Introduction
The Aedes aegypti mosquito (A. aegypti) is responsible for major public health problems in our current society, as it is described as the main vector of Dengue, Chikungunya, and Zika viruses (Terra et al., 2017). A. aegypti adapts very easily to different types of artificial breeders and is highly anthropophilic, which favors the emergence of epidemics of arboviruses transmitted by this vector (Morais et al., 2020). The high incidence of viral diseases transmitted by female mosquito, has represented a major economic, social, and public health problem. The difficulty in controlling Ae. aegypti can be attributed to its biological characteristics, such as adaptability to different environmental spheres, temperature, and seasonality (Morais et al., 2020).
Vector control is one of the most important ways to prevent the spread of these diseases. Several studies demonstrate that the continuous application of chemical agents has led to mosquito resistance. Furthermore, the use of these products can have effects on human health and the environment, making it necessary to seek safer and more effective alternatives. In response to this problem, natural synthetic compounds have become tools with great functionality and the potential to serve as palliative and effective methods for the environmental control of vectors. (Aguiar et al., 2017; Mendes et al., 2017; Souza et al., 2019).
In this way, new possibilities arise for combating the vector. Based on organic synthesis, the need to search for new bioactive constituents with insecticidal potential, such as chalcones, has been assessed. Chalcones are one of the most important classes of flavonoids associated with secondary metabolism in plants. Precursors for the biosynthesis of flavonoids, chalcones are mainly polyphenolic compounds that have a variety of biological activities, such as antimicrobial, antiparasitic, and others. Therefore, despite their positive characteristics, there is still a lack of information on how much these compounds could help in vector population control (Souza et al., 2019; Gomes et al., 2020).
Based on this context, the general objective of this study is to investigate the effects and activities of heterocyclic chalcone in the analysis of larvicidal activity, and using computational tools in Aedes aegypti.
Aims
The present study aims to analyze and elucidate the larvicidal activity of a heterocyclic chalcone using computational tools and in vivo studies in Ae. aegypti.
Methods
1. Synthesis of heterocyclic chalcone and compound preparation
The heterocyclic chalcone was obtained from the Laboratory of Organic and Medicinal Synthesis (LASOM)/UFPB. The compound was obtained through the Claisen Schmidt condensation between 3,4-methylenedioxyacetophenone and thiofurfural benzaldehyde. Potassium hydroxide and ethanol were used as catalysts. The product was formed with the precipitation of a yellowish solid, which was filtered and washed with etanol. The heterocyclic chalcone were diluted in stock solution with common diluents to facilitate manipulation in experimental bioassays. On a balance, 0.1g (or 100 ?L) of the test substance was weighed, and then mixed with diluents (e.g. Tween 80, Tween 20) using a pestle until a homogeneous state is apparent. Then, water will be added slowly until a final working solution of 10 mL is obtained. This solution will be used to dilute lower concentrations for use in biological tests.
3. Molecular docking
Molecular Docking simulation was used to investigate the mechanism of the chalcones under study that contribute to the larvicidal effect through the binding affinity of the compound to Sterol Transport Protein-2 complexed with palmitic acid (PDB: 1PZ4), with a resolution of 1.35 Å. The 3D structure of the protein was obtained from the Protein Data Bank (PDB) (Bernstein et al., 1977).
Prior to the start of the computer simulation, the chemical structure of the compound was drawn in the Marvin Sketch Software, ChemAxon. And, the chemical structure was standardized in 3D and the energy of the compound was minimized by molecular mechanics methods and the semi-empirical Austin Model 1 (AM1) method using the Spartan 14 software, WaveFunction .
All water molecules were removed from the crystalline structure, then a “template” was created between the enzyme and the co-crystallized ligand with the purpose of demarcating the active site of the macromolecule. The procedure continued with the insertion of the test molecules, and finally the molecular docking simulation was carried out. The root mean square deviation (RMSD) was calculated from the poses, indicating the degree of reliability of the fit. The RMSD provides the connection mode close to the experimental structure and is considered successful if the value is less than 2.0 Å.
The Molegro Virtual Docker v.6.0.1 (MVD) software was used with the predefined parameters in the same software. The complexed ligand was used to define the active site. Then, the compound was inserted to analyze the stability of the system through the interactions identified with the active site of the enzyme, taking as reference the energetic value of the MolDock Score. The MolDock SE (Simplex Evolution) algorithm was used with the following parameters: A total of 10 runs with a maximum of 1,500 interactions using a population of 50 individuals, 2,000 minimization steps for each flexible residue, and 2,000 global minimization steps per simulation. The function MolDock Score (GRID) was used to calculate the energy values of fitting. A GRID was set at 0.3 Å, and the search sphere was set at 15 Å radius. To analyze the ligand energy, internal electrostatic interactions, internal hydrogen bonds, and sp2-sp2 torsions were evaluated.
4. Prediction model
The larvicidal potential of the chalcones under study was evaluated using the Molpredict X database (Scotti et al., 2022). The aforementioned prediction model was built using fingerprint-type RDKit descriptors, with compounds that have a pIC50 value ? 4.05 being considered active and present global validation determined by the Mathews Correlation Coefficient of 0.68, indicating a good prediction. The mentioned model presents experimental validation described in the research by Fernandes and collaborators (2019).
5. Bioassays
Bioassays with the mosquito Ae. aegypti (LAPAVET-SD) will be carried out at the Laboratory of Biotechnology Applied to Parasites and Vectors at the Biotechnology Center of the Federal University of Paraíba.
6. Larvae rearing and maintenance
The Laboratory of Biotechnology Applied to Parasites and Vectors (LAPAVET) possesses a cyclic colony of Ae. aegypti mosquitoes through insectary cages used for controlled space and ease of handling. The insectary cages are stored in B.O.D. (Biochemical Oxygen Demand) incubators configured to promote an inner environment of constant temperature (28 ± 1 ºC) and humidity (75 ± 5 ºC). Rearing and maintenance methods are applied so the colony is able to be perpetuated.
7. Larvicidal bioassay
L3 larvae of Aedes aegypti (n = 25) were collected from incubation trays and transferred to 15 mL falcon tubes with 10 mL of dechlorinated water. Then, the groups created were exposed to the established substance at determined concentrations of 5-100 ppm using a micropipette. For the negative control, the larvae were exposed to distilled water and Tween 80 and 20 (2%). The tubes were stored on racks and, after 24 and 48 hours of exposure, mortality were analyzed. The test were performed in duplicate.
8. Statistical analysis
The data were analyzed using GraphPad Prism software for Windows (GraphPad Software, San Diego, CA). Values were expressed as mean ± standard deviation. The results underwent ANOVA (analysis of variance) one-way or two-way, followed by Tukey’s post-test for multiple comparison procedure when needed. Differences will be considered significant when p < 0.05.
Results and discussion
1. Molecular docking
The heterocyclic chalcone under study was subjected to molecular docking simulations on the sterol transport protein-2 complexed with palmitic acid (PDB: 1PZ4). Results were generated using the MolDock Score scoring function. Values more negatives indicated a greater affinity for the target under study.
Before carrying out the molecular docking simulation, validation of the compound under study was continued through re-docking between the ligand and the co-crystallized protein. The value of the RMSLD (Root Mean Square Ligand Deviation), indicates the root-mean-square distance between the ligand atoms in the crystal structure and the corresponding atoms in the anchored pose. Observing the value (best score) of RMSLD is a good way to evaluate the ability of a method in find the binding mode of a ligand at a set of positions. For a docking to be considered reliable, the RMSLD value must be equal to or less than 2.0 Å. The RMSLD value for the co-crystallized ligand palmitic acid corresponded to 0.8037, indicating that the generated poses positioned the ligand correctly in the active site and that the program provided values considered satisfactory for docking validation.
The docking results regarding the binding energy of the heterocyclic chalcone derivative obtained for the sterol transport protein-2 complexed with the palmitic acid (PDB: 1PZ4) according to the MolDock Score scoring function was -117.151 kcal.mol-1. It can be observed that the heterocyclic chalcone presented a lower binding energy when compared to co-crystallized ligand (-116.139 kcal.mol-1), thus demonstrating a greater affinity to the target under study and corroborating the results demonstrated in the prediction model, which possibly indicates a high probability of larvicidal potential. In addition to evaluating the energy score, the chemical interactions established between the heterocyclic chalcone derivative and the sterol transport protein-2 complexed with palmitic acid (PDB: 1PZ4) were evaluated.
The interactions derived from heterocyclic chalcone with sterol transporter protein-2 complexed to palmitic acid (PDB: 1PZ4) are hydrophobic interactions and hydrogen bond interactions. Hydrophobic interactions were the most prevalent, being observed mainly in cyclic groups of the chemical structure of the compounds, particularly the thiophene ring. The interactions corresponded to: Ile 12 (1 interaction), Leu 102 (1 interaction), Leu 109 (1 interaction), Leu 48 (1 interaction), Ile 19 (1 interaction), Arg 24 (1 interaction), Val 26 (1 interaction), and Phe 105 (1 interaction).
Only one hydrogen bond interaction was observed, corresponding to Val 26. It is worth highlighting that the residues Leu 102 and Arg 24 are key residues in maintaining the hydrophobic chain of the protein under study. The co-crystallized ligand palmitic acid showed steric interactions, hydrophobic interactions, and hydrogen bonding interactions. The ester group has steric interactions through residue Arg 24 (1 interaction) and hydrogen bond-type interactions through residues Gln 25 (1 interaction) and Val 26 (1 interaction).
The long hydrocarbon chain of the fatty acid was responsible for establishing the other interactions that corresponded to residues Val 26 (1 interaction), Leu 48 (1 interaction), Phe 105 (1 interaction), Arg 15 (1 interaction), Ile 106 (1 interaction), Leu 102 (1 interaction), Leu 109 (1 interaction), and Ile 12 (1 interaction), these being hydrophobic interactions and the exclusion of Gly 75 from interactions of the interaction type.
2. Prediction model
The prediction model developed against compounds with larvicidal activity already reported and experimentally validated (Scotti et al., 2022) demonstrated that the heterocyclic chalcone under study presented an activity probability value corresponding to 1, which indicates a high probability of possible larvicidal activity, as compounds are classified on a scale ranging from 0 to 1.
3. Larvicidal bioassay
In the results arising from the larvicidal bioassays, we were able to observe that in the 24-hour period of exposure of the larvae to the designated compound, there was a higher mortality rate at the concentration of 100 ppm in comparison to all other tested concentrations. During the 48-hour exposure period, we observed a 32% increase in mortality rate at a concentration of 10 ppm and 24% at a concentration of 5 ppm. In this sense, we can assess that, as the exposure duration of the larvae to the given substance increases, there is a substantial rise in organism mortality. This phenomenon may be attributed to a mechanism of action involving palmitic acid, as previously indicated in the molecular docking study, which assessed the binding of the sterol transport protein-2 complexed with palmitic acid (PDB: 1PZ4) with the heterocyclic chalcone. The protein acts together with the palmitic acid in the transport and metabolism of fatty acids (Dyer et al., 2003). As it is a saturated fatty acid, palmitic acid participates in lipid metabolism pathways to generate Acetyl-CoA and ATP (adenosine triphosphate) (Dyer et al., 2003). The interference of heterocyclic chalcone in this process could justify the increase in larval mortality rate when exposed to the substance. Hence, for further insights, an optimal approach would involve a combination of future studies to elucidate whether the molecule can indeed disrupt the mechanism involving palmitic acid or if an alternative pathway is established during the interaction process.
Conclusion
In conclusion, it is evident that the heterocyclic chalcone exhibits larvicidal activity at the various concentrations tested. Furthermore, its binding energy when compared to the co-crystallized compound is lower. Similarities were observed between the residues established by the heterocyclic chalcone and the co-crystallized ligand that corresponded to the hydrophobic interactions of the residues: Leu 109, Leu 102, Ile 12, Leu 48, Val 26, and Phe 105, thus indicating that the compounds may share the active site. Finally, the combination of future studies is important to understand the role of the molecule in the organisms tested and the mechanisms involved with the co-crystallized ligand and the transporter protein. This is vital for mitigating potential risks arising from vector resistance and enhancing vector control.
Acknowledgment
I would like to thank the Federal University of Paraíba (UFPB), the Laboratory of Biotechnology Applied to Parasites and Vectors (LAPAVET), the Research Support Foundation of the State of Paraíba (FAPESQ-PB), and the UFPB Institute for Development of Paraíba (IDEP- PB) for their support and funding, which made this research possible.
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