The potential for histone deacetylase (HDAC) inhibitors as cestocidal drugs

Abstract

Background: Echinococcosis and cysticercosis are neglected tropical diseases caused by cestode parasites (family Taeniidae). Not only there is a small number of approved anthelmintics for the treatment of these cestodiases, but also some of them are not highly effective against larval stages, such that identifying novel drug targets and their associated compounds is critical. Histone deacetylase (HDAC) enzymes are validated drug targets in cancers and other diseases, and have been gaining relevance for developing new potential anti-parasitic treatments in the last years. Here, we present the anthelmintic profile for a panel of recently developed HDAC inhibitors against the model cestode Mesocestoides vogae (syn. M. corti).

Methodology/principal findings: Phenotypic screening was performed on M. vogae by motility measurements and optical microscopic observations. Some HDAC inhibitors showed potent anthelmintic activities; three of them -entinostat, TH65, and TH92- had pronounced anthelmintic effects, reducing parasite viability by ~100% at concentrations of ≤ 20 μM. These compounds were selected for further characterization and showed anthelmintic effects in the micromolar range and in a time- and dose-dependent manner. Moreover, these compounds induced major alterations on the morphology and ultrastructural features of M. vogae. The potencies of these compounds were higher than albendazole and the anthelmintic effects were irreversible. Additionally, we evaluated pairwise drug combinations of these HDAC inhibitors and albendazole. The results suggested a positive interaction in the anthelmintic effect for individual pairs of compounds. Due to the maximum dose approved for entinostat, adjustments in the dose regime and/or combinations with currently-used anthelmintic drugs are needed, and the selectivity of TH65 and TH92 towards parasite targets should be assessed.

Conclusion, significance: The results presented here suggest that HDAC inhibitors represent novel and potent drug candidates against cestodes and pave the way to understanding the roles of HDACs in these parasites.

Fig 1. Effect of Class I- and Class II-HDAC inhibitors on Mesocestoides vogae TTy (tetrathyridia) viability.

In vitro anthelmintic activity was determined for the Class I-HDAC inhibitors: (A) entinostat, (B) TH65, (C) EG18, and (D) TB87; and the Class II-HDAC inhibitors: (E) JS16, (F) JS28, (G) KK16, and (H) ST36. The compounds were evaluated at concentrations of 2, 20 and 50 μM (TH65 was not evaluated at 50 μM due to low solubility) and at different incubation times, using the M. vogae TTy motility assay. Parasites incubated with the drug vehicle (DMSO 1%) were used as a negative control. Relative motility indices were measured from three independent biological replicates, each one in quadruplicate. Error bars represent the standard deviation and the asterisks indicate those values that showed differences with statistical significance compared to the negative control, according to two-way ANOVA test and Dunnett’s post-tests (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

Fig 2. Effect of SmHDAC8 inhibitors (series TH, EG, and TB) on Mesocestoides vogae viability.

In vitro anthelmintic activity was determined for compounds of the series TH: (A) TH60, (B) TH71, (C) TH74, (D) TH85, (E) TH92, (F) TH119, (G) TH120, (H) TH138, and (I) TH139; series EG: (J) EG13 and (K) EG20; and series TB: (L) TB98. All compounds were evaluated at concentrations of 2, 20, and 50 μM and at different incubation times using the M. vogae TTy motility assay. Parasites incubated with the drug vehicle (1% DMSO) were used as a negative control. Relative motility indices were measured from three independent biological replicates, each one in quadruplicate. Error bars represent the standard deviation and the asterisks indicate those values that showed differences with statistical significance compared to the negative control, according to two-way ANOVA test and Dunnett’s post-tests (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

Fig 3. Anthelmintic dose-dependent effect of selected HDAC inhibitors and albendazole (ABZ).

The anthelmintic dose-dependent effect was determined for the selected HDAC inhibitors (A) TH65, (B) TH92, and (C) entinostat, and (D) the current anthelmintic drug ABZ at 6 days of incubation from three independent biological replicates, each one in quadruplicate. Error bars represent the standard deviation. In each graph, the compounds’ chemical structure and the half-maximal inhibitory concentration (IC₅₀) are shown. IC₅₀ is expressed in μM, with the corresponding standard deviation.

Fig 4. Reversibility in vitro test of selected HDAC inhibitors and albendazole (ABZ).

In vitro anthelmintic activity was determined for entinostat, TH65, TH92, and ABZ at different incubation times, using the Mesocestoides vogae TTy (tetrathyridia) motility assay. Relative motility indices were measured from three independent biological replicates, each one in quadruplicate. Error bars represent the standard deviation. The compounds were tested at their respective IC₉₀ concentrations for 6 days, then culture medium was removed, and TTy were gently washed and incubated with a fresh culture medium without adding the compounds for 8 additional days. Parasites incubated with the drug vehicle (1% DMSO) were used as a negative control.

Fig 5. Effect in parasite morphology of selected HDAC inhibitors and trichostatin A (TSA).

Scanning electron microscopy images of Mesocestoides vogae tetrathyridia (TTy) treated with the individual HDAC inhibitors -entinostat, TH65, and TH92- or the pan-HDAC inhibitor TSA at 20 μM after 6 days of incubation and at different magnifications (indicated in each figure). Extensive damage was observed on M. vogae TTy treated with the HDAC inhibitors, with marked alterations on the tegument and a disintegration of parasite morphology, as well as a significant number of vesicle-like structures of different sizes on the tegument (arrows) and a complete loss of microtriches. The sizes of the scale bars are shown in each image. TTy incubated with the drug vehicle 1% DMSO were used as a negative control.

Fig 6. Effect of pairwise drug combinations of selected HDAC inhibitors with albendazole (ABZ) on parasite viability.

In vitro anthelmintic activity was determined for individual HDAC inhibitors plus ABZ: (A) entinostat, (C) TH65, and (E) TH92; as well as entinostat in combination with (B) TH65, and (D) TH92. (F) Concentrations used for each compound, corresponding to their respective IC₂₅ values. The compounds were tested alone and in combination at different incubation times, using the Mesocestoides vogae tetrathyridia (TTy) motility assay. Relative motility indices were measured from three independent biological replicates, each one in quadruplicate. Error bars represent the standard deviation. Proportional effect indices were calculated at 6 days of incubation (marked with a box). TTy incubated with the drug vehicle (1% DMSO) represented a negative control.

Fig 7. Classification of the anthelmintic effect determined for pairwise drug combinations of selected HDAC inhibitors and albendazole (ABZ).

The proportional effect indices were determined for all binary drug combinations of HDAC inhibitors and ABZ tested. The values are displayed in a heat-map format to display the deviation away the different combinations’ merely additive predicted effects. Index values range from -1 to +1. The predicted additive effect index (0) is depicted in yellow. If the effect determined for the pair of compounds is higher than the predicted outcome, it is indicated in green; if the determined effect is lower, it is indicated in red. The proportional effect indices were calculated from three independent biological replicates, each in quadruplicate.