Diisopropylphenyl-imidazole (DII): A new compound that exerts anthelmintic activity through novel molecular mechanisms

Abstract

Nematode parasites cause substantial morbidity to billions of people and considerable losses in livestock and food crops. The repertoire of effective anthelmintic compounds for treating these parasitoses is very limited, as drug development has been delayed for decades. Moreover, resistance has become a global concern in livestock parasites and is an emerging issue for human helminthiasis. Therefore, anthelmintics with novel mechanisms of action are urgently needed. Taking advantage of Caenorhabditis elegans as an established model system, we here screened the nematicidal potential of novel imidazolium and imidazole derivatives. One of these derivatives, diisopropylphenyl-imidazole (DII), is lethal to C. elegans at both mature and immature stages. This lethal effect appears to be specific because DII concentrations which prove to be toxic to C. elegans do not induce significant lethality on bacteria, Drosophila melanogaster, and HEK-293 cells. Our analysis of DII action on C. elegans mutant strains determined that, in the adult stage, null mutants of unc-29 are resistant to the drug. Muscle expression of this gene completely restores DII sensitivity. UNC-29 has been largely reported as an essential constituent of the levamisole-sensitive muscle nicotinic receptor (L-AChR). Nevertheless, null mutants in unc-63 and lev-8 (essential and non-essential subunits of L-AChRs, respectively) are as sensitive to DII as the wild-type strain. Therefore, our results suggest that DII effects on adult nematodes rely on a previously unidentified UNC-29-containing muscle AChR, different from the classical L-AChR. Interestingly, DII targets appear to be different between larvae and adults, as unc-29 null mutant larvae are sensitive to the drug. The existence of more than one target could delay resistance development. Its lethality on C. elegans, its harmlessness in non-nematode species and its novel and dual mechanism of action make DII a promising candidate compound for anthelmintic therapy.

Fig 1. Anthelmintic activity screening of new imidazole-derivative compounds.

(A) Structures of imidazolium salts and neutral compounds synthesized (compounds 1–11). (B) To evaluate nematicidal effect L4/young adult C. elegans were exposed to the compounds (150 μg/ml) for 72 h. Animal survival was subsequently evaluated. Only compounds 10 and 11 induced animal death.

Fig 2. Selective toxicity of DII.

(A) The effect of MI (10) and DII (11) in bacteria was evaluated using E. coli and S. aureus cultures. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were obtained using microdilution methods. As control of antibiotic activity ciprofloxacin (cipro.) was used. (B) DII lethal dose curve. 40 L4 wild-type worms were exposed to a range of DII concentrations and animal death was evaluated after 96 h of exposure. Data was fitted with a 4PL curve. LC50 = 23.44 ± 1.06 μM, Hill slope = 2.15 ± 0.25, R² = 0.99. Each concentration point represents the mean value ± SEM of three independent experiments. (C) DII effect on human cell cultures was evaluated using HEK-293 cells. Cells were exposed to DII (0–100 μM) and after 96 h of incubation, cell death was quantified using Propidium Iodide (PI) staining. Red staining accounts for dead cells. DMSO (0.1%) and chloroquine (50 μM) were used as negative and positive control, respectively. Bar scale: 100 μm. Results are presented as mean ± SEM (ns: no statistically significant, p > 0.05, ***p<0.001; n = 3) (D) DII effect on Drosophila melanogaster. Fly larvae were exposed to DII (300–1200 μM) until they left the food to pupariate. Larval survival was calculated as the percentage of larvae that reached the pupal stage. Results are presented as mean ± SEM (ns: no statistically significant, p > 0.05; n = 3).

Fig 3. Nematicidal effect of DII on wild-type and mutant C. elegans strains.

(A) DII lethal dose curves at 8 (black circle) and 24 (white triangle) h of exposure. ~40 L4 wild-type worms were exposed to a range of DII concentrations and animal death was evaluated at different time points. Data was fitted with a 4PL curve. (black circle) LC50 = 448.5 ± 1.1 μM, Hill-slope = 4.4 ± 0.9, R² = 0.91 and (white triangle) LC50 = 342.3 ± 1.0 μM, Hill-slope = 6.7 ± 1.3, R² = 0.95. Each concentration point represents the mean value ± SEM of three independent experiments (B) DII nematicidal effect on C. elegans mutant strains previously reported as resistant to currently used anthelmintic agents. ~80 L4 mutant animals were exposed to DII (600 μM) and worm survival was scored at each indicated time (4, 8 and 24 h). Only CB1072 unc-29(e1072)I strain was resistant to DII anthelmintic activity.‬ Results are presented as mean ± SEM. Statistical significance compared to wild-type worms (**p<0.01, ***p<0.001; n = 3).

Fig 4. DII effect on L-AChR-deficient mutants.

(A) DII-anthelmintic activity in different L-AChR subunit mutants. ~80 L4 mutant animals were exposed to DII (600 μM) and after the indicated time (4, 8, 24, 48 and 72 h) survival was scored. Results are presented as mean ± SEM. Statistical significance compared to wild-type worms (*p<0.05, **p<0.01, ***p<0.001; n = 3). (B) Acute paralysis assays. 30–40 L4 worms were exposed to different levamisole or DII concentrations (0–800 μM) and 10 minutes later paralysis was evaluated. Data was fitted with a 5PL curve. (black circle) EC50 = 63.6 ± 1.1 μM, Hill-slope = 2.39 ± 1.37, R² = 0.97 and (grey square) Hill-slope = 1.96 ± 2.31, R² = 0.49. Each concentration point represents the mean value ± SEM of three independent experiments. (C) Body length measurement after levamisole and DII (600 μM) 2 h treatment. Results are presented as mean ± SEM (***p<0.001; n = 20) Bar scale: 100 μm. (D) Egg laying rate in levamisole and DII-treated animals. 12 gravid worms were incubated for 1 h with the drugs (100 and 300 μM) and the number of laid eggs was counted. Results are expressed as the mean number of laid eggs per animal in 1 h ± SEM (*p<0.05, **p<0.01; n = 4). (E) Levamisole dose-response curves in presence of DII. Animals were exposed to a range of levamisole concentrations (1–600 μM) in the presence of constant DII concentrations (100 or 200 μM). After 10 minutes of incubation, paralysis was evaluated. No differences were observed among the three dose-response curves. Data was fitted with a 4PL curve. (black circle) EC50 = 60.00 ± 1.12 μM, Hill-slope = 1.78 ± 0.36, R² = 0.91, (dark pink triangle up) EC50 = 66.03 ± 1.15 μM, Hill-slope = 2.65 ± 0.81, R² = 0.90, (light pink triangle down) EC50 = 62.25 ± 1.14 μM, Hill-slope = 2.36 ± 0.61, R² = 0.93. Each concentration point represents the mean value ± SEM of three independent experiments. (F) UNC-29 muscle expression restores DII sensitivity. unc-29(e1072)I animals were injected with Pmyo-3::UNC-29; to restore muscle UNC-29 expression. Wild-type, unc-29 null mutants and muscle rescue were exposed to DII (600 μM) and worm survival was evaluated at different time points (4, 6, 8, 12 and 24 h). Data are presented as mean ± SEM. Statistical significance compared to wild-type worms (*p<0.05, ***p<0.001, ns: no statistically significant, p > 0.05; n = 3).

Fig 5. DII anthelmintic activity in C. elegans immature stages.

(A) Left. Isolated eggs were exposed to DII (0–600 μM). After 48 h of exposure, living animals were counted. Survival percentages are relative to the number of eggs at the beginning of the assay. Data are presented as mean ± SEM. Statistical significance compared to control (**p<0.01; n = 3) Right. DII effect on C. elegans developmental rate. Eggs were exposed to DII (600 μM) and animal stages were evaluated at each indicated time point (24, 48 and 72 h). L1-L3: early larval stages, L4: last larval stage. Animal stage percentages are relative to the number of living animals at the indicated time point. (B) Left. L1 larvae were exposed to DII (100, 300 and 600 μM) and after 24, 48 and 72 h animal viability was evaluated. Right. DII (600 μM) larvicidal effect on unc-29 mutant larvae. Data are presented as mean ± SEM. Statistical significance compared to control at the same time point (*p<0.05, **p<0.01, ***p<0.001, ns: no statistically significant, p > 0.05; n = 3).