The adversities presented by treatment with drugs from the 5-nitroimidazole class demonstrate the problem involving dependence on only one pharmaceutical class. Adverse reactions, allergies and resistant isolates described in literature, showcase the need for more reliable alternatives (Dunne et al. 2003; Leitsch 2016).
With this in mind, chalcones emerge as an interesting pharmacological target. They are widely distributed in nature and have numerous biological activities already reported, in their natural or synthetic form. Chalcones are easy to synthesize and highly versatile in their main structure, allowing new derivatives to arise from different substituents and conformations in their main chain (Zhuang et al. 2017).
Previous studies have already demonstrated the antiparasitic potential of chalcones against T. vaginalis. Singh et al. (2016) demonstrated the antiparasitic efficacy of chalcones with trizole and organosilatranes substituents in T. vaginalis and Giardia Lamblia (Singh et al. 2016). Trein et al. (2018) highlighted the tricomonicidal potential of 3-aminochalcone, which inhibitory effect, at a concentration of 100 µM, did not differ from that of positive control with MTZ (Trein et al. 2019). Das Neves et al. (2019) showed promising results of 2-hydroxychalcones associated with metronidazole, demonstrating reduced viability at low concentrations, in addition to lower rates of cytotoxicity (das Neves et al. 2020).
A total of 10 molecules, with different substituents, were evaluated regarding their anti-T. vaginalis potential in this study. Six molecules exhibited satisfactory results in the pharmacological screening at 100 µM. Among them, four had their MIC established at concentrations below the used by the positive control (MTZ 100 µM) and proceeded to other assays previously described. Compounds 3f and 3j presented significant differences from the positive control (MTZ) until the concentration of 120 µM, however; their MIC values could not be obtained. The reason, most likely, being the low stability of these molecules.
In the cytotoxicity assays, the results presented by the VERO cell line corroborate to the indices found in the HMVII cell line. Strategies can be adopted to reduce these levels, such as association with other molecules with anti-T. vaginalis that have already been evaluated, providing a synergistic effect of both molecules at lower concentrations (Hübner et al. 2016). Alternatively, different routes of administration and drug delivery systems can be adopted. Among the possibilities, the use of topical ointments or nanoencapsulated drugs can promote a more directed action, providing more efficiency and reducing the side effects. (Frank et al. 2015; Bouchemal et al. 2017).
As for the antioxidant assays, none of the compounds elicited antioxidant activity in the three assays presented (DPPH, FRAP and ABTS). Some molecules presented levels lower than the negative control, indicating a possible pro-oxidant activity (data not shown). These results align with the ones obtained by Sena-Lopes et al. (2017), indicating no antioxidant defences in the metabolism of T. vaginalis (Sena-Lopes et al. 2017). It can be inferred that an increase in concentration of oxidant radicals inside the parasite is a proficient way of acting against it.
Even though the results indicate that there was no accumulation of ROS produced by T. vaginalis after incubation with chalcone analogues, the hypothesis of parasite death due to the accumulation of oxidant radicals should not be discarded. The assay executed by Trein et al. (2018) also presented no change in the production of ROS by the parasite after treatment with compounds (Trein et al. 2019). However, when performing the incubation together with neutrophils, there was a significant increase in the production of ROS. Given the fact that neutrophils represent the host's first line of immune defence in T. vaginalis infection, it is more likely that this accumulation is related to the host organism immune response than to the pathogen metabolism (Song et al. 2008).
In the TBARS assays, only the treatment with the compound 3g showed a significant increase level in production of MDA. Analysing the chemical structure of the active molecule, it is possible to observe the presence of fluorine in the ramification. This molecule has already been related to the increase of MDA in plants and animal tissues (Inkielewicz-Stêpniak 2011; Tkachenko et al. 2021). There are few studies describing this methodology in T. vaginalis. However, supposed that the increase in MDA produced is directly related to the interaction between the molecule and the lipoprotein membrane of this parasite, it may destabilize the membrane and culminate in the parasite death (Grotto et al. 2009). Factors such as incubation time and concentration of compounds can interfere with the presented levels, requiring a different exposure or changes in the evaluated concentrations (Perjsi and Rozmer 2011).
Molecular docking is an important tool for screening new drugs for infectious diseases and predicting possible pharmacological targets. In silico analyses indicate a stronger interaction between enzymes and the chalcone analogues tested, aligning with other results shown. We emphasize the good performance of TvLDH and TvPNP, in order to highlight a possible mechanism of action for these molecules from tested enzymes.
Purine nucleoside phosphorylase is an enzyme of metabolic importance in T. vaginalis that participates on the purine recovery pathway. The auxotrophic capacity of this protozoan, which lacks de novo synthesis capability, makes the action of TvPNP and nucleoside kinase fundamental for the recovery of purine bases (Rinaldo-Matthis et al. 2007).
Lactate dehydrogenase is related to biochemical processes, through catalysing the conversion of lactate into pyruvate, with generation of NAD+, used in the glycolysis pathway for ATP production. The interaction of chalcone analogues with TvLDH can interfere with the parasite's energetic metabolism, causing critical damage to its energy production and reduced survival capacity (Kayamba et al. 2021)
At this point, we can suggest two pathways of action with the interactions pointed out: the first corresponds to interference in the metabolism of hydrogenosomes through action of TvLDH enzyme, directly affecting the energy production of the organism. The second occurs by interfering with the purine recovery pathway, performed by interference of TvPNP.
It is notable the high TvMGL enzyme score with the presented compounds. This enzyme plays a role in regulating sulfur-containing amino acids, responsible for numerous biological processes in the parasite. Its absence in mammals makes this enzyme an interesting target regarding its selectivity (Sato and Nozaki 2009).
The high scores presented by these three enzymes corroborate with results presented by Das Neves et al (2019). In their study, the link between hydroxychalcones and the active site of the enzymes TvLDH, TvPNP and TvMGL is highlighted, indicating a possible inhibition of these sites, interfering with the survivability of parasites (das Neves et al. 2020). Another interesting point is the difference or absence found in the structure of these enzymes in T. vaginalis from the form found in the organism of mammals. This fact contributes to a more selective profile of interaction of these molecules with only with the parasite, providing more safety in treatment (Setzer et al. 2017).