 0.05, **p-value < 0.001 and ***p-value < 0.0001.

[0053] FIG.12. Representative images of body wall muscle mitochondria in C. elegans strain SJ4103 after 48-hour treatment with 0.5% DMSO (control) or 5µM Avocadyne Acetate, 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). l, Distribution of mitochondrial defects in control and treated animals, N=12). 50 muscles from each group from two independent experiments were analyzed (top graph: p-value = 0.0056, Fisher’s Exact test). Controls for all treatments were DMSO.

[0054] FIG.13. Knockdown of carnitine shuttle rescues avocadyne acetate phenotype in C. elegans. The histogram illustrates the effect on development of N2 L1 stage worms grown in the presence of 5µM avocadyne acetate or 1% DMSO and exposed with RNAi for acs-2, cpt-1, cpt-4, dif-1, cpt-2 or empty vector L4440. Survival scores represent the development of the worms upon 5 days of treatment, with 0 being 100% arrested / lethal and 5 being 100% developed / alive. Each bar in the histograms represent mean ± S.E.M.; N=3 replicates; >50 individual worms. Comparison between groups were done using unpaired t- test and statistical significance indicated by stars above the compared samples, *p-value < 0.05, **p-value < 0.001 and ***p-value < 0.0001.

[0055] FIG.14A. Overlay of1H DOSY map and corresponding1H NMR region of avocadene acetate in CDCl3 highlighting the uniformity of the translational diffusion coefficient (D) of the molecule. The circled signals of the acetyl methyl (1’) and the terminal CH2(17) (FIG.14B) exhibit the same D value, i.e.7.66±0.04 ^10^10m2 / s and 7.56±0.13 ^10^10m2 / s, respectively. The molecular species employed for the experiments was synthesized with13C labeling at the acetyl moiety.

[0056] FIG.14B. Overlay of1H DOSY map and corresponding1H NMR region of avocadene acetate in CDCl3highlighting the uniformity of the translational diffusion coefficient (D) of the molecule. The circled signals of the acetyl methyl (1’) and the terminal CH2 (17) (FIG.14B) exhibit the same D value, i.e.7.66±0.04 ^10^10m2 / s and 7.56±0.13 ^10^10m2 / s, respectively. The molecular species employed for the experiments was synthesized with13C labeling at the acetyl moiety.

[0057] FIG.14C. Overlay of1H DOSY map and corresponding1H NMR region of avocadene acetate in CDCl3. Theoverlay as that in panel (FIG.14A) is shown for the chloroform extracts of C.elegans embryos, following 30 minutes treatment with avocadene acetate. The D values measured for the chloroform extracts were 6.15±0.50 ^10^10m2 / s and 5.34±1.92 ^10^10m2 / s, for the acetyl methyl (1’) and the CH2 (17) of avocadene acetate. Despite the errors due to the low intensity of the avocadene acetate signals, the reliability of the determinations is ensured by the observed D value of CDCl3(invariably 1.90±0.02 ^10^9m2 / s, vs.2.01 ^10^9m2 / s expected).

[0058] FIG.14D. Overlay of1H DOSY map and corresponding1H NMR region of avocadene acetate in CDCl3. The same overlay as that in panel (FIG.14A) is shown for the chloroform extracts of C.elegans embryos, following 120 minutes treatment with avocadene acetate. The D values measured for the chloroform extracts were 6.26±0.58 ^10^10m2 / s and6.43±2.71 ^10^10m2 / s (D), for the acetyl methyl (1’) and the CH2 (17) of avocadene acetate. Despite the errors due to the low intensity of the avocadene acetate signals, the reliability of the determinations is ensured by the observed D value of CDCl3 (invariably 1.90±0.02 ^10^9m2 / s, vs.2.01 ^10^9m2 / s expected).

[0059] FIG.14E. Overlay of1H DOSY map and corresponding1H NMR region of avocadene acetate in CDCl3. The same overlay as that in panel (FIG.14A) is shown for the chloroform extracts of C.elegans L4 larvae, following 30 minutes treatment. The D values measured for the chloroform extracts were, 6.23±1.79 ^10^10m2 / s and 8.56±2.22 ^10^10m2 / s, for the acetyl methyl (1’) and the CH2 (17) of avocadene acetate. Despite the errors due to the low intensity of the avocadene acetate signals, the reliability of the determinations is ensured by the observed D value of CDCl3 (invariably 1.90±0.02 ^10^9m2 / s, vs.2.01 ^10^9m2 / s expected).

[0060] FIG.14F. Overlay of1H DOSY map and corresponding1H NMR region of avocadene acetate in CDCl3. The overlay as that in panel is shown for thechloroform extracts of C.elegans L4 larvae, following 120 minutes treatment. The D values measured for the chloroform extracts were 5.78±0.41 ^10^10m2 / s and 5.98±2.81 ^10^10m2 / s for the acetyl methyl (1’) and the CH2(17) of avocadene acetate. Despite the errors due to the low intensity of the avocadene acetate signals, the reliability of the determinations is ensured by the observed D value of CDCl3 (invariably 1.90±0.02 ^10^9m2 / s, vs.2.01 ^10^9m2 / s expected).

[0061] FIG.15A. Malonyl-CoA supplementation alleviates the embryonic lethality phenotype of pod-2(ye60) mutant and partially rescues the avocadene acetate lethality phenotype in both N2 and pod-2 worms. Dose response and LD50 shift curves showing that pod-2(ye60) mutant is more sensitive to avocadene acetate and 50 µM malonyl-CoA supplementation partially rescues the lethality. Three biological replicate experiments with three technical replicates per data point are shown with average and SEM plotted. Each bar in the histograms represent mean ± SEM.; N=5 replicates; >50 individual worms. Comparison between groups were done using unpaired t-test and statistical significance indicated by stars above the compared samples, *p-value < 0.05 and ****p-value < 0.0001.

[0062] FIG.15B. Malonyl-CoA supplementation alleviates the embryonic lethality phenotype of pod-2(ye60) mutant and partially rescues the avocadene acetate lethality phenotype in both N2 and pod-2 worms. Representative images showing partial rescue of avocadene acetate lethality phenotype by malonyl CoA, in both WT and pod-2 mutant. Eachbar in the histograms represent mean ± SEM.; N=5 replicates; >50 individual worms. Comparison between groups were done using unpaired t-test and statistical significance indicated by stars above the compared samples, *p-value < 0.05 and ****p-value < 0.0001.

[0063] FIG.15C. Malonyl-CoA supplementation alleviates the embryonic lethality phenotype of pod-2(ye60) mutant and partially rescues the avocadene acetate lethality phenotype in both N2 and pod-2 worms. Average LD50of wild type, bpl-1::AID and pod- 2::AID strains pre-treated with Auxin followed by treatment with avocadene acetate at various concentrations. Each bar in the histograms represent mean ± SEM.; N=5 replicates; >50 individual worms. Comparison between groups were done using unpaired t-test and statistical significance indicated by stars above the compared samples, *p-value < 0.05 and ****p-value < 0.0001.

[0064] FIG.16A. Sequence and structure alignments of CT domains of POD-2 and human ACC proteins. Alignments of the BC domain: structure alignment of the modeled POD-2 BC domain and solved ACC2 BC domain with bound ND-646 compound at the active site (PDB: 5kkn, RMSD=1.8 Å); (bottom) multiple sequence alignment of corresponding POD-2 (SEQ ID NO:1) and human ACC1 (SEQ ID NO:2) and ACC2 sequences (SEQ ID NO:3).

[0065] FIG.16B. Sequence and structure alignments of CT domains of POD-2 and human ACC proteins. Alignments of the CT domain: (top) structure alignment of the modeled POD-2 CT domain and solved ACC2 CT domain with bound CP-640186 compound at the active site (PDB: 3ff6, RMSD=3.2 Å); (bottom) multiple sequence alignment of corresponding POD-2 (SEQ ID NO:4) and human ACC1 (SEQ ID NO:5) and ACC2 sequences (SEQ ID NO:6).

[0066] FIG.17. Stacked plot highlighting the constancy of the acetyl methyl peak of acetyl CoA (AcCoA) at 2.20 ppm in the1H NMR spectra of 50 µM AcCoA dissolved in D2O with 50 µM ATP and 30 mM (NH4)HCO3, without (left) and with (right) 50 µM avocadene acetate. The time intervals spanned by the displayed plots are 2.5 hours (left) and 1 hour (right).

[0067] FIG.18A.1H NMR stacked plot highlighting the decrease of the acetyl methyl peak of acetyl CoA (AcCoA) after addition of a C. elegans protein fraction containing POD- 2.

[0068] FIG.18B. Time course of normalized concentrations assessed from NMR measurements after POD-2 addition. The data represent the experimental trends of the reference solution – 50 µM AcCoA in D2O + 50 µM ATP + 30 mM (NH4)HCO3 – (red), thereference solution with 20 µM (violet) or 50 µM (green) avocadene acetate, and the reference solution devoid of ATP (purple). The correction applied in the lower plot concerns the red, violet and green data sets that are scaled with respect to the data collected without ATP. The AcCoA reduction in the absence of ATP is due to mitochondrial AcCoA-acetyltransferase and AcCoA-hydrolase, according to the proteomic assessment of the NMR samples (not shown). The error bars represent the signal-area maximal uncertainty. The double- exponential fitting curves follow the equation: ^^ ൌ ^^^^ ^^^^^ି^భ௧^ ^^ଶ^^ି^మ௧or, for the solution without ATP: ^^ ൌ ^^^^ ^^^^^ି^భ௧where t is time, A0, A1and A2are k1and k2are slow and fastkinetic constants featuring the consumption and the carboxyl transfer to AcCoA, respectively (see Table 3).

[0069] FIG.19. MALDI-FTICR mass spectra of reference solutions (50 µM AcCoA, 50 µM ATP, 30 mM (NH4)HCO3in D2O) after completion of the NMR acquisitions without (upper trace) and with (lower trace) a C. elegans protein fraction containing POD-2. The upper trace shows the AcCoA [M+H]+molecular ion at 810.2 Th (Th = thompson, i.e. mass- over-charge ratio unit), as expected without the conversion catalyzed by POD-2. The lower trace contains the weak [M+H]+molecular peak of MalCoA, at 856.1 Th, as expected from POD-2 activity, along with a peak of unconverted AcCoA at 810.2 Th. Note that the MalCoA molecular ion occurs with an increment of 2 Th because of the incorporation of 2 deuterium atoms from the keto-enol equilibrium in D2O. The low intensity of MalCoA [M+H]+molecular ion is related to inherently weak ionization and fragmentation, with a step involving CO2neutral loss leading to AcCoA. The fragmentation-derived AcCoA occurs at 812.1 and 813.1, with the incorporation of 2 or 3 deuterium atoms which is the signature of the malonyl precursor in D2O.

[0070] FIG.20. Onset of malonyl CoA (MalCoA) inferred from NMR spectroscopy. Though the most characteristic signal from MalCoA, i.e. the methylene between the two carbonyls of the molecule, is not observable by1H NMR in D2O because of isotopic exchange in keto-enolic equilibria, a couple offrom the pantothenic acid portion of the coenzyme A moiety of MalCoA spectrum are different with respect to the corresponding signals of the parent AcCoA. In particular the chemical shifts of the CH3at 0.73 ppm (left stacked plot, red circle on the structure) and the adjacent CH at 3.87 ppm(right stacked plot, green circle on the structure) change as the product accumulates while reaction proceeds. Therefore, a decrease of the parent substrate signal can be observed together with the simultaneous increase of the corresponding product signal. The low resolution within those signal pairs hampers quantification, but the qualitative significance of the pattern can still be appreciated. No similar effects could be observed to differentiate the adenosine moiety signals of AcCoA and ATP to highlight ATP hydrolysis because the external phosphate loss of the latter has no detectable consequence on the1H signal chemical shifts and intensity of the adenosine moiety.

[0071] FIG.21. (Top) Dehydrovariabilin impairs development and survival in C. elegans and P. pacificus. (middle) Dose-response curves of Dehydrovariabilin. Percentage of adults surviving after 48 hours of treatment. Percentage of animals developing past the L1 stage after 5 days of treatment beginning at the L1 stage. Percentage of eggs hatched after 48 hours of treatment. (bottom) The table shows the calculated LD50 values (concentration at which development or survival was reduced by 50%) of Dehydrovariabilin. Each data point represents mean±S.E.M.; N=3 replicates; >50 individual worms / eggs per concentration. In all experiments, DMSO was used as a negative control.

[0072] FIG.22. Dehydrovariabilin is effective against a multi-drug resistant parasitic nematode strain (H. contortus UGA). Table. Rates of embryonic and larval lethality of parasitic worms after egg treatment with Dehydrovariabilin. Panel: Dehydrovariabilin is non- toxic to mammalian U2OS cells up to 100 µM.

[0073] FIG.23. Anthelmintic-resistant mutants are sensitive to Dehydrovariabilin. Dehydrovariabilin is effective against ivermectin-resistant mutant strain avr-14(ad1302); avr- 15(ad1051) glc-1(pk54), levamisole-resistant mutant strain unc-29(e193), monepantel resistant mutant strain acr-23(ok2804), benzimidazoles resistant mutant strain ben-1(e1880), emodepside resistant strain slo-1(js379).

[0074] FIG.24A. Dehydrovariabilin causes disorganization of cell junctions in the epidermis of arrested C. elegans embryos. A. Confocal brightfield and fluorescence images (DLG-1::GFP) of C. elegans embryos at early elongation (comma stage), mid-elongation (1.5-fold), two-fold, and late elongation (3-fold) that are untreated or treated with D1(dehydrovariabilin 50 µM).

[0075] FIG.24B. Dehydrovariabilin causes actin disorganization in the epidermis of arrested embryos in C. elegans. Lateral view of 2-fold embryos imaged in brightfield or fluorescentce showing LifeAct epidermal F- actin. DMSO control embryos at two fold stageshow normal circumferentially oriented actin bundles compared to disorganized actin filaments after treatment with D1 (dehydrovariabilin 50 µM).

[0076] FIG.25. ACC inhibitor synthetic compound CP-640186 shows a similar phenotype to Avo and SCD inhibitor MK-8245 causes embryonic arrest at 10 µM and synergizes with Avo. DETAILED DESCRIPTION OF THE DISCLOSURE

[0077] Although claimed subject matter will be described in terms of certain examples, other examples, including examples that do not provide all of the benefits and features set forth herein, are also within the scope of this disclosure. Various structural, logical, process step, and electronic changes may be made without departing from the scope of the disclosure.

[0078] As used herein, unless otherwise indicated, “about”, “substantially”, or “the like”, when used in connection with a measurable variable (such as, for example, a parameter, an amount, a temporal duration, or the like) or a list of alternatives, is meant to encompass variations of and from the specified value including, but not limited to, those within experimental error (which can be determined by, e.g., a given data set, an art accepted standard, etc. and / or with, e.g., a given confidence interval (e.g.90%, 95%, or more confidence interval from the mean), such as, for example, variations of + / -10% or less, + / -5% or less, + / -1% or less, and + / -0.1% or less of and from the specified value), insofar such variations in a variable and / or variations in the alternatives are appropriate to perform in the instant disclosure. As used herein, the term “about” may mean that the amount or value in question is the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, compositions, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error, or the like, or other factors known to those of skill in the art such that equivalent results or effects are obtained. In general, an amount, size, composition, parameter, or other quantity or characteristic, or alternative is “about” or “the like,” whether or not expressly stated to be such. It is understood that where “about,” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0079] Ranges of values are disclosed herein. The ranges set out a lower limit value and an upper limit value. Unless otherwise stated, the ranges include the lower limit value,the upper limit value, and all values between the lower limit value and the upper limit value, including, but not limited to, all values to the magnitude of the smallest value (either the lower limit value or the upper limit value) of a range. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “0.1% to 5%” should be interpreted to include not only the explicitly recited values of 0.1% to 5%, but also, unless otherwise stated, include individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5% to 1.1%; 0.5% to 2.4%; 0.5% to 3.2%, and 0.5% to 4.4%, and other possible sub-ranges) within the indicated range. It is also understood (as presented above) that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about, it will be understood that the particular value forms a further disclosure. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0080] The articles “a” and “an” are used in this disclosure to refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0081] As used herein, unless otherwise stated or indicated, “s” refers to second(s), “min” refers to minute(s), and “h” refers to hour(s).

[0082] The phrase “therapeutically effective amount” is used herein to mean an amount sufficient to reduce by at least about 15 percent, preferably by at least 50 percent, more preferably by at least 90 percent, and most preferably prevents oxidative stress in the individual. Alternatively, a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in the individual.

[0083] As used herein, unless otherwise stated, the term “group” refers to a chemical entity that is monovalent (i.e., has one terminus that can be covalently bonded to other chemical species), divalent, or polyvalent (i.e., has two or more termini that can be covalently bonded to other chemical species). The term “group” also includes radicals (e.g., monovalent and multivalent, such as, for example, divalent radicals, trivalent radicals, and the like). Illustrative examples of groups include:. the term “alkyl” or “alkyl group”groups and / or cyclic hydrocarbon groups. Examples of alkyl groups include, but are not limited to, methyl groups, ethyl groups, propyl groups, butyl groups, isopropyl groups, tert-butyl groups, cyclopropyl groups, cyclopentyl groups, cyclohexyl groups, and the like. Alkyl groups are saturated groups, unless it is a cyclic group. For example, an alkyl group is a C1 to C40 alkyl group, including all integer numbers of carbons and ranges of numbers of carbons therebetween (e.g., C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12,C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, C30, C31, C32, C33, C34, C35, C36, C37, C38, C39, and C40). The alkyl group may be unsubstituted or substituted with one or more substituents. Examples of substituents include, but are not limited to, halogens (-F, -Cl, -Br, and -I), aliphatic groups (e.g., alkyl groups, alkenyl groups, alkynyl groups, and the like), halogenated aliphatic groups (e.g., trifluoromethyl group), aryl groups, halogenated aryl groups, alkoxide groups, amine groups, nitro groups, carboxylate groups, carboxylic acids, ether groups, alcohol groups, alkyne groups (e.g., acetylenyl groups and the like), and the like, and combinations thereof.

[0085] The present disclosure provides methods of inhibiting fatty acid synthesis and / or fatty acid oxidation pathways, methods of inhibiting nematode growth, and methods of treating a subject having a nematode-based infections. Also provided are compounds for uses thereof.

[0086] In an aspect, the present disclosure provides compounds. The compounds may be used to inhibit fatty acid synthesis and / or fatty acid oxidation pathways, inhibit nematode growth, treat a subject having or suspected of having a nematode-based infection, or generally as an anthelmintic.

[0087] The compounds of the present disclosure may have the following structure: orere . R' is OH or O. R is H or -COCH3. R'' is H or an alkoxy(e.g., methoxy). Each R'' may be the same or different.

[0088] In various examples, a compound of the present disclosure has the following structure: ,. closure has the following structure: oror .

[0090] In various examples, compounds may be administered as a composition comprising any combination of the compounds disclosed herein. For example, when the combination of compounds is: , A. In various examples, when thecombination of compounds is: ,B.

[0091] The present disclosure includes all possible stereoisomers and geometric isomers of a compound of the present disclosure. The present disclosure includes both racemic compounds and optically active isomers. When a compound of the present disclosure is desired as a single enantiomer, it can be obtained either by resolution of the final product or by stereospecific synthesis from either isomerically pure starting material or use of a chiral auxiliary reagent, for example, see Z. Ma et al., Tetrahedron: Asymmetry, 8(6), pages 883- 888 (1997). Resolution of the final product, an intermediate, or a starting material can be achieved by any suitable method known in the art. Additionally, in situations where tautomers of a compound of the present disclosure are possible, the present disclosure is intended to include all tautomeric forms of the compounds.

[0092] Prodrugs of a compound of the present disclosure also can be used as the compound in a method of the present disclosure. It is well established that a prodrug approach, wherein a compound is derivatized into a form suitable for formulation and / or administration, then released as a drug in vivo, has been successfully employed to transiently (e.g., bioreversibly) alter the physicochemical properties of the compound (see, H. Bundgaard, Ed., "Design of Prodrugs," Elsevier, Amsterdam, (1985); R.B. Silverman, "TheOrganic Chemistry of Drug Design and Drug Action," Academic Press, San Diego, chapter 8, (1992); K.M. Hillgren et al., Med. Res. Rev., 15, 83 (1995)).

[0093] Compounds of the present disclosure can contain one or more functional groups. The functional groups, if desired or necessary, can be modified to provide a prodrug. Suitable prodrugs include, for example, acid derivatives, such as amides and esters. It also is appreciated by those skilled in the art that N-oxides can be used as a prodrug.

[0094] Compounds of the disclosure may exist as salts. Pharmaceutically acceptable salts of the compounds of the disclosure generally are preferred in the methods of the disclosure. As used herein, the term "pharmaceutically acceptable salts" refers to salts or zwitterionic forms of a compound of the present disclosure. Salts of compounds of the present disclosure can be prepared during the final isolation and purification of the compounds or separately by reacting the compound with an acid having a suitable cation. The pharmaceutically acceptable salts of a compound of the present disclosure are acid addition salts formed with pharmaceutically acceptable acids. Examples of acids which can be employed to form pharmaceutically acceptable salts include inorganic acids such as nitric, boric, hydrochloric, hydrobromic, sulfuric, and phosphoric, and organic acids such as oxalic, maleic, succinic, and citric. Nonlimiting examples of salts of compounds of the disclosure include, the hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, 2- hydroxyethansulfonate, phosphate, hydrogen phosphate, acetate, adipate, alginate, aspartate, benzoate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerolphsphate, hemisulfate, heptanoate, hexanoate, formate, succinate, fumarate, maleate, ascorbate, isethionate, salicylate, methanesulfonate, mesitylenesulfonate, naphthylenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3- phenylproprionate, picrate, pivalate, propionate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, paratoluenesulfonate, undecanoate, lactate, citrate, tartrate, gluconate, methanesulfonate, ethanedisulfonate, benzene sulphonate, and p-toluenesulfonate salts. In addition, available amino groups present in the compounds of the disclosure can be quatemized with methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dimethyl, diethyl, dibutyl, and diamyl sulfates; decyl, lauryl, myristyl, and steryl chlorides, bromides, and iodides; and benzyl and phenethyl bromides. In light of the foregoing, any reference to compounds of the present disclosure appearing herein is intended to include a compound of the present disclosure as well as pharmaceutically acceptable salts, hydrates, or prodrugs thereof.

[0095] The compounds may exhibit wide variability in pharmacokinetic and physicochemical properties while still retaining desirable biological activity as described herein. For example solubility, e.g., log P, is variable while still retaining desirable biological activity as described herein.

[0096] In an aspect, the present disclosure provides compositions comprising one or more compound(s) of the present disclosure. The compositions may further comprise one or more pharmaceutically acceptable carrier(s).

[0097] The compositions may include one or more pharmaceutically acceptable carrier(s). The composition may be for administration to a subject in need of treatment.

[0098] Non-limiting examples of compositions include solutions, suspensions, emulsions, solid injectable compositions that are dissolved or suspended in a solvent before use, and the like. Injections may be prepared by dissolving, suspending, or emulsifying one or more of the active ingredient(s) in a diluent. Non-limiting examples of diluents include distilled water (e.g., for injection), physiological saline, vegetable oil, alcohol, and the like, and combinations thereof. Injections may contain, for example, stabilizers, solubilizers, suspending agents, emulsifiers, soothing agents, buffers, preservatives, and the like, and combinations thereof. Injections may be sterilized in the final formulation step or prepared by sterile procedure. A pharmaceutical composition of the disclosure may also be formulated into a sterile solid preparation, for example, by freeze-drying, and may be used after sterilized or dissolved in sterile injectable water or other sterile diluent(s) immediately before use. Additional examples of pharmaceutically acceptable carriers include, but are not limited to, sugars, such as, for example, lactose, glucose, and sucrose; starches, such as, for example, corn starch and potato starch; cellulose, such as, for example, sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as, for example, cocoa butter and suppository waxes; oils, such as, for example, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as, for example, propylene glycol; polyols, such as, for example, glycerin, sorbitol, mannitol, and polyethylene glycol; esters, such as, for example, ethyl oleate and ethyl laurate; agar; buffering agents, such as, for example, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; other non-toxic compatible substances employed in pharmaceutical formulations, and the like, and combinations thereof. Non-limiting examples of pharmaceutically acceptable carriers are found in: Remington: The Science and Practice of Pharmacy (2012) 22nd Edition, Philadelphia, PA. Lippincott Williams & Wilkins. In variousexamples, the composition may be suitable for injection. Parenteral administration includes infusions and injections, such as, for example, intramuscular, intravenous, intraarterial, intraperitoneal, subcutaneous administration, and the like.

[0099] The compositions may be administered systemically. Compositions may be administered orally, may be administered parenterally, and / or intravenously. Compositions suitable for parenteral, administration may include aqueous and / or non-aqueous carriers and diluents, such as, for example, sterile injection solutions. Sterile injection solutions may contain anti-oxidants, buffers, bacteriostatic agents and solutes, which render the composition isotonic with the blood of the intended recipient. Aqueous and / or non-aqueous sterile suspensions may include suspending agents and thickening agents.

[0100] The compositions of the present disclosure may be administered systemically. The term “systemic” as used herein includes parenteral, topical, oral, spray inhalation, rectal, nasal, and buccal administration. The term “parenteral” as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial administration. Preferably, the compositions are administered orally, intraperitoneally, or intravenously.

[0101] Compositions of the disclosure can comprise more than one pharmaceutical agent. For example, a first composition comprising a compound of the disclosure and a first pharmaceutical agent can be separately prepared from a composition which comprises the same compound of the disclosure and a second pharmaceutical agent, and such preparations can be mixed to provide a two-pronged (or more) approach to achieving the desired prophylaxis or therapy in an individual. Further, compositions of the disclosure can be prepared using mixed preparations of any of the compounds disclosed herein.

[0102] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0103] Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0104] Compositions of the disclosure suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and / or as mouth washes and the like, each containing a predetermined amount of a compound of the present disclosure as an active ingredient. A compound of the present disclosure may also be administered as a bolus, electuary or paste.

[0105] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent.

[0106] The tablets, and other solid dosage forms of the pharmaceutical compositions of the present disclosure, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and / or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient can also be in micro- encapsulated form, if appropriate, with one or more of the above-described excipients.

[0107] Liquid dosage forms for oral administration of a compound of the present disclosure include pharmaceutically-acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

[0108] Suspensions, in addition to a compound of the disclosure, the composition may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0109] In various examples, the pharmaceutically acceptable formulation is such that it provides sustained delivery of a compound of the present disclosure to an individual for at least 12 hours, 24 hours, 36 hours, 48 hours, one week, two weeks, three weeks, or four weeks after the pharmaceutically-acceptable formulation is administered to the individual.

[0110] The compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the individual being treated, the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of a compound of the present disclosure which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient.

[0111] Methods of preparing these compositions include the step of bringing into association a compound of the present disclosure with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present disclosure with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0112] In an aspect, the present disclosure provides various methods. The methods may be methods of inhibiting fatty acid synthesis and / or fatty acid oxidation pathways, methods of inhibiting nematode growth, and / or methods of treating a subject having a nematode-based infections.

[0113] In various examples, a method for treating a subject having or suspected of having a nematode-based infection comprises administering to the subject a therapeutically effective amount of one or more of the following compounds:or ; R' is OH or O; R is H or -COCH3; and R'' is H or an group (e.g., methoxy). Each R'' may be the same or different. In various examples, a combination of compounds of the present disclosure may be administered. Examples of compounds further include: , , ,,or. may be administered. For example, a subject may be administered Avocatin A and / or Avocatin B. In various examples, the subject does not have a hematological cancer (e.g., leukemia, such as, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), or myeloid leukemia). In various examples, the subject does not have malaria. The administration may be in combination with other treatments or therapies. The method may further comprise administering: . fatty acid synthesis and / or fattyacid oxidation pathways in a subject comprises administering to the subject a therapeutically effective amount of one or more of the following compounds: or;R' is OH or O; R is H or -COCH3; R'' is H or an alkoxy (e.g., methoxy). Each R'' may be the same or different. In various examples, acombination of compounds of the present disclosure may be administered. Examples of compounds further include:,or .may be administered. For example, a subject may be administered Avocatin A and / or Avocatin B. In various examples, the subject does not have a hematological cancer (e.g., leukemia, such as, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), or myeloid leukemia). In various examples, the subject does not have malaria. The administration may be in combination with other treatments or therapies. The method may further comprise administering: .nematode growth and / or reproduction comprises contacting a nematode with one or more of the following compounds:or ; R' is OH or O; R is H or -COCH3; and R'' is H or angroup (e.g., methoxy). Each R'' may be the same or different. In various examples, a combination of compounds of the present disclosure may be administered. Examples of compounds further include: , ,, , examples includeor .may be contacted. For example, Avocatin A and / or Avocatin B may be used. The method may further compriseadministering: . infected or suspected of beingthe nematode may be a drug-resistant nematode. In various other examples, a nematode may be parasitic or non-parasitic. Examples of nematodes that may be affected by a method of the present disclosure include, but are not limited to, filarial nematodes (e.g., Brugia pahangi), gastrointestinal nematodes, Teladorsagia circumcincta, Haemonchus contortus, Heligmosomoides polygyrus, and free- living nematodes, Pristionchus pacificus, Caenorhabditis elegans, or any combination thereof. The treatment may result in death of at least a portion of the nematodes and / or reduction in reproduction of the nematodes and / or reduction of adult worm load. Nematodes may be affected various developmental stages. For example, nematodes may be eggs, embryos, L1 stage worms, young adults, or dauers.

[0117] The subject to be treated by the method of the disclosure may be human or non-human (e.g., mammal). Non-human animals include ungulates such as bovines. Additional on-limiting examples of non-human mammals include pigs, mice, rats, rabbits, cats, dogs, or other agricultural mammals, pet, or service animals, and the like.

[0118] Compositions comprising a compound of the disclosure and a pharmaceutical agent for administration can be prepared at a patient’s bedside, or by a pharmaceutical manufacturer. In the latter case, the compositions can be provided in any suitable container, such as a sealed sterile vial or ampoule, and may be further packaged to include instruction documents for use by a pharmacist, physician or other health care provider. The compositions can be provided as a liquid, or as a lyophilized or powder form that can be reconstituted if necessary when ready for use. In particular, the compositions can be provided in combination with any suitable delivery form or vehicle, examples of which include, for example, liquids, caplets, capsules, tablets, inhalants or aerosol, etc. The delivery devices may comprise components that facilitate release of the pharmaceutical agents over certain time periods and / or intervals, and can include compositions that enhance delivery of the pharmaceuticals, such as nanoparticle, microsphere or liposome formulations, a variety of which are known in the art and are commercially available.

[0119] The dose of the composition comprising a compound of the present disclosure and a pharmaceutical agent generally depends upon the needs of the individual to whom the composition of the disclosure is to be administered. These factors include, for example, the weight, age, sex, medical history, and nature and stage of the disease for which a therapeutic or prophylactic effect is desired. The compositions can be used in conjunction with any other conventional treatment modality designed to improve the disorder for which a desired therapeutic or prophylactic effect is intended, non-limiting examples of which include surgical interventions and radiation therapies. The compositions can be administered once, or over a series of administrations at various intervals determined using ordinary skill in the art, and given the benefit of the present disclosure.

[0120] The compounds of the present disclosure can be therapeutically administered as the neat chemical, but it is preferred to administer a compound of the present disclosure as a pharmaceutical composition or formulation. Thus, the present disclosure provides a pharmaceutical composition comprising a compound of the present disclosure together with a pharmaceutically acceptable diluent or carrier therefor. Also provided is a process of preparing a pharmaceutical composition comprising admixing a compound of the present disclosure with a pharmaceutically acceptable diluent or carrier therefor.

[0121] When a compound of the present disclosure are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically-acceptable carrier.

[0122] In certain examples, the methods of the disclosure include administering to an individual a therapeutically effective amount of a compound of the present disclosure in combination with another pharmaceutically active ingredient. Pharmaceutically active ingredients that may be used can be found in Harrison’s Principles of Internal Medicine, Thirteenth Edition, Eds. T.R. Harrison et al. McGraw-Hill N.Y., NY; and the Physicians Desk Reference 50th Edition 1997, Oradell New Jersey, Medical Economics Co.

[0123] Methods delineated herein include those wherein the individual is identified as in need of a particular stated treatment. Identifying an individual in need of such treatment can be in the judgment of an individual or a health care professional and can be subjective (e.g., opinion) or objective (e.g., measurable by a test or diagnostic method). In other methods, the individual is prescreened or identified as in need of such treatment by assessment for a relevant marker or indicator of suitability for such treatment.

[0124] The compounds and compositions disclosed in the present disclosure can also be used for prophylaxis in an individual who is at risk of being exposed to nematode infection. A prophylactic use may be useful, for example, in an individual who is about to undertake a journey to a region where nematode infection has been reported or is known to occur. For the prophylactic use, one or more doses of a composition comprising the compounds of the present disclosure may be administered. Dosing for prophylactic treatment may vary compared to dosing of known nematode infections. In one example treatment may be given 1-3 times a day for 1 to 10 days or longer. In one example, treatment is given for up to three times (such as 1 to 3 times) daily for up to 10 days (such as 1- 10 days) in immunocompetent hosts. For immunosuppressed hosts such as those with AIDS, long-term suppressive treatment may be warranted.

[0125] The identification of those patients who are in need of prophylactic treatment for a nematode infection can readily identify such candidate patients, by the use of, for example, clinical tests, physical examination and medical / family history. The individual may have a nematode infection, may be at risk of developing a nematode infection, or may need prophylactic treatment prior to anticipated or unanticipated exposure to a condition(s) capable of increasing susceptibility to a nematode infection. In an example, those in need of prophylactic treatment for a nematode infection can take a therapeutically effective amount of a compound of the present disclosure from 1 to 30 days prior to an anticipated or unanticipated exposure to a condition(s) capable of increasing susceptibility to a nematode infection. In another example, those in need of prophylactic treatment for a nematode infection can take a therapeutically effective amount of a compound of the present disclosure from 1 to 24 hours prior to an anticipated or unanticipated exposure to a condition(s) capable of increasing susceptibility to a nematode infection. In an example, an individual can take a therapeutically effective amount of a compound of the present disclosure shortly after exposure to nematode infection.

[0126] In an aspect, the present disclosure provides a method for identifying if a compound / drug is an anthelmintic. The method may be performed on known libraries or libraries made via OBOC (one bead one compound) methods.

[0127] In an example, the method comprises contacting a culture comprising one or more nematodes with the compound or drug; incubating the culture for a period of time; imaging at least a portion of the plurality of the nematodes after the incubating; and comparing the images of the at least a portion of the plurality of nematodes from the culture with a control, wherein the control comprises nematodes that have not been contacted withthe compound / drug. In various examples, the culture further comprises E. coli. In various examples, if there is a plurality of nematodes, the nematodes may be a plurality of different nematode species or a single species of nematode. In various examples, if there is a plurality of nematodes, the nematodes may be at different developmental stages or roughly around the same developmental stage or the same developmental stage.

[0128] The culture may be incubated for one or more days. For example, the culture may be incubated for 5 or more days.

[0129] Various types of imaging may be done and at various stages during the incubation. For example, imaging may be done to collect comparative data to compare the efficacy of the drug / compound over a period of time.

[0130] Comparison of images may be done over a period of time and against one or more controls.

[0131] In an aspect, the disclosure provides a kit for treating a nematode infection in an individual is provided and includes a compound of the present disclosure, pharmaceutically acceptable esters, salts, and prodrugs thereof, and instructions for use. In certain embodiments, the disclosure provides: a kit for treating a nematode infection, in an individual, the kit comprising a compound of the present disclosure. The kit may also include instructions for administration of the compound or composition. The instructions may include details on one or more of the following: dosage, frequency, number of administrations to be carried out (such as number of tablets to be consumed), whether the composition needs to be taken with food, water etc., storage of the composition, and the like.

[0132] The steps of the method described in the various examples and examples disclosed herein are sufficient to carry out the methods of the present invention. Thus, in an example, the method consists essentially of a combination of the steps of the methods disclosed herein. In another example, the method consists of such steps.

[0133] The follow are Statements of the present disclosure. Statement 1. A method of treating a subject having or suspected of having nematode-based infection, comprising administering to the subject a therapeutically effective amount of one or more of the following compounds: ,ion X a group formed from an anthelmintic compound; ;is OH or O; R is H or -COCH3; and each R'' is independently H or an alkoxy group (e.g., methoxy). Statement 2. A method according to Statement 1, wherein the one or more of the following compounds are: , , ,, , Statement 3. A method according to Statement 1 or Statement 2, wherein the administration further comprises administering: .Statement 2, wherein at least a combination of the following compounds is administered: .1 or Statement 2, wherein at least a combination of the following compounds is administered:and .of the preceding Statements, wherein the nematode-based infection is associated with a drug-resistant nematode species. Statement 7. A method according to any one of the preceding Statements, wherein the nematode-based infection is associated with a parasitic or non-parasitic nematode species. Statement 8. A method according to any one of the preceding Statement, wherein the nematode-based infection is associated with one or more of gastrointestinal nematodes Teladorsagia circumcincta, Haemonchus contortus, Heligmosomoides polygyrus, filarial nematodes (e.g., Brugia pahangi), free-living nematodes Pristionchus pacificus, Caenorhabditis elegans, or any combination thereof. Statement 9. A method according to any one of the preceding Statements, wherein the administration results in death of at least a portion of nematodes associated with the nematode-based infection. Statement 10. A method according to any one of the preceding Statements, wherein at least a portion of the nematodes associated with the nematode-based infection are dauer or embryos. Statement 11. A method according to any one of the preceding Statements, wherein the administration results in a reduction of adult worm load. Statement 12. A method according to any one of the preceding Statements, wherein administration results in reduction of nematode reproduction. Statement 13. A method according to any one of the preceding Statements, wherein the subject is a mammal. Statement 14. A method according to any one of the preceding Statements, wherein the subject is a human. Statement 15. A method according to any one of the preceding Statements, wherein the subject is an agricultural animal, service animal, or pet. Statement 16. A method according to any one of the preceding Statements, wherein the subject does not have a hematological cancer.Statement 17. A method according to any one of the preceding Statements, wherein the subject does not have leukemia. Statement 18. A method according to any one of the preceding Statements, wherein the subject does not have malaria. Statement 19. A method according to Statement 17, wherein the leukemia is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), or myeloid leukemia. Statement 20. A method according to any one of the preceding Statements, wherein the one or more compounds is administered in conjunction with another therapy. Statement 21. A composition comprising a pharmaceutically acceptable excipient, ,Statement 22. A composition comprising a pharmaceutically acceptable excipient and Avocatin A. Statement 23. A compound having the following structure: oris a single bond or double bond;is OH when is a single bond and O when is a double bond; R is H or -andX is a group formed from an anthelmintic compound. Statement 24. A compound according to Statement 23, wherein the compound has the following structure:or . Statements 23 or 24. Statement 26. A method of treating a subject having or suspected of having nematode-based infection comprising, comprising administering to the subject a therapeutically effective amount of one or more compounds of Statements 23 or 24 or composition of Statement 25. Statement 27. A method according to Statement 26, further comprising administering one or more of the following compounds: ,, Statement 28. A method for inhibiting fatty acid synthesis and / or fatty acid oxidation pathways in a subject, comprising administering to the subject a therapeutically effective amount of one or more compounds of Statements 23 or 24 or a composition of Statement 25. Statement 29. A method of Statement 28, further comprising administering one or more of the following compounds: , , ,, Statement 30. A method for inhibiting fatty acid synthesis and / or fatty acid oxidation pathways in a subject, comprising administering to the subject a therapeutically effective amount of one or more of the following compounds: ,X is a group formed from an anthelmintic compound;Y ;H or -COCH3; and each R'' is independently H or an alkoxy group (e.g., methoxy). Statement 31. A method according to Statement 30, further comprising administering .or Statement 31, wherein at least a combination of the following compounds is administered: .30 or Statement 31, wherein at least a combination of the following compounds is administered: .any one of Statements 30 to 33, wherein the subject is a mammal. Statement 35. A method according to any one of Statements 30 to 34, wherein the subject is a human.Statement 36. A method according to any one of Statements 30 to 35, wherein the subject is an agricultural animal, service animal, or pet. Statement 37. A method according to any one of Statements 30 to 36, wherein the subject does not have a hematological cancer. Statement 38. A method according to any one of Statements 30 to 37, wherein the subject does not have leukemia. Statement 39. A method according to any one of Statements 30 to 38, wherein the subject does not have malaria. Statement 40. A method according to Statement 38, wherein the leukemia is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), or myeloid leukemia. Statement 41. A method according to any one of Statements 30 to 40, wherein the one or more compounds is administered in conjunction with another therapy. Statement 42. A method for identifying if a compound / drug is an anthelmintic, comprising contacting a culture a plurality of nematodes with the compound or drug; incubating the culture for a period of time; imaging at least a portion of the plurality of the nematodes after the incubating; and comparing the images of the at least a portion of the plurality of nematodes from the culture with a control, wherein the control comprises nematodes that have not been contacted with the compound / drug. Statement 43. A method according to Statement 42, wherein the culture further comprises E. coli. Statement 44. A method according to Statement 42 or Statement 43, wherein the plurality of nematodes are eggs, L1 stage worms, young adults, and / or dauers.

[0134] The following example is presented to illustrate the present disclosure. It is not intended to be limiting in any matter. EXAMPLE

[0135] This example provides a description of compositions of the present disclosure.

[0136] 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 tothe few anthelmintic drugs is increasing. Described herein is a set of avocado fatty acids / alcohols (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 showed significant efficacy in H. polygyrus infected mice. In C. elegans, AFA exposure affected all developmental stages, caused paralysis, impaired mitochondrial respiration and increased reactive oxygen species production and mitochondrial damage. Genetic and biochemical tests revealed 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.

[0137] Described herein is a multispecies HTS approach using C. elegans and P. pacificus to screen a commercial small molecule library containing FDA-approved drugs and natural products for potential broad-spectrum anthelmintic activity. Most molecules in this library are expected to be well tolerated in humans, which was confirmed by measuring toxicity in a human cell line. Among the bioactive compounds identified in the screen was a group of structurally similar 17-carbon fatty alcohol compounds present in extracts of the avocado Persea americana. It was found that these compounds, which are collectively referred to as avocado-derived fatty alcohols (AFAs), elicit dose-dependent lethality in parasitic nematodes and target C. elegans acetyl-CoA carboxylase ACC (POD-2), the rate limiting enzyme in lipid biosynthesis.

[0138] Novel class of anthelmintics. To identify new compounds with potential as anthelmintics, a library of 2,300 small molecules containing FDA-approved drugs was screened, other known bioactive compounds, and natural products (Spectrum Collection, MicroSource Discovery Systems, Inc.). Because parasitic nematodes are not amenable to high throughput screening (HTS) due to their complex life cycle, to identify potential broad- spectrum anthelmintics compounds that affect both of two distantly related free-living model species, C. elegans and P. pacificus (FIG.1a), were searched for. To avoid non-specific cytotoxicity, especially in human cells, it was additionally required that there was low toxicity in U2-OS human bone osteosarcoma cells (FIG.6).

[0139] The screen resulted in the identification of a family of related compounds isolated from the avocado Persea americana. These AFA compounds represent structurally similar 17-carbon fatty alcohols and their acetate variants (FIG.1B): avocadene ((2R,4R)- 1,2,4-trihydroxyheptadec-16-ene), avocadyne ((2R,4R)-1,2,4-trihydroxyheptadec-16-yne), avocadene acetate ((2R,4R)-2,4-dihydroxyheptadec-16-en-1-yl acetate), avocadyne acetate((2R,4R)-2,4-dihydroxyheptadec-16-yn-1-yl acetate), avocatin B (a mixture of avocadene and avocadyne), and avocatin A (a mixture of their respective acetate forms).

[0140] Nematodes are known to be highly resistant to harsh environmental conditions. Infective and adult stages are surrounded by a thick protective cuticle and embryos are protected by multiple layers that include a chitin layer, a vitelline layer and a chondroitin proteoglycan layer. Using the model C. elegans, it was looked to see if AFAs accumulate inside embryos or post-embryonic stages. Using NMR, it was found that extracts of both embryos and larvae contained significant amounts of AFAs following 10-20 µM treatments, as shown by the presence of the characteristic signals of the terminal olefinic group of avocadene acetate (FIG.1C).

[0141] To characterize the activity profile of AFAs, they were assayed at different concentrations (from 1 µM to 100 µM) in liquid culture across C. elegans developmental stages (FIG.2). It was found that all six of these (avocadene, avocadyne, their acetate forms, and the two mixtures avocatin A and B) yielded a concentration-dependent toxic effect on L1 larval development (FIG.2A), adult survival (FIG.2B) and egg hatching (FIG.2C). In contrast, two structurally related compounds, avocadenofuran and avocadynofuran, which contain a 17-carbon chain but differ in having a furan ring (FIG.1B), showed no bioactivity in any assays at concentrations up to 100 µM (FIG.2A–D). Comparison of LD50 values showed that avocadene acetate, avocadyne acetate, and avocatin A exhibited similar potency and showed stronger effects than the non-acetate forms (FIG.2D).

[0142] To further explore this class of compounds, it was asked if related molecules would have similar effects. It was found that two other purified molecules extracted from Avocado, persenone A ((2R,5E,12Z,15Z)-2-hydroxy-4-oxoheneicosa-5,12,15-trienyl acetate) and persin ((2R, 12Z,15Z)-2-hydroxy-4-oxoheneicosa-12,15-dienyl acetate), as well as a fully saturated synthetic molecule, avocadane acetate ((2R,4R)-2,4-dihydroxyheptadecan-1-yl acetate) (FIG.1B), had similar activities (FIG.8). Because persin and its derivatives are known to be toxic to animals, novel AFAs found in the screen were focused on.

[0143] The egg hatching assay was performed on isolated embryos, and it was confirmed the presence of AFAs within treated embryos using NMR (FIG.1C), demonstrating that these lipid alcohols can cross the eggshell barrier. To compare potency, C. elegans and P. pacificus egg hatching was measured in the presence of avocadyne acetate or a selection of other anthelmintics (FIG.2E). It was found that albendazole, mebendazole, and levamisole had no effect on egg hatching in either species; praziquantel (used to treat infections of the skin-penetrating worm Schistosoma) reduced egg hatching by 25% (n = 517)in C. elegans and by 90% (n = 416) in P. pacificus at both 5 µM and 10 µM, and ivermectin decreased egg hatching by only 25% in both species (n = 594 and 751, respectively). In contrast, avocadyne acetate led to 100% inhibition of egg hatching in both worm models at 10 µM (n = 466 in C. elegans and n = 447 in P. pacificus). AFAs thus show a rare ability among known anthelmintics to directly interfere with embryogenesis (independently of the maternal provisioning of fat molecules during oogenesis), which would be desirable for the treatment of parasitic nematodes.

[0144] Nematodes, such as C. elegans, can enter a dauer stage, during which they reduce their metabolism in order to adapt to harsh environments such as dryness, reduced nutrients, and altered temperature. The dauer stage is thought to resemble the infective L3 stages of many parasitic nematodes. To determine whether dauer larvae are also sensitive to AFAs, dauers from both wild-type N2 (obtained by starvation) and the daf-2(m41) strain (which induces dauers at 25°C) were treated with avocadyne acetate for 48 h at 25 °C, and found that it elicited lethality in N2 and daf-2(m41) dauer larvae with an LD50of 8.5 µM and 13.5 µM, respectively (FIG.2F).

[0145] AFAs show a new mode of action. To test the effect of the AFA compounds on parasitic nematodes, assays were conducted in four veterinary parasites: H. contortus, T. circumcincta, H. polygyrus and B. pahangi. Avocatin B at both 10 µM and 50 µM induced strong larval lethality in all three parasitic species tested: H. contortus, T. circumcincta, and H. polygyrus (FIG.3A). Importantly, Avocatin A acts as macrofilaricidal as it kills adult B. pahangi and is effective at against L3 and microfilarial stages starting at 10µM (Fig.3b). Furthermore, AFAs are also active in the multidrug resistant H. contortus UGA strain at 10 µM (FIG.3A). Furthermore, AFAs are also active in the multidrug resistant H. contortus UGA strain at 10 µM (FIG.3A and FIG.3B). In H. contortus, all six bioactive AFA compounds from the primary screen impaired egg hatching and caused larval lethality; among these, the acetate forms elicited the strongest phenotypes (Table 1).

[0146] To test the possibility that AFAs represent a new class of anthelmintics, we screened them in C. elegans mutants that are resistant to the known anthelmintics ivermectin / abamectin, levamisole, benzimidazoles and amino-acetonitrile derivative (AAD) monepantel. All mutant strains were susceptible to AFA toxicity at the same level as wild type N2, indicating that AFA compounds act through a different mechanism (FIG.3C, FIG.8). To further characterize the mode of action of AFAs, random mutagenesis was performed in C. elegans using ethylmethanesulfonate (EMS). Despite performing several large rounds of screening and testing the resistance of ~10 million genomes, no strains wererecoverable with a reproducible level of resistance. The results of this forward genetic screen suggest either that evolving resistance to AFAs may require changes in multiple loci, or that their anthelmintic effects arise from physical damage to cellular structures.

[0147] AFAs are active in vivo. To determine whether the anthelmintic effects of AFAs tested directly on nematodes would translate to an in vivo parasitic nematode system, mice infected by H. polygyrus, a model for gastrointestinal infection, were used. The effect of Avocatin A was tested on infected mice that were pre-inoculated and allowed to develop a high titer of infection for a week. Infected mice treated with Avocatin A showed consistent and significant reduction compared with a placebo control in both H. polygyrus fecal egg count (FEC) and in the number and health of adult worms dissected from the mice on day 21 (FIG.3d). Three of the ten tested mice were essentially cured of the H. polygyrus infection within the two-week trial period and four out of ten produced no eggs.

[0148] AFAs inhibit respiration in C. elegans. To explore the mode of action of AFAs, the phenotypic effects of treating C. elegans embryos and adults with avocadene acetate was examined, one of the most potent AFA compounds. Adults displayed dose- dependent paralysis and inhibition of pharyngeal pumping within ~5 minutes of treatment (FIG.4A, FIG.4B, FIG.4C, and FIG.9). In embryos, acute treatment with 50 µM led to developmental arrest of all embryonic stages within ~30 minutes (FIG.5D, FIG.10).

[0149] To further characterize the paralysis, oxygen consumption rates (OCR) in whole nematodes were measured using a Seahorse XFe96 Analyzer (FIG.4D). Basal OCR, a measure of baseline mitochondrial respiration, was significantly lower in avocadene acetate- treated worms in comparison with DMSO controls (FIG.4D). Maximal OCR was measured in the presence of carbonyl cyanide-p-trifluoromethoxy phenylhydrazone (FCCP). FCCP is an uncoupler of mitochondrial oxidative phosphorylation that collapses the mitochondrial inner membrane proton gradient, which inhibits ATP synthesis and causes the electron transport chain to work at full capacity. These experiments showed that avocadene acetate- treated worms exhibited significantly lower maximal respiration and spare capacity (maximal OCR - basal OCR) in comparison with controls (FIG.4D, FIG.11A).

[0150] The electron transport chain is the main source of ROS in the cell, and defects in mitochondrial respiration are known to lead to oxidative stress. ROS levels were measured in the presence of avocadene acetate using two different fluorescent reporters: a general ROS sensor, chloromethyl-2’,7’ dichlorodihydrofluorescein diacetate (CM-H2DCFDA), and MitoSOX Red, which specifically senses superoxide within mitochondria. MitoSOX is targeted to the mitochondria-rich pharyngeal bulb upon ingestion by C. elegans, where itdisplays red fluorescence upon oxidation by superoxide. In comparison with DMSO-treated controls, the CM-H2DCFDA sensor showed a 30% increase in ROS (p < 0.0001, unpaired t- test) (FIG.4E), while MitoSOX showed a 50% increase in fluorescence intensity in the posterior pharyngeal bulb region (p = 0.0125, unpaired t-test) (FIG.4F and FIG.4G). The elevated levels of ROS observed using both assays indicate that treatment with avocadene acetate leads to either increased production or impaired neutralization of ROS.

[0151] It was next explored whether oxidative stress is an important contributor to the anthelmintic effect of AFAs by pre-treating animals with antioxidants. Since glutathione (GSH) is a well-known antioxidant and a substrate of several antioxidant enzymes, worms were pre-treated with the GSH precursor N-acetylcysteine (NAC), which both promotes production of GSH and neutralizes certain oxidizing species directly. L4 worms pre-treated with 10 mM NAC showed a 40% reduction in avocadene acetate-induced larval lethality compared to controls (FIG.4H, FIG.11B, and FIG.11C). Animals were also pre-treated with the essential vitamin B12, which has been shown to reduce ROS levels in both the cytoplasm and mitochondria. Vitamin B12 deficiency leads to oxidative stress, and supplementation with vitamin B12 improves mitochondrial homeostasis in worms. It was found that pre- treatment with vitamin B12 alleviates the effect of avocadene acetate-induced lethality (FIG. 11D and FIG.11E). These results support the idea that administration of AFAs leads to oxidative stress and that this contributes to their anthelmintic activity.

[0152] Because oxidative stress is associated with mitochondrial damage, it was tested whether avocadene acetate causes defects in mitochondrial morphology that could be a sign of mitochondrial dysfunction. Using a C. elegans strain that expresses GFP in the mitochondrial matrix in body wall muscle, it was found that avocadene acetate-treated worms displayed a spectrum of mitochondrial defects (FIG.12). Whereas mitochondria normally form an elongated, tubular network, those in treated animals became fragmented and formed isolated spherical structures of varying sizes that appeared to undergo progressive loss, becoming barely detectable in the most extreme cases. The severity of these effects was significantly higher overall in comparison with DMSO-treated controls (p=0.0056, Fisher’s Exact test) (FIG.12).

[0153] AFAs target ACC / POD-2. The structures of AFA compounds, coupled with their mitochondrial phenotypes and effects on oxygen consumption and ROS, suggested the possibility that they may interfere with enzymes involved in lipid metabolism. To test this idea, we performed a chemical genetic screen was performed with candidate genes in the fatty acid synthesis (FAS) and fatty acid oxidation (FAO) pathways.

[0154] The first set of genes tested related to the shuttling of fatty acids into the mitochondria. Medium- and long-chain fatty acids (up to C20) are first activated by acyl-CoA synthetase in the cytosol (ACS-2 in C. elegans) and are then actively translocated into mitochondria by the carnitine shuttle. This system comprises the outer mitochondrial membrane protein CPT1 (carnitine palmitoyl transferase I, which has five homologs in C. elegans) and two inner membrane components, carnitine-acylcarnitine translocase (CACT, DIF-1 in C. elegans) and carnitine palmitoyl transferase II (CPT2, C. elegans CPT-2). Once inside mitochondria, fatty acids undergo multiple rounds of beta-oxidation, producing electron carriers for oxidative phosphorylation and acetyl-CoA, which can be converted to other metabolites and used to extract additional energy via the tricarboxylic acid (TCA) cycle.

[0155] If AFA compounds interfere with fatty acid metabolism, then disrupting the shuttle pathway would impact AFA-induced phenotypes. Consistent with this expectation, it was found that RNAi knockdown of key carnitine shuttle genes – including acs-2, the CPT1 homolog cpt-4, dif-1, and cpt-2 – partially rescued the effect of 5 µM avocadyne acetate (p = 0.0271, p = 0.0377, p = 0.0151, p = 0.0039, unpaired t-test) (FIG.13). However, RNAi of the CPT1 homolog cpt-1 had no significant effect (FIG.13 and Table 2). These results suggest that lowering the activity of the carnitine shuttle by RNAi can reduce, but not eliminate, sensitivity to AFA compounds.

[0156] Chemical genetic interactions of AFAs with a subset of genes involved in downstream FAO reactions were explored. The first step in each cycle of beta-oxidation is mediated by long-, medium-, and short-chain acyl-CoA dehydrogenases. Previous work using acute myeloid leukemia (AML) cells suggested a direct interaction of avocadyne with the human very long chain acyl-CoA dehydrogenase (VLCAD). RNAi of 12 known C. elegans acdh genes (acdh-1 to 4 and acdh-6 to 13) had no impact on avocadyne acetate lethality (Table 2), suggesting that if AFA compounds directly inhibit one or more of these, this can be compensated by some level of functional redundancy. No genetic interaction using the acdh- 12(gk5631) C. elegans mutant was found, the homolog with highest sequence similarity to the human VLCAD. Similarly, only inconsistent effects were uncovered when testing the propionate pathways, which we could partly explain by the level of B12 in the food (Table 2). Consistent with the possibility that AFAs are not metabolized, NMR studies showed that C13- labeled Avocadene Acetate was not degraded in C. elegans (FIG.14).

[0157] In the process of testing genetic interactions with enzymes involved in fatty acid metabolism, it was found that the C. elegans Acetyl CoA Carboxylase (ACC) pod-2(ye60) temperature-sensitive (ts) mutant is significantly more susceptible to avocadene acetate treatment than wild type (FIG.5A, FIG.15A, and FIG.15B), showing an LD50of 7.91 µM vs.12.57 µM for WT (p=0.003, unpaired t-test). A strain carrying an auxin-induced degron (AID) fused to POD-2 protein (pod-2::AID) was also significantly more sensitive to avocadene acetate: young adults incubated with auxin for 24h to deplete the POD-2 protein showed an LD50of 2.12 µM vs.15.14 µM in untreated controls (FIG.15C). POD-2, like all carboxylases, is covalently biotinylated by BPL-1 (biotin protein ligase 1) to catalyze the ATP-dependent, rate-limiting first step in fatty acid synthesis through the carboxylation of acetyl-CoA to malonyl-CoA. Consistent with AFAs acting through inhibition of POD-2, reducing BPL-1 levels by treating bpl-1::AID worms with auxin enhanced the AFA effect (LD50 of 8.1 µM vs.15.05 µM in untreated controls) (FIG.15C).

[0158] Malonyl-CoA, the product of the ACC / POD-2 enzyme, plays a major coordinating function in both synthesis and oxidation of fatty acids. If AFAs inhibit the synthesis of malonyl-CoA, exogenous addition of malonyl-CoA could counteract their effect. Indeed, malonyl-CoA supplementation lowered avocadene acetate toxicity in both wild type and pod-2(ye60) mutants, shifting the LD50 from 12.57 µM up to 16.30 µM in WT and from 7.9 µM up to 12 µM in a pod-2(ye60) strain (FIG.5a, FIG.15A, and FIG.15B).

[0159] Human ACC, which is conserved by sequence alignment and structural modeling with POD-2 (FIG.16), is reported to be inhibited by the same AFAs in vitro. Therefore it was tested whether AFAs could directly inhibit C. elegans POD-2 by purifying a POD-2::6xHis-tagged protein from worms and measuring the enzymatic activity by real-time1H NMR. POD-2 converts acetyl-CoA to malonyl-CoA in an ATP-dependent manner, and the addition of avocadene acetate to this reaction inhibited acetyl-CoA depletion in a concentration-dependent manner (FIG.5B, FIG.17, and FIG.18, Table 3). Because of the keto-enolic equilibrium and the ensuing deuteration in D2O, we could not quantify the appearance of the malonyl moiety methylene protons using NMR. However, the production of malonyl-CoA could be qualitatively confirmed by both mass spectrometry (FIG.19) and NMR signal resolution of the panthotenic acid moieties (FIG.20). These data showed a significant and concentration-dependent inhibition of C. elegans POD-2 by avocadene acetate (FIG.18, Table 3).

[0160] POD-2 / ACC functions as a homodimer via a two-step process guided by the biotin carboxylase (BC) and carboxyltransferase (CT) domains to carboxylate acetyl-CoA into malonyl-CoA. Given that the BC and CT domains are both affected by other ACC inhibitors, docking-based in silico searches for targets of avocado-derived compounds in bothdomains (described herein) were performed. Using structural alignment with the solved yeast ACC complexes (PDB: 1w96, 5csl), the search was centered in the vicinity of the soraphen A and biotin binding sites in the BC and CT domains, respectively. The computationally modeled affinities (Kdvalues) of avocadene acetate and avocadyne acetate compounds at the CT site (2.53 μM for avocadene acetate and 1.66 μM for avocadyne acetate) were two orders of magnitude stronger than predicted values at the BC site (~400 μM); persin also has a similar binding affinity (3.49 μM) (FIG.5C). Further, avocadynofuran essentially does not bind these areas of POD-2 (>100,000 μM), consistent with the functional results (FIG.1 and FIG.2). Together, the in silico docking results suggested that the CT active site is the primary target of avocadene / avocadyne acetate and persin inhibitors. Due to the position of the acetate group, this analysis could not reveal mutations that would simultaneously maintain enzymatic activity and significantly reduce avocadene / avocadyne acetate binding. Docking analyses also revealed that CP-640186 and sethoxydim, two known ACC inhibitors, could act on POD-2 in the same manner. While both CP-640186 and AFA strongly inhibit embryonic development (FIG.5D), CP-640186 does not appear to cross the eggshell barrier: AFA-like phenotypes required breaking the eggshell when mounting embryos under a coverslip for microscopy and were not observed in liquid cultures of embryos, nor in larvae. Together these data support the conclusion that these different pharmacological interventions affect the worms in a remarkably similar way, but they differ in their ability to penetrate the worm eggshell and cuticle.

[0161] Due to the limited classes of anthelmintic drugs in use, widespread anthelmintic resistance is particularly severe in the livestock industry worldwide and has also been documented in humans and companion animals. Presented herein is a novel class of related natural lipid alcohols, AFAs, that show anthelmintic effects in vitro and in vivo. AFAs are effective in eliciting lethality across all life stages tested across multiple species, including a field-derived H. contortus multi-drug resistant strain. When tested in vivo, Avocatin A treatment caused a significant parasite load reduction of both adults and eggs. No overt host toxicity was observed in mice treated with Avocatin A, consistent with these data in cell lines, as well as published reports describing a favorable safety profile for Avocatin B (the non-acetylated version of Avocatin A) in mice and preliminary results from phase I clinical trials.

[0162] Avocado extracts are a rich source of compounds with diverse bioactivities, antimicrobial and medicinal properties, and safety profiles. Isolated avocadene and avocadyne were recently shown to have mosquito larvicidal activity. While purified AFAshave not previously been described to affect nematodes, the polyphenols quercetin and epicathecin from seeds of Persea americana are reported to affect larvae of the parasitic nematode H. contortus in goats, and extracts from the related species Persea willdenovii also show anthelmintic properties. Thus, avocado fruits appear to have evolved a variety of ways to resist infections from insects, nematodes, microbes, and potentially other pests.

[0163] Multiple lines of evidence indicate that AFA compounds exert their anthelmintic effects by interfering with lipid homeostasis in C. elegans via the lipid biosynthesis enzyme ACC / POD-2: (1) The enzymatic activity of purified POD-2 is directly inhibited by AFAs. (2) Depleting POD-2 or its upstream biotin ligating protein BPL-1 enhances AFA-induced lethality. (3) Because POD-2 converts acetyl-CoA to malonyl-CoA, the rate-limiting substrate for de novo fatty acid biosynthesis, targeting either POD-2 or BPL- 1 depletes the endogenous malonyl-CoA pool. Consequently, supplementation with malonyl- CoA in the presence of AFA partially rescues worm development and survival. (4) Malonyl- CoA also serves a key role in balancing lipid biosynthesis and catabolism by negatively regulating CPT1 and thus FAO, and RNAi knockdown of several carnitine shuttle genes partially alleviates AFA-induced toxicity.

[0164] Lipid metabolism enzymes have been proposed as “chokepoints” for worm survival and potential targets for anthelmintic drug discovery, and recently an insecticide that acts as an ACC inhibitor (spirotetramat) was shown to be active at high concentration against a plant parasitic nematode Heterodera schachtii (50mM) and C. elegans (200 µM).

[0165] The embryonic defect is more puzzling. While an effect was observed in early embryos, POD-2 is not reported to be highly expressed in the early C. elegans embryos; rather, malonyl-CoA is thought to be supplied maternally by the soma to the germline. However, transcriptome analysis of early embryos detects pod-2 transcripts, which could be maternally inherited. The rapidly dividing cells in the non-feeding early embryo require a substantial supply of lipid entities for new cell membrane formation and for energy production. AFAs and other pharmacological ACC inhibitors, CP-640186 and sethoxydim, both halt embryonic development at any stage including in very early (4-cell) embryos, suggesting either that POD-2 activity is inhibited in those stages, or that another molecular target yet to be identified is present in embryos. The POD-2 paralog T28F3.5 is not reported to be expressed in the early stages of embryogenesis and is thus unlikely to be a major contributor to early embryonic phenotypes.

[0166] Focusing on the POD-2 CT active site (FIG.5C-i), both avocadene acetate (FIG.5C-ii) and avocadyne acetate (FIG.5C-iii) are predicted to bind in a location thatoverlaps both the biotin and CoA binding sites (especially near its sulfur atom) in solved structures (FIG.5C-iv). The acetyl group of avocadene / avocadyne acetate binds near CoA, whereas the compound’s tail region extends to the biotin site and beyond. Moreover, all top three favorably docked conformations of these AFAs show significant consistency in the position of the acetyl group; most conformational variations are observed near the tail ends. This analysis suggests that the inhibitory mechanism of avocadene / avocadyne acetate differs somewhat from the synthetic compound CP-640186, which competes with biotin binding but not with CoA.

[0167] The anthelmintic effect of AFA across a broad range of free-living and parasitic nematodes and their in vivo efficacy at treating infected mice make them great candidates for further development as anthelmintics and point to the conclusion that fatty acid metabolism / homeostasis in nematodes is an interesting avenue for future studies of potential anthelmintic targets.

[0168] In summary, the AFA compounds described here have potential to serve as new anthelmintic drugs, either alone or in combination with other anthelmintic compounds. Synergy between compounds with different mechanisms of action could decrease the risk of toxicity and potential off-target effects by reducing the concentration of individual compounds needed to achieve potent anthelmintic activity.

[0169] Chemical sources. The library presenting 2320 compounds, including FDA- approved drugs, bioactive compounds and some natural products, was purchased from Microsource Discovery Systems, Inc. Additional quantities of individual AFAs were purchased from Microsource Discovery Systems and Sigma-Aldrich. Indole-3-acetic acid, malonyl-coenzyme A, N-acetyl cysteine, serotonin and vitamin B12 were purchased from Sigma-Aldrich. The ACC inhibitors used in the study are CP-640186 (Cayman Chemicals), spirotetramat (Sigma) and sethoxydim (Dr. Ehrenstorfer). NEOBEE 1053 oil for mice experiments was purchased from Stepan. Avocadane acetate and labeled C13Avocadene acetate were synthesized by ChiroBlock GmbH.

[0170] Persenone A and persin were extracted and purified from Colombian Hass avocado according to a previously reported recipe. The purification was performed with a 1260 Infinity series Agilent HPLC system (Santa Clara) coupled to a G4212B photodiode array detector (PDA) and self-collector. The PDA detector was set at 235 nm. A Zorbax C18 column (4,6 x 250 mm x 5 µm, Phenomenex) kept at 25 °C was employed. The mobile phase compositions were 0.1% trifluoroacetic acid (TFA) in water (A) and 0.1% TFA in CH3CN (B). Solvents were pumped at 1 ml min-1using the gradient program: 0-6 min, 30% B; 6-12min, 30%-90% B; 12-28 min, 90% B; 28-35 min, 30% B; followed by 5 minutes re- equilibration. Persenone A and persin were eluted at 18.6 and 19.5 minutes, respectively.

[0171] Worm strains. P. pacificus PS312 and C. elegans N2 Bristol wild-type strains, SJ4103 (zcls14 [myo-3::GFP(mit)], CB1370 (daf-2(e1370)), HY520 (pod-2(ye60)), JD608 (avr-14(ad1302); avr-15(ad1051) glc-1(pk54)), CB3474 (ben-1(e1880)), RB2119 (acr- 23(ok2804)), CF1038 (daf-16(mu86)), NM1968 ((slo-1(js379)), PR1152 (cha-1(p1152)), VC731 (unc-63(ok1075)), CB193 (unc-29(e193)), VC4560 (acdh-12(gk5631[loxP + myo- 2p::GFP::unc-543' UTR + rps-27p::neoR::unc-543' UTR + loxP]) II) were obtained from the Caenorhabditis Genetics Center (CGC). Murat Artan and Mario de Bono kindly provided the PHX1772 (pod-2(syb1772[pod-2::His10]), AX8465 (pod-2(db1461[pod- 2::degron::3XFLAG])II; ieSi57 II) and AX8273 (bpl-1(db1372[bpl-1::degron(AID)]) ieSi57[eft-3p::TIR1::mRuby::unc-543’UTR+Cbr unc-119(+)] II) strains. Antony P. Page kindly provided the EG175 (dyf-17 (ox175)) strain.

[0172] Chemical screening. Newly hatched C. elegans and P. pacificus larvae cultivated in 96-well plates were treated with either DMSO (as a control) or library compounds. The primary screen was performed using the NYUAD high-throughput screening automated platform at a concentration of 10 µM in duplicates. Briefly, an overnight culture of OP50 Escherichia coli was concentrated 2.5-fold using S-complete medium.0.5 µL of the chemical was dispensed using the Bravo automated liquid handling platform (Agilent) into each well of the 96-well plate, followed by the addition of 30 µL of bacterial suspension using the MultiFlo FX Multi-Mode Dispenser platform (BioTek). Approximately 10 synchronized L1 stage worms diluted in S-complete were added to each of these wells using the Multiflo Fx device. The plates were incubated at 20 °C for 5 days for C. elegans and 22.5 °C for 7 days for P. pacificus in Cytomat automated incubators (Thermo Scientific). These conditions allow animals to develop into the adult stage and give rise to newly hatched progeny. Brightfield images were captured using a Cellinsight CX5 high-throughput microscope (Thermo Scientific) and archived in a custom in-house database. Images were inspected by visual comparison of treated and mock-treated animals to identify compounds that affect larval development or survival, adult fertility, paralysis and / or embryonic lethality in the next generation.

[0173] Dose-response experiments for AFAs were carried out in a similar manner at 0, 1.25, 2.5, 5, 10, 25, 50 and 100 µM concentrations. Worms of 4 stages were used: eggs, L1 stage worms, young adults and dauers. Eggs were collected by straining a synchronized egglaying population using a 40-micron strainer that captured the adults and allowed the eggs to pass through. L1 stage worms were obtained by bleaching a synchronized egg laying population and allowing the eggs to hatch for 24 hours. Adults were harvested by allowing a synchronized L1 stage population to grow up to the young adult stage. To culture dauers, a few hermaphrodites were transferred to an NGM plate seeded with OP50 E. coli and incubated at 22 °C. Plenty of dauers developed after 10 days. For daf-2 dauers, a chunk of worms was transferred onto an NGM plate with OP50 E. coli and incubated at 25°C for 48 hours to develop into dauers. Images of eggs, young adults and dauers before treatment and 48 hours post-treatment were captured to quantify embryonic and larval lethality. For L1 stage worms, images before treatment and 5 days post-treatment were captured to quantify the percentage of larvae that developed to later larval and adult stages. These experiments were performed in quadruplicate in 3 independent experiments. The effect of avocadyne acetate and existing anthelmintics on C. elegans and P. pacificus egg hatching (n>100) was quantified after 48-hour treatment at 5 µM and 10 µM on bleached eggs.

[0174] Anthelmintic-resistant mutant strains JD608, CB3474, RB2119, CF1038, EG175, NM1968, PR1152, VC731 and CB193 were synchronized and L1s were tested for AFA toxicity in a dose dependent manner. Avocadene acetate LD50 for the anthelmintic- resistant mutants were obtained in a dose response manner as described above.

[0175] Phenotype scoring. For the primary screen, phenotypes were classified as follows: larval lethal, larval arrest, slow growth, sterile and embryonic lethal. The extent of phenotypic severity was assigned as weak, medium or strong based on an approximate estimate of the percentage of the initial count. For dose-response and egg hatching experiments, worm / egg counts were noted pre- and post-treatment, to quantify % alive, % developed and % hatched. For RNAi experiments, a score between 0 to 5 was given based on the extent of development, with 0 being 100% arrested / lethal and 5 being 100% developed / alive.

[0176] High-content screening in mammalian cells. U2-OS cells were cultured in ATCC-formulated McCoy's 5a Medium Modified, supplemented with 10% fetal bovine serum (F7524, Sigma), 100 U / ml penicillin and 100 µg / ml streptomycin (P4333, Sigma), in a humidified incubator with 5% CO2 at 37°C. The day before compound addition, U2-OS cells were trypsinized and cell density was determined using a Countess II Automated Cell Counter (Thermo Fisher Scientific).2 x 103U2-OS cells were seeded in each well of a 384- well plate in 32 µl complete culture medium. The next day, 8 µl culture medium containing compounds at different concentrations were added to the cell culture to achieve 1 µm to 100µm final concentrations using a Bravo Automated Liquid Handling Platform.4 biological replicates in 4 different plates for each concentration were included in the experiment, with DMSO as control. The plates were briefly centrifuged for 30 seconds at 500 x g and incubated for 24 hours before staining. For staining, 20 µl Mitotracker Deep Red (1:1500 dilution, M22426 Thermo Scientific) was added to the cell culture using a Matrix® WellMate liquid dispenser (Thermo Scientific). After a brief centrifuge, the cells were incubated at 37°C for 30 minutes. Next, 20 µl formaldehyde (13% in PBS) was added to the cells for fixation at room temperature for 15 minutes. The fixative was removed, and the fixed cells were washed twice in 60 µl PBS, using a BioStack Microplate Stacker and MultiFlo FX Multi-Mode Dispenser. After washing, cells in each well were treated with 25 µl of staining solution containing Syto14 Green nucleic acid stain (1:2000 dilution, S7576 Thermo Scientific), Wheat Germ Agglutinin_Alexa Fluor™ 555 Conjugate (1:200 dilution, W32464 Thermo Scientific) and Hoechst 33342 (1:4000 dilution, 62249 Thermo Scientific) for 45 minutes at room temperature. After staining, cells were washed twice with 60 ul PBS and wells refilled with 50 ul PBS using the BioTek platform. Plates were sealed with an aluminum foil cover and stored at 4°C until imaging. Stained cells were scanned with the Cellinsight CX7 High-Content Screening (HCS) Platform (Thermo Scientific), with a 20× objective. Compartmental Analysis Bio Application was applied for the image analysis. The nuclei with Hoechst-staining were identified as primary objects (Circ), and simulated cytoplasm (Ring) was created according to nuclear shape and neighboring cells.

[0177] NMR Experiments. NMR spectra were always recorded at 20 °C, on the 14.0 T Bruker Ascend instrument of New York University Abu Dhabi Core Technology Platform, equipped with a single-axis gradient cryoprobe operating at 600.19 MHz (1H). The spectra were recorded at 20 °C in deuterated chloroform. C. elegans embryos (50,000) or L4 larvae (10,000) were treated with 10 and 20 µM avocadene acetate, respectively (Sigma). The treatment times were 0.5 and 2 hours for embryos and 0.5, 2, 4 and 8 hours for L4 larvae. Subsequently, the treated and the corresponding control batches were centrifuged to collect the pellets after removal of the supernatant. The pellets were washed 3 times with distilled water prior to extraction. To check for external avocadene acetate complete removal, the intermediate washing was also carried out with 10% aqueous ethanol, leading to the same results as obtained with the triple water washing.

[0178] The metabolite extraction from the embryo and larvae pellets was carried out using a variant of the Folch method. The pellet samples, resuspended in a minimal amount of water (100-150 µL) and kept on ice, were sonicated in vials floating in water / ice bath with anultrasound transducer immersed in the sample (20 irradiation / pause cycles (2 s / 5 s), frequency 20 kHz, power 25 Watts, applied with a Qsonica Q125-220 instrument). The sonicated pellets were then transferred in glass vials and vortexed for 120 s after a 2 mL addition of a CHCl3 / CH3OH / H2O (8:4:3) mixture. A second sonication step was subsequently carried out for 15 minutes, with the sample vials floating in ultrasonic bath (20 kHz, 40 Watts applied with an Elmasonic P instrument) prefilled with ice and water. The samples were then centrifuged (15 minutes, 1,250g at 4°C) and the distinct organic and aqueous phases were separated and dried under N2 stream. The dried organic phase, re-dissolved in deuterated CDCl3 (Sigma) was ready for NMR analysis, whereas the aqueous phase, devoid of methanol after drying, was lyophilized and redissolved in D2O (Sigma) for the NMR assessment.

[0179] The samples for NMR kinetic data determinations were prepared by adding to 30 mM (NH4)HCO3(Sigma) dissolved in D2O microliter aliquots of acetyl-CoA (Sigma) and ATP (Sigma) mother solutions, to reach final concentrations of 50 µM for both species. A sample without ATP was also prepared for the corresponding control experiment. For the assessment of AFA compound inhibitory effect, a few microliters of concentrated avocadene acetate preparation (Sigma) in DMSO-d6 (Cambridge Isotope Laboratories) were also added to the mentioned D2O solutions, setting avocadene acetate concentration at 20 µM and 50 µM. Careful adjustments of the NMR acquisition conditions were preliminarily carried out on each specific substrate or substrate and inhibitor D2O solution, before adding a 15 µL aliquot of POD-2-containing solution from the pool previously obtained by affinity chromatography and subsequent buffer exchange with deuterated Tris (Cambridge Isotope Laboratories). Typically, acquisitions started after a 6-8 minute dead-time delay for equilibration and field homogeneity stabilization. The experimental data were collected by issuing automatic consecutive acquisitions of one-dimension1H spectra (128 scans / spectrum requiring 6.1 minutes) for 10-12 hours. The optimized acquisition sequence included residual HOD suppression obtained by pairing WATERGATE elements in the excitation sculpting mode. The experimental data were processed with Topspin 4.0.6 (Bruker) and the acetyl-CoA acetyl methyl peak (2.20 pm) of each spectrum was carefully integrated using the value of the first acquired spectrum in each series as normalization reference.

[0180] Experiments in worm parasites. All parasitic nematode (H. polygyrus, T. circumcincta and H. contortus (ISE- drug sensitive and UGA- drug resistant)) stages and species were tested by placing either 50 freshly prepared eggs or 50 L1 larvae in microwell and testing as described for C. elegans and scoring survival after 24-48 hours. For parasitic nematode egg purification, fresh faeces from an infected host were blended in distilled water,passed through a 250 µm sieve and then centrifuged at 1100g for 5 minutes. The pellet was then resuspended in saturated NaCl and re-centrifuged for 5 minutes at 1100g and the top layer of supernatant was retained from a 38 µm sieve, washed in distilled water before following the bleach and culture methods described for C. elegans. T. circumcincta eggs were isolated from fresh fecal samples collected from naturally infected lambs and purified by salt flotation, cleaned with bleach, placed on OP50 NGM plates for 4 days to hatch. Cleaned embryos or hatched larvae were picked into 100 µl M9 with bacteria and no more than 1% DMS0 (much less – 0.1%) either alone or containing one compound. Scoring of larval or embryonic lethality was performed after 24-hour incubation at 22 °C. H. contortus infected fecal samples collected from Moredun, strain MHC03 (ISE) were purified by salt flotation and bleaching, then placed on OP50 NGM plates. Samples were aliquoted in 100 µl total M9 and incubated with compound or DMS0 (1%). Scoring was performed after 24-hour exposure at 22 °C. H. polygyrus samples were similarly collected from infected mouse feces. For the egg assays the above purification was carried out and eggs were washed in M9 buffer, exposed directly to compounds in M9 buffer and hatching was assessed over 2 days.

[0181] The lifecycle of Brugia pahangi was maintained by serial passage through mosquitoes (Aedes aegypti) and jirds (Meriones unguiculatus), as described elsewhere. Adults and microfilariae (Mfs) were obtained from the peritoneal cavity and cultured in 2ml of RPMI 1640 media with 2% glucose and 100U / ml penicillin, 100µg / ml streptomycin antibiotics at 37°C with 5% CO2. Each well of a 24 well plate contained either 4 adults or 1000 Mfs and were supplemented with either 1% DMSO or 1-50uM Avo A. Worms were scored for viability and motility after 24 hours and experiments were repeated at least 3 times.

[0182] Forward genetics screens. Forward genetic screens were adapted from previous methods. Wild-type L4 stage parental (P0) worms were mutagenized in 50 mM ethyl methanesulfonate for 4 hours. Synchronized L1 progeny from either F1 or F2 generations were dispensed onto 96-well plates containing 40 µM lethal dose of avocadene acetate in OP50 resuspended in S-complete. Worms were dispensed at a density of 50 L1s per well. Plates were scanned by eye under the dissecting scope for viable worms. In total, 2.1 million F1 larvae (i.e 4.2 million independently mutagenized haploid genomes) and 2.5 million F2 larvae (i.e 5 million independently mutagenized haploid genomes) were screened for avocadene acetate resistance. Therefore, a total of 9.2 million mutagenized genomes were screened.

[0183] Mouse Experiments. The H. polygyrus life cycle was maintained and the L3 larvae obtained as previously described57. H. polygyrus infection: C57BL / 6 female mice, aged 6–8 weeks, were infected by oral gavage with 200 H. polygyrus L3 in 200 µl water. Oral drug administration: Mice were given Avocadene acetate (Microsource discovery systems) at 2.5 mg in 200 µl SEDDS emulsion (89% water, 0.2% tween80 (Sigma) and 0.8% NEOBEE (Stephan), 10% DMSO) by gavage from the day 9 of infection, and treatment was repeated on days, 11, 13, 15, 17 and 19. Control mice were treated identically with SEDDS. On day 21, worm and egg counts were performed. The egg burdens of individual mice were assessed by collecting 2–3 faecal pellets for each H. polygyrus–infected mouse at the specified time intervals. Faeces were weighed before being solubilized in 2 ml PBS followed by the addition of 2 ml saturated sodium chloride solution. Egg counts were then carried out with the use of a McMaster chamber and the average number of eggs / g faeces calculated per sample. At the end point of each study, the intestinal adult worms were also counted to give the total worm burden of each individual mouse; the small intestinal tissues were recovered, and total worm burdens enumerated with the aid of a dissecting microscope. Counts and statistics were plotted using Prism graph.

[0184] Microscopy. To obtain early embryos, 5-10 young adult worms were dissected in a 7 μL droplet of 1% DMSO or the respective chemical treatment on a coverslip. The embryos were left in the droplet for 5-10 minutes to ensure sufficient exposure to the treatment. The coverslip was then mounted on a 2% agarose pad on a glass slide. DIC images were acquired every 2 seconds for the duration of 1 hour at 63x magnification for all the time lapse movies. The embryos were treated with avocadene acetate, CP-640186 and sethoxydim at concentrations of 50 μM, 100 μM and 100 μM respectively. A droplet of M9 containing 1% DMSO was used as a control. All images were acquired using Leica DMi8 microscope and processed using ImageJ.

[0185] Motility assessment and scoring. Assays were set up by dispensing approximately 10 synchronized young adult worms in a volume of 50 µl M9 buffer and 10 µl of OP50 bacterial suspension in each well of a 96-well flat bottom plate. Worms were treated with different concentrations (10 µM, 25 µM, 50 µM and 100 µM) of avocadene acetate or DMSO control. Plates were shaken thoroughly using the Multiflo Fx and images of entire wells captured by the Cellinsight CX5 high-throughput microscope. The protocol was set up to capture two images of each well, taken 500 ms apart from each other. This was repeated at 20 minutes intervals for a duration of 160 minutes. Image analysis and fractional mobility score was adapted from a previous study.

[0186] Electropharyngeogram recordings. The ScreenchipTM(InVivoBiosystems) was used to record electropharyngeograms (EPG) and quantify pharyngeal pump frequency in worms. Disposable microfluidic cartridges of size SC40 were used for all experiments. Synchronized young adult worms were washed 3 times with M9 buffer and incubated with 10 mM serotonin at 20 °C for 30 minutes, followed by treatment with avocadene acetate or DMSO control for approximately 5 minutes. Worms were loaded into the SC40 cartridge using a syringe and allowed to settle for 30-60 seconds. Their EPG recordings were taken for 60 seconds using the NemAquire software (InVivoBiosystems). The entire process was monitored under the Leica M125C inverted microscope to ensure that the worm was correctly positioned between the electrodes and only one worm occupied the channel at a time. Readings of 15 animals were taken for each treatment. Mean pump frequencies were calculated by the NemAnalysis software (InVivoBiosystems).

[0187] Oxygen Consumption Rate (OCR) measurements using the Seahorse XFe96 Analyzer. Synchronized L1 nematodes were treated with 0.05% DMSO or 5 µM avocadene acetate at 20°C, until they developed to the L4 stage (42-44 hours). Animals were washed three times with M9, and 20-25 animals in 20 µl M9 were transferred to each well of a 96-well Seahorse cell culture plate containing 180 µl of M9. The drugs FCCP and sodium azide were preloaded to the cartridge injection ports to a final concentration of 10 µM and 40 mM respectively. Baseline respiration was measured initially for 30 minutes followed by injection of FCCP (to measure maximal respiration) and sodium azide to measure non- mitochondrial respiration (Basal OCR was derived by subtracting the non-mitochondrial oxygen consumption induced by sodium azide from the measured baseline respiration levels). Basal respiration, maximal respiration and spare capacities were determined for each well and normalized to the number of worms per well.

[0188] ROS measurement. Intracellular ROS levels were measured using chloromethyl-2,7’ dichlorodihydrofluorescein diacetate (CM-H2DCFDA). Synchronized L1s were treated with DMSO (0.1%) or avocadene acetate (10 µM) for 48 hours at 20 °C. Worms were washed to remove OP50 and 10 - 15 animals were incubated with 50 µM CM- H2DCFDA for 4 hours in a 96-well plate. Fluorescence emitted at 535 nm upon excitation at 480 nm was measured using an EnSpire Multimode Plate Reader. Fluorescence levels were normalized to animal numbers by measuring absorbance at 600 nm. Fluorescence peaked after 4 hours, so absorbance values at this time point were compared among the control and treated groups.

[0189] MitoSOX™ Red staining. Animals at the L1 stage were treated with DMSO (0.1%) or avocadene acetate 10 µM for 42 hours. They were then washed off plates and MitoSOX staining was performed using around 100 animals in 500 µl M9 + cholesterol and 50 µl of OP50. The animals were incubated with 1 µl MitoSOX (final concentration 10 µM) in a rocker at 20°C in the dark for 24 hours. They were washed 3 times with M9, allowed to forage in a fresh OP50 plate for 1 hour and imaging of the pharyngeal region was performed using a Leica DMi8 microscope, using a 63× objective with 100 ms exposure using fluorescent filters 510 / 580.

[0190] NAC and Vitamin B12 treatment. Approximately 10 L1 stage worms per well in 96-well plates were treated with or without 10 mM N-acetyl cysteine (NAC) until they reached the L4 stage. Animals were then treated with 15 µM avocadene acetate or DMSO and survival was scored after 2 days. Similarly, L1 stage worms were treated in the presence or absence of 64 nM vitamin B12, followed by treatment with 6 µM, 8 µM and 10 µM of avocadene acetate upon reaching L4 stage.

[0191] Mitochondrial morphology scoring. C. elegans strain SJ4103, expressing mitochondrial matrix GFP in body wall muscles, was used to measure the mitochondrial morphology changes associated with avocadyne acetate treatment. Animals at the L1 stage were treated with 0.5% DMSO or 0.5% DMSO + avocadyne acetate (5 µM final concentration) for 48 hours. The animals were transferred to 2% agarose pads containing 1 mM levamisole, covered with a cover slip, and imaged using a Leica TCS SP8 confocal microscope with a 63× objective and 488 nm scanning laser.

[0192] RNAi experiments. All RNAi clones were obtained from the Ahringer library (Source BioScience) and their sequence confirmed. The experiments were carried out on L1 stage worms using the same method as described in the ‘Chemical screening’ section, using overnight RNAi bacterial cultures induced with 1 mM IPTG for 4 hours at 37 °C, instead of OP50. For avocadyne acetate and acs-2 / cpt-1 / cpt-4 / dif-1 / cpt-2 / acdh-1 / acdh-2 / acdh-3 / acdh-4 / acdh-6 / acdh-7 / acdh-8 / acdh-9 / acdh-10 / acdh-11 / acdh-12 / acdh-13 screens, 5 µM avocadyne acetate was used.

[0193] Malonyl-CoA rescue experiments on WT and pod-2(ye60). N2 and pod- 2(ye60) cold-sensitive animals were grown at 24 °C. When they reached the L3 stage, they were transferred to 15 °C for a duration of 16 hours. Subsequently, the animals were exposed to different concentrations of avocadene acetate. Additionally, one experimental set received a supplementation of 50 μM Malonyl-CoA. The surviving animals were then counted after a span of 48 hours.

[0194] Auxin-inducible degron (AID) experiments. AX8465, AX8273 and N2 strains were used in this assay. Age synchronized L1 larvae were grown on OP50 at a density of approximately 10 - 15 worms per well in 96-well plates as described above. Upon reaching the L4 larval stage, 1 mM auxin (indole-3-acetic acid) was added to plates containing AX8273 and incubated for 48 hours, followed by the addition of various concentrations of avocadene acetate. For AX8465 strain, treatment of auxin at a concentration of 50 µM took place at the young adult stage and avocadene acetate was added 24 hours later. Live and dead worms were quantified after 48 hours.

[0195] POD-2 biochemistry. C. elegans strain, PHX1772 pod-2(syb1772[pod- 2::His10]) II was used for the pull down experiments. Worm protein extract was prepared from 500,000 L4 stage worms, grown in liquid culture. The worms were pelleted, washed 3 times with M9 and resuspended in the lysis buffer. The worm suspension was then added dropwise to a pre-cooled mortar with liquid nitrogen. The frozen beads were ground to a fine powder. This powder was thawed again and homogenized using a QSonica sonicator. The sample was then centrifuged at 50,000 rpm using a Beckman Coulter optima XPN-90 Ultracentrifuge using Type 90 Ti rotor at 4 °C. The clear supernatant was then transferred to equilibrated Ni-NTA agarose resin slurry column and the mix was incubated for 1 hour at 4 °C. After collecting the flow through, the column was washed 5 times with the wash buffer and the elution of the protein was done using the elution buffer containing 250 mM imidazole. The eluted fraction was then concentrated by passing through a centrifugal filtration column (100 K molecular weight cut off) and the purified fraction was used for NMR assays.

[0196] Mass spectrometry and Proteomics. Preparation of in-solution digests. Bradford assay was performed to measure the protein concentration. Thirty µg protein per sample were reduced with 20 mM DTT (dithiothreitol, Sigma), at room temperature (RT), for 1 h and S-alkylated with 50 mM IAA (iodo-acetamide, Sigma), for 1 h in dark room. The remaining IAA was quenched with 20 mM DTT for 1 h in the dark. Prior to digestion, urea (Sigma) was diluted to a concentration of 1 M with 50 mM (NH4)HCO3 (ABC, Sigma) to prepare the solvent. The proteins were digested with 1:50 (w / w enzyme:protein) MS-grade trypsin / lys-C mix (Thermo Scientific) for 24 h at RT. The digestion was quenched by lowering the pH with formic acid (Fisher) and the sample was desalted with a ZipTip C18 (Merck-Millipore) column. Samples were resuspended in 10 µl of 0.2% formic acid prior LC- MS2(Liquid Chromatography tandem Mass Spectrometry) analysis.

[0197] LC-MS2analysis. The LC was performed on a fully automated EASY-nLC 1200 (Thermo Scientific). The system was fitted with a C18 column (PepMapTMRSLC, Thermo Scientific) with an inner diameter of 75 μm and length of 15 cm. The column was kept at a constant temperature of 40 ºC. Mobile phases consisted of 0.1% formic acid for solvent A and 0.1% formic acid in 95% acetonitrile for solvent B. Samples were loaded in solvent A. A linear gradient was set as follows: 0% B for 3 minutes and then a gradient up to 35% B in 45 minutes and to 60% B in 60 minutes. A 10-minute wash at 90% B was used to prevent carryover, and a 15-minute equilibration with 0% B completed the gradient. The LC system was coupled to a Q-Exactive high field orbitrap (Thermo Scientific) equipped with an Easy spray ion source and operated in positive ion mode. The spray voltage was set to 1.8 kV, S-lens RF level to 35 and ion transfer tube to 275 °C. The full scans were acquired in the orbitrap mass analyzer that covered a mass-over-charge (m / z) range of 350–1800 at a resolution of 120,000. The AGC target was set to 3E6 and maximum ion time to 50 ms. The MS2analysis was performed under data-dependent mode to fragment the top 20 most intense precursors using HCD fragmentation. The MS2parameters were set as follows: resolution, 15,000; AGC target, 1E5; minimum AGC target, 8.0E3, intensity threshold, 4.0E5; maximum ion time, 20 ms; isolation width, 1.4 m / z; precursor charge state, 2-5; peptide match, preferred; dynamic exclusion, 30 s, and fixed first mass of 80 m / z. The normalized collision energy for HCD was set to 32%.

[0198] Protein identifications were determined using Thermo Proteome Discoverer 2.4.1.15 (http: / / thermofischer.com / ) software’s with following settings: enzyme, trypsin; instrument, orbitrap; fragmentation, HCD; acquisition, DDA without merging the scans; and precursor and fragment mass tolerance, 10 ppm and 0.2 Da, respectively. Correct precursor was detected using mass only. Peptide identifications were performed using the UniProtKB_SwissSprot database (release 2021_02, https: / / www.uniprot.org / uniprot) with C. elegans taxonomy. The proteome discoverer search tool was used to search for peptides with fixed carbamidomethylation and variable oxidation of methionine (M) and N-terminal acetylation modifications. FDR threshold was set to 1 %, fold change was set to 2, with 2 unique peptides per protein used to filter the identified protein lists.

[0199] After undergoing real-time NMR analysis, the samples were lyophilized, resuspended in 50 µL of water and thoroughly mixed at 1:1 volumetric ratio with matrix solution (2,5-Dihydroxybenzoic acid) (Merck KGaA, Darmstadt, Germany). One µl of each sample was deposited onto the MALDI (Matrix Assisted Laser Desorption Ionization) target plate and the spots were left to dry at ambient condition prior FT-ICR-MS (FourierTransform Ion Cyclotron Resonance Mass Spectrometry) analysis. FT-ICR-MS allows the collection of high-resolution, accurate mass and isotopic fine structure for direct determination of a compound. The measurements were carried out on a Bruker SolariX FT- ICR mass spectrometer (Bruker Daltonics GmbH, Germany). The system consists of an ESI / MALDI dual ion source and is equipped with a 7.0T superconducting magnet (Magnex Scientific Inc., UK). Measurements were carried out in the MALDI positive ion mode. The spectra were acquired with a time domain of 4 mega words over a m / z range of 100 to 900. The laser parameters for ion generation were: wavelength, 400 µm; relative power, 55%; shots, 20; focus setting, ‘medium’; frequency, 200 Hz. The plate offset and deflector plate were set at 100 and 220 V, respectively. Data were acquired using ftmsControl software and subsequently processed with DataAnalysis software (Bruker Daltonics). One hundred scans were acquired for each sample.

[0200] Computational modeling. Modeling of POD-2 structures. The POD-2 (isoform c) structure predicted by AlphaFold (downloaded from UniProt) was used. Isoform c, with 2165 residues, includes the BC, CT and BCCP domains. The POD-2 dimer and its bound ligands biotin and CoA were modeled by structural alignment using solved yeast ACC homodimer complexes (PDB ID: 1w2x, 5csl). Specifically, the model POD-2 CT domain (residues 1438-2165) deviates from 1w2x and 5csl by 2.8 and 2.5 Å, respectively, suggesting a high degree of structural similarity to solved structures.

[0201] The Schrodinger software was used to perform induced-fit docking of chemical compounds to protein receptors. The induced-fit docking method accounts for the flexibility of ligand and protein receptor site conformations to find low binding-energy complexes. The significance of the results is determined by the binding free energy (∆G) and structural consistency of the low-energy complexes. The experimentally measurable dissociation constant (Kd) can be computed from the binding free energy (∆G) using the formula Kd = exp(∆G / RT), where R is the gas constant and T is the temperature (25 °C). For avocado-derived compounds, the ligand size parameter was set to 25 Å to ensure that the search space around the target is sufficiently large. Besides this, dockings were performed using default parameters. Molecular structures of the ligands of interest were downloaded from PubChem and then prepared for docking using Schrodinger’s LigPrep module to generate energy-minimized structures. The method was used to reproduce 3 solved ligand / ACC complexes for CP-640186 (PDB ID: 1w2x), ND-646 (5kkn) and soraphen A (1w96). The compound conformations were reproduced with high fidelity (complex RMSD~0.2 Angstrom), and the predicted favorable affinities are 6, 36 and 139 nM, respectively, for CP-640186, ND-646 and soraphen A.

[0202] Statistics and reproducibility. For dose-response, egg hatching and combinatorial chemical and RNAi screens, N=3 independent biological replicates were performed, with >50 individuals per replicate. Data visualizations and statistical comparisons between groups were performed using Graphpad Prism (v8.3.0) or R. Best fit dose-response curves and LD50 values were generated using nonlinear regression analysis. Unpaired two- tailed t-tests were used to compare groups and p-value<0.05 was considered as statistically significant.

[0203] Table 1. AFA compounds affect the parasites Haemonchus contortus. Embryonic and larval lethality counts after treatment with different AFA compounds. Haemonchus contortus Total eggs % emb lethality % larval lethality DMSO 1% 857 40 10AFA. *mild effect. **strong effect.Genes RNAi + AFA Mutant + AFA Mutant genotype Mitochondrial β-oxidation acs-2 rescue* cpt-1 no effect

[0205] Table 3. Fitting parameters for the experimental data reported in FIG.18. The experimental data fitting was obtained using Prism 10 (GraphPad Software LLC). All parameters are reported with the respective 95% confidence interval (profile likelihood). The leftmost column (Sample) lists the data source solutions, namely the reference solution – 50 µM AcCoA dissolved in D2O + 50 µM ATP + 30 mM (NH4)HCO3–, the reference solution with 20 µM or 50 µM avocadene acetate, and the reference solution devoid of ATP. The reported parameter values refer to the fitting for normalized AcCoA concentration data, further corrected by scaling with respect to the corresponding data collected without ATP. The p values given in the rightmost column were obtained from two-tail t test of two samples (experimental data and corresponding fitting), to express the probability of the experimental data to fit the exponential model by chance.Sample^^^^^^^^ଶ^^^^^ଶp valueReference 0.223±0.0150.551±0.041 0.394±0.0417.21±0.80 ^10^35.66±1.05 ^10^22 ^10^5^3^3^3

[0206] Although the present disclosure has been described with respect to one or more particular embodiments and / or examples, it will be understood that other embodiments and / or examples of the present disclosure may be made without departing from the scope of the present disclosure.

Claims

CLAIMS:

1. A method of treating a subject having or suspected of having nematode-based infection, comprising administering to the subject a therapeutically effective amount of one or more of the following compounds: ,X a group formed from an anthelmintic compound; ;is OH or O; R is H or -COCH3; and each R'' is independently H or an alkoxy group.

2. The method according to claim 1, wherein the one or more of the following compounds are: , ,, ,3. The method according to claim 2, wherein at least a combination of the following compounds is administered: .

4. The method according to claim 2, wherein at least a combination of the following compounds is administered: .

5. The method according to claim 1, wherein the nematode-based infection is associated with a drug-resistant nematode species.

6. The method according to claim 1, wherein the nematode-based infection is associated with a parasitic or non-parasitic nematode species.

7. The method according to claim 1, wherein the nematode-based infection is associated with one or more of Brugia pahangi, Teladorsagia circumcincta, Haemonchus contortus, Heligmosomoides polygyrus, Pristionchus pacificus, Caenorhabditis elegans, or any combination thereof.

8. The method according to claim 1, wherein the administration results in death of at least a portion of nematodes associated with the nematode-based infection.

9. The method according to claim 1, wherein at least a portion of the nematodes associated with the nematode-based infection are dauer or embryos.

10. The method according to claim 1, wherein the administration results in a reduction of adult worm load.

11. The method according to claim 1, wherein administration results in reduction of nematode reproduction.

12. The method according to claim 1, wherein the subject is a mammal.

13. The method according to claim 12, wherein the subject is a human.

14. The method according to claim 1, wherein the subject is an agricultural animal, service animal, or pet.

15. The method according to claim 1, wherein the subject does not have a hematological cancer.

16. The method according to claim 15, wherein the subject does not have leukemia.

17. The method according to claim 1, wherein the subject does not have malaria.

18. The method according to claim 17, wherein the leukemia is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), or myeloid leukemia.

19. The method according to claim 1, wherein the one or more compounds is administered in conjunction with another therapy.

20. A composition comprising a pharmaceutically acceptable excipient and Avocatin A.

21. The composition according to claim 20, further comprising: .

22. A compound having the following structure: or,a or bond; is OH when is a single bond and O when is a double bond;R is H or - andX is a group from an anthelmintic 23. The compound according to claim 22, wherein the compound has the following structure: or .

24. A composition comprising a compound according to claim 22.

25. A method of treating a subject having or suspected of having nematode-based infection comprising, comprising administering to the subject a therapeutically effective amount of one or more compounds of claim 22 or a composition comprising one or more compounds according to claim 22.

26. The method according to claim 25, further comprising administering one or more of the following compounds: ,, 27. A method for inhibiting fatty acid synthesis and / or fatty acid oxidation pathways in a subject, comprising administering to the subject a therapeutically effective amount of one or more compounds of claim 22 or a composition comprising one or more compounds according to claim 22.

28. A method of claim 27, further comprising administering one or more of the following compounds: ,29. A method for identifying if a compound / drug is an anthelmintic, comprising contacting a culture a plurality of nematodes with the compound or drug; incubating the culture for a period of time; imaging at least a portion of the plurality of the nematodes after the incubating; and comparing the images of the at least a portion of the plurality of nematodes from the culture with a control, wherein the control comprises nematodes that have not been contacted with the compound / drug.

30. A method according to claim 29, wherein the culture further comprises E. coli.

31. A method according to claim 29, wherein the plurality of nematodes are eggs, L1 stage worms, young adults, and / or dauers.

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