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).
PARASITE PREPARATION: The larval stages -tetrathyridia (TTy) were maintained in the laboratory by alternate intraperitoneal infection of adult Wistar female rats (3 months old) and adult Balb/c female mice (3 months old). TTy were collected from the peritoneal cavity using standard aseptic techniques and washed three times with sterile phosphate-buffered saline solution (PBS) pH 7.2 with levofloxacin (20 μg/mL).Before being employed in experiments, TTy were size-selected using monofilament polyester meshes to a final size of 150 to 250 μm and incubated for 24 h in 5 mL of MvRPMI medium-a modified RPMI 1640 medium without phenol red complemented at 37˚C under 5% CO2 atmosphere. COMPOUNDS: The HDAC inhibitors stock solutions were prepared at 10mM in 100% dimethyl sulfoxide (DMSO) and stored at -20˚C until use. The Class I-HDAC inhibitors entinostat, TH65, and TH92 were selected for further characterization based on their potent anthelmintic effect, as they were able to kill all TTy at 20 μM concentration before 9 days of treatment. ABZ at 20 μM was evaluated as positive control. M. Vogae TTy MOTILITY ASSAY: The effect of each compound on parasite viability was determined using a motility assay employing the WMicrotracker device. Briefly, 5 TTy were incubated in U-shape 96-well microplates with 200 μL of MvRPMI medium per well at 37˚C under 5% CO2 atmosphere. Parasites pre-treated with ethanol 70% for 30 min were used as a positive control. All motility assays were performed using an equal amount of the drug vehicle (1% DMSO final concentration) and corresponding negative controls (1% DMSO). The anthelmintic dose-dependent effect was determined for the selected HDAC inhibitors at 6 days of incubation from three independent biological replicates, each one in quadruplicate. The evaluation of irreversibility of the anthelmintic effect was determined incubated TTy with the compounds at their respective IC90 concentrations for 6 days; then, culture medium was removed and TTy were gently washed four times in PBS (pH 7.2) at 37°C. Finally, TTy were incubated for 8 additional days in a fresh culture medium without adding the compounds.
Dose-response curves generated for entinostat, TH65, TH92, and ABZ revealed a concentration-dependent effect. activity.The individual IC50 values of these HDAC inhibitors were significantly lower than that of ABZ (20.58 ± 0.38 μM), being entinostat the most potent with a very low IC50 (1.22 ± 0.07 μM, p < 0.0001), followed by TH92 (6.27 ± 0.58 μM, p < 0.0001) and TH65 (11.45 ± 1.20 μM, p < 0.0001), Table 1. Moreover, the irreversibility of the in vitro anthelmintic effect observed for these compounds was assessed. No increase on parasite viability was observed after the compounds were removed from the culture medium. Moreover, the reduction of M. vogae TTy viability reached 100% before or at 10 days of incubation (Fig 4). These findings indicate that the compounds´ anthelmintic effects persisted after induction during the incubation period and were irreversible. The evaluation and characterization of various inhibitors against HDAC enzymes should help explore the roles of these enzymes in cestodes, to aid in the development of new cestocidals.
Hugo R Vaca, Ana M Celentano, María A Toscanini, Tino Heimburg, Ehab Ghazy, Patrik Zeyen, Alexander-Thomas Hauser, Guilherme Oliveira , María C Elissondo, Manfred Jung, Wolfgang Sippl, Federico Camicia, Mara Cecilia Rosenzvit.