## 2 Results
In this way, we avoid relying on molecular docking results that use simplified scoring functions designed to process large amounts of compounds in a reasonable time, and scoring values should not be compared across different molecular targets. Computational target fishing approaches are based on the similarity principle, which states that similar chemical compounds should have similar bioactivity profiles. These methods are often trained with activity data available on public databases such as ChEMBL. Unfortunately, such databases are biased toward interactions of chemicals with human proteins. Thus, in this study, all probable targets of compound **17** are first predicted, and the homolog proteins of these in _T. cruzi_ are identified. In this way, we address the limitations in bioactivity data deposited in the databases for the parasite while narrowing the space of possible molecular targets of the compounds in _T. cruzi_. This approach serves to propose an initial set of possible targets of the compound for further molecular docking calculations and MD simulations. Table 2 lists the potential molecular targets of compound **17** in _T. cruzi_ that were identified using the homology-based target fishing approach. The information in Table 2 includes the UniProt accession number, the ID used in the manuscript, and a brief functional description for each protein. Compound **17** was docked to the proteins listed in Table 2 using the methodology described in the Materials and Methods section. In the case of  
Figure 2: Scatter plots of cells from the cell death study. (**A**) Control group; (**B**) group treated with compound **16**; (**C**) group treated with compound **17**; (**D**) group treated with compound **18**; (**E**) group treated with compound **20**; (**F**) group treated with compound **15**. the DHFR-TS enzyme, both the folate and dUMP were explored separately. On the other hand, TUBA and TUBB polymerize to form microtubules, and the inhibition of this process can influence the normal development of cells. Thus, the binding of compound **17** to the interface of the TUBA-TUBB dimer was explored. The assembly of this dimer was carried out as proposed in a previous report [18] and will be referred to as TUB from here on. The molecular docking results are provided in Table 3.  
Figure 3: Relative fluorescence intensity box plot (**A**,**C**,**E**,**G**,**I**) and histogram graphs (**B**,**D**,**F**,**H**,**I**) of Rho123 dye in epimastigote forms treated with compounds **15 (A**,**B**), **16 (C**,**D**), **17 (E**,**F**), **18 (G**,**H**) and **20 (I**,**J**). The black, dark blue, and light blue lines represent the fluorescence of the control groups, the highest, and the lowest compound concentrations, respectively. * \(p<0.05\) vs. control group. Molecular docking resulted in 20 different compound-receptor complexes that were then subject to the calculation of the MD-based free energies of binding according to the procedure described in the Section 4. The results of these calculations are given as Supplementary Materials in Table S1, while the lowest (best) free energy of binding computed for compound **17** to each target is represented in Figure 4.  
According to these results, the most probable target of the chemical in _T. cruzi_, among those evaluated in our research, is HDAC (histone deacetylase). Hence, a more detailed analysis of the predicted binding mode of compound **17** to HDAC was performed, as shown in Figure 5. For this enzyme, the ligand is predicted to orient with the tri-methoxyphenyl ring at the entrance of the binding cavity, forming \(\pi\)-\(\pi\) stacking interactions with F152 and H180 while also interacting with F208 and L274. The carbonyl oxygen points directly to the Zn\({}^{2+}\) ion. On the other hand, the amide nitrogen serves as a hydrogen bond donor to G151. These \(\pi\)-\(\pi\) stacking, electrostatic, and hydrogen bond interactions are proposed as the main factors stabilizing the binding of compound **17** to HDAC. Finally, the isobutyl tail