2.2.3. Pharmacokinetic and Toxicological Properties
Medicinal chemistry has as main aim the development of drugs for human beings, however, our approach was to find, using ligand-based virtual screening, a novel compound that shows higher potential larvicidal than temephos. Our pharmacokinetic selection was designed to determine a molecule with physicochemical properties equal to or better than thse of the temephos molecule. According to Burt [
17], the mechanism of action of insecticides has two main steps to achieve a concentration that affects the central nervous system (CNS) of the insect. In the first stage the cuticle is divided into a biphasic system, comprising an external phase with lipophilic elements, and an internal phase of associated hydrophilic elements, and another pathway is hemolymph, which displays low penetration, so a alternative strategy used is via high penetration of the integument of the wall of the body, such as the trachea, becoming an important pathway for the CNS [
18].
It has been observed that insecticides of the organophosphate class, like temephos, require a solvent to ensure better absorption in the body cavity of the insect [
18]. Webb and Green [
19], Hurst [
20] and Läuger, Martin and Müller [
21] have shown that insecticides whose molecules contain both an oil soluble group and a water soluble group, by orienting themselves suitably at an oil/water partition system, pass rapidly through the cuticle of the insect by diffusion along this interface. According to Matthews [
22], the three major neurotransmitters found in both males and females are acetylcholine, glutamate and GABA. An ideal designed insecticide would be extremely toxic to
Aedes aegypti, either by inhibiting acetylcholinesterase or juvenile hormones, but not to other species in the environment, although the toxicity will depend on the physiological and biochemical differences of the organisms to which they are targeted [
5]. The pharmacokinetic properties that characterize the parameters required for a good insecticidal action are described in
Table 1.
The parameters used for screening were established in the QikProp environment, and thus previously selected for using the Derek software, for searching molecules in the database with potential biocidal effect, in particular for
Aedes aegypti, and not active against any other organism or the environment. Parameters were used to evaluate if the molecule can overcome the barriers and reach the CNS (Central Nervous System), so that the “star” parameter or drug-like properties were analyzed, which are compared with 95% commercial and common drugs [
23]; MW, corresponding to the molecular weight of the molecule in (g·mol
−1), ClogP/w, which corresponds to the octanol and water partition coefficient; HBD means hydrogen bonds that would be donated to the water solvent; HBA means the number of hydrogen bonds that would be accepted from a solvent [
24,
25,
26,
27,
28]. The temephos molecule and those selected from the database were within the recommended values regarding the drug-like as well as the physicochemical properties, in order to show potential CNS action.
Another parameter available in QikProp is the biological prediction that shows the same focus of the study, i.e., the passage of the molecule to reach the enzymes under study: BB log, which establishes if the drug can overcome the blood/brain barrier, and MDCK, apparent cellular permeability, in nm/s, of two Madin-Darby canine kidney cell lines. MDCK cells are considered a good model for the blood-brain barrier [
29,
30]; Lipinski’s Rule of 5 predicts the most probable absorption and permeation rates [
31]. Following our hieratic ligand-based virtual screening, 11 compounds were selected according to the criteria and parameters mentioned above. According to Voutchkova et al. [
29], who used some QikProp parameters based on drugs that had not been evaluated in the EPA Toxic Release Inventory (TRI) reference parameters, correlating the toxic effect with recommended values of commercial drugs, a selection was performed in the present work. The temephos molecule showed the expected values for the toxic molecule and also showed a high value as recommended by TRI, confirming its high toxicity, and our molecules are also within the recommended TRI standard, as observed in
Table 1.
The screening parameters were established through the QikProp program that facilitated the filtering in the DEREK software, searching for database molecules with potential biocidal effects and no toxic action in any other organism or the environment as well. The temephos molecule in the DEREK program, obtained as prediction cholinesterase inhibition, hepatotoxicity and skin sensitization. None of the 11 molecules analyzed showed any kind of plausible toxic alert.
According to the WHO [
28], temephos can be used to control mosquitoes in potable water, but should not exceed an amount of 1 mg/L. Temephos was tested for toxicity and mutagenicity against
Escherichia coli and
Salmonella typhimurium, and at concentrations above 3.33 μM it was mutagenic and genotoxic, with and without metabolic activation [
32]. In a study by Lee with fish of the species
Fundulus heteroclitus, it was found that temephos was the compound that presented the highest acute toxicity among the compounds analyzed at a concentration of 0.04 mg/L [
33]. A toxicity study also conducted on survival and subsequent emergence of
Arnitus hesperidurn and
Prospaltella opulenta found no toxic effect of the 14 insecticides evaluated along with temephos, but correlated that a high solubility of compounds above 2000 ppm in water directly influences the life of the species analyzed [
34].
A direct relationship between the degree of AChE inhibition and toxicity might not always be expected [
35]. A study was conducted to determine the median lethal concentration of an emulsifiable formulation of temephos as well as the response of BChE and AChE enzymes and to study their effects on cholinesterase behavior and activity in green frog tadpoles (
Rana clamitans). A concentration of 10 μL/L caused death of all the subjects, but there was no favorable expression in respect to the enzymes [
36]. A more complete study carried out by Junges [
37] investigated the lethal and sublethal effects of three insecticides (temephos,
Bacillus thuringiensis var. Israelensis and permethrin) on amphibians (
Rhinella arenarum, Rhinella fernandezae and
Physalaemus albonotatus) and temephos was the second most toxic and all insecticides were observed to produce behavioral changes in tadpoles independent of the dose-response. The toxic effects of temephos in cichlid fish (
Tilapia melanopleum) and dragonfly larvae (Odanata)
Neurocordulia virginiensis, which established a estimated safe concentration for the fish of 3.0 mg/L, were analyzed in the renewal toxicity test, giving a results of 0.2 mg/L for insect larvae [
38].
Despite many studies about toxicity of temephos, the degree of expected adverse effects on living beings as well as the environment remains inconclusive. For the ligand-based virtual screening approach here used for selection of molecules with potential activity in the CNS of Aedes aegypti, molecular docking for only the best-ranked and selected 11 molecules was performed, in order to complete our study with the design of molecules that could minimize or cause no affects to other species or the environment, but with specific biocidal action in inhibiting the acetylcholinesterase and juvenile insect hormone enzymes.
2.2.4. Molecular Docking Procedures
Molecular docking was performed using a validation protocol in order to determine the pose (conformation + orientation) of the ligands (I40, GNT and JHIII) inside the enzyme active sites (PDB IDs 1QON, 4EY6 and 5V13, respectively, to the mentioned ligands order). The mean square root deviation (RMSD) between the reference ligands and the experimental ligands of I40 (0.85 Å), GNT (0.34 Å) and JH31 (1.33 Å) were calculated. The RMSD analysis was determined by considering the most adequate position of the initial structure around the X-ray crystallographic complex, analyzing experimentally the most stable position so that there is no structural change between the proteins and the reference ligand when complexed with the macromolecule under study. The best molecular fit is determined by an RMSD less than or equal to 1.5 [
39,
40,
41], as shown in
Figure 2.
According to Harel [
42], a study with a potent inhibitor of the enzyme acetylcholinesterase called I40 for
Drosophila melanogaster (PDB ID 1QON) was conducted, noting that interactions occur mainly between Trp-83 and, Trp-472, Phe-330, Tyr-71, around of the α-helix between the amino acid residues Tyr-370 and Tyr-374. He also specified that the active site modifications occur in nine residues in the throat of the active site (Tyr-71, Trp-83, Tyr-324, Phe-330, Tyr-370, Phe-371, Tyr-374, Trp-472 and His-480).
The active site of the human AChE has an extension of 20 Å, with a catalytic active site (CAS) and a peripheral anionic site (PAS). Since the functionality of AChE’s active site depends on the specificity of the catalytic triad of amino acid residues Ser-203, His-447 and Glu-334, it involves reactions of substrates and catalyze the hydrolysis of acetylcholine in acetic acid and choline, by an oxyanion orifice consisting of Gly-121, Gly-122 and Ala-204. In the anionic subsite, site-directed mutagenesis indicates that the aromatic residues Trp-86, Trp-9, Trp-10, Glu-202 cationic moiety is often attributed to being electrostatic and Phe-337 plays an important role in terminal trimethylammonium attachment [
43,
44,
45].
The first step to initiate molecular docking is to perform binding affinity analysis of all the ligands deposited in the PDB and the ligands under study, i.e., the acetylcholinesterase inhibitors of the insect Drosophila melanogaster, fruit fly (PDB ID 1QON, I40), human acetylcholinesterase (PDB ID 4EY6, GNT) and juvenile homone (PDB 5V13, JHIII), for interactions that show higher binding affinity than the specific ligand (I40, GNT), but for JHIII the L46, L66 and L68 ligands results were better than for the enzyme refiner ligand.
After analysis of the 11 compounds submitted to docking, only the ligands L46, L66 and L68 showed higher values than those observed for temephos, our template. The ligands did not show any of the characteristic features of the temephos molecule, such as the phosphorus atom, with a double bond to the sulfur. However, molecules L46, L66 and L68 demonstrated excellent binding affinity and interaction with the enzyme acetylcholinesterase, higher than that found for the temephos molecule. Validations were used as parameters for molecules that are complexed in the PDB, which do not present such groups and have effectiveness in the inhibition of the enzyme acetylcholinesterase according to Srivastava et al. [
46], who performed design and development of some phenyl benzoxazole derivatives as potent acetylcholinesterase (AChE) inhibitors by in vivo and ex vivo analyses, and revealed the true nature and competitive type of the AChE inhibition among their analyzed molecules, even though they did not have a phosphorus atom and sulfur neither.
The binding affinity indicates a strong bond when it is the lowest, signaling that designed ligands will have excellent interaction with the receptor [
47,
48]. The binding affinity of the analyzed molecules in the macromolecule PDB ID 1QON of the fruit fly did not present values higher than I40 (−12.71 kcal/mol), but all the ligands evaluated presented values adapted
p <0,0206, in respect to temephos (−8.11 kcal/mol), which showed a difference of −4.92, −5.72, −2.42, L46, L68 and L66, respectively. Compound L66 (−10.68 Kcal/mol) had a value closer to I40 with a significant difference of only 2.4, followed by L46 (−10.96 kcal/mol), with a significant difference of 3.2, according to
Figure 3.
For human acetylcholinesterase (PDB ID 4EY6) the ligands showed high binding affinity when compared to the controls used in the molecular docking study (temephos), values that approximate the GNT binding affinity values (−9.72 kcal/mol), whereas the ligands show −6.25 kcal/mol, ligand 46 shows −6.05 Kcal/mol and temephos shows −3.18 kcal/mol, each one showing a difference of 1.65, 2.6, 2.8 and 5.63. Compound L68 shows −7.2 Kcal/mol, a value close to the found for GNT, according to
Figure 4.
In order to carry out the molecular docking with PDV 5V13, the ligands L46, L66 and L68 were used, even though the ligands had values higher than temephos or JHIII. The results of the affinity values can be observed in
Figure 5.
Binding affinity of the compounds here investigated have shown values higher than those observed for JHIII (−8.53 kcal/mol) and temephos (−6.6 kcal/mol), whereas L46 ligands (−9.2 kcal/mol), L66 (−10.96 kcal/mol) and L68 (−8.16 Kcal/mol) presented adjusted values (p <0.0001), in respect to the two molecules. Compound L66 shows a value above that found for the juvenile hormone ligand (JHIII), with a significant difference of −2.43, followed by L46, with a significant difference of −0.67, and there was no significant difference in respect to the L68 molecule, however all the ligands had equal or higher binding affinity than to JHIII and temephos.
Results found after the molecular docking was in accordance with the reported by Harel [
42], where the PDB ID 1QON macromolecule and I40, L46, L66 and L68 ligands showed similar interactions between all ligands and the β-sheet containing Trp-83 and the α-helix between amino acid residues Tyr-370, Tyr-374 (except L46), Tyr-71 (except L66), confirming our selection for a potential inhibitor of the insect acetylcholinesterase enzyme. Compounds L46 and L66 showed the same interactions observed for compound I40, indicating strong interaction with the acetylcholinesterase catalytic site, around the helix located between amino acid residues Trp-83, Tyr-370, Tyr-71, Phe-371, see
Figure 6.
Organophosphates are insecticides that have as mechanism of action the ability to irreversibly inhibit the enzyme acetylcholinesterase, which acts directly in the post-ganglionic synaptic cleft, and has as a function the elimination and reuptake of the neurotransmitter acetylcholine, so that there is no excess of nerve impulse and cell death. In addition many substrates are hydrolyzed through a nucleophilic attack generating an acyl enzyme or a phosphoryl-enzyme intermediate and then deacylation or dephosphorylation, becoming an irreversible reaction [
35,
49].
Compounds L46, L66 have interactions with Ser-203 and L66 and L68 with His-447, showing that our ligands were able to interact only with two amino acids of the AChE catalytic triad, residues Ser-203, His-447, but did not interact with Glu-334. However, Glu-202 appeared in our study, which is also part of the amino acid residues with modification in the active site enzyme, analyzing interaction with ligands L66 and L68 and also with the galatamine refining ligand. The main mechanism of inhibition involving the amino acid residue Ser-203, in which it covalently binds between the central phosphorus side chain [
50]
According to Han et al. [
45] who performed a study of potent inhibitors of the enzyme acetylcholinesterase analyzing nine residues (Tyr-72, Tyr-124, Tyr-341, Tyr-286, Phe-295, Phe-297, Trp-236, Tyr-337 and Phe-338) with potential modifications in the enzyme and possible inhibition, L66 ligands showed interactions with Tyr-124, Phe-338, L46 interacted with Trp-236 and all ligands under study demonstrated interaction with Tyr-337.
Interactions were observed for compounds L46, L66 and L68 at the catalytic site of the enzyme, confirming the binding affinity values, and thus achieving a better inactivation of the enzyme and allowing them to be considered as potential inhibitors of the enzyme acetylcholinesterase with insecticidal action as well, even presenting a lower binding affinity value, see
Figure 7.
The amino acid residues of the juvenile hormone protein interacting with JHIII in
Aedes aegypti were described by Kim et al. [
51], who determined that the epoxy group forms a hydrogen bond with the phenolic hydroxyl of Try-129, and we can observe that the ligand L66 shows this interaction and the rest of the isoprenoid chain is surrounded by hydrophobic side chains including those of Phe-144, Try-64, Trp-53, Val-65, Val-68, Leu-72, Leu-74, Val-51 and Tyr-33. The L66 ligand did not only show an interaction with Leu-72, but could also be an excellent modulator of the juvenile hormone protein in its active site, followed by the ligands L46 and L68 which have shown interactions with Try-64, Trp-53, Val-53, Val-65, Val-68, whereas interactions with Val-51 and Try-33 were found only on L46, with protein modification, see
Figure 8.
The juvenile hormones, together with the 20E, are pleiotropic molecules produced by the corpus allatum, that need to maintain their homestasi. As an example, the juvenile hormone must maintain a certain index in the hemolymph, so that the insect displays adequate growth during the phases of its development. The hormones determine the development of the insect, acting mainly in its growth process, controlling critical physiological events, performing repairs, being an important insect metamorphosis manager, and play a role in reproductive maturation (ovary) in adults [
6,
52].
The L66 molecule show higher binding affinity than the L68 molecule, with several residues reported in the literature that may cause modification in the active site as a consequence of the enzymatic inhibition in the active site, for which the docking here performed show that the interactions mainly occur with Trp-83, Try-370, Phe-372 and Try-71—the same interactions encapsulated in I40—the ligand complexed in the PDQ ID 1QON. On the other hand, for the molecule L68, which shows a lower binding affinity and with only Try-370 and Try-374, similar to I40, the bond-free energy is very low, −1.33, which shows that factors such as the number of uncharged hydrogen bonds, the size of the polar or non-polar surface portions, the number of rotational bonds or the enthalpy required to desolvate the ligand or protein, are physicochemical parameters that may characterize a low free energy value [
53]-see
Table 2.
The molecules L46 and L66 form π-alkyl type interactions with Trp-83, similar to those that occur with the reference molecule - I40. The molecule L66 shows a very low free energy (−3.88 kcal/mol), but the interactions with Ser-203, at a distance of 2.93 Å, through a π-donor type bond with His-447, make interactions of π-π-stacked type with the Tyr residue, forming a π-alkyl type interaction similar to those occurring with GNT (see
Table 3).
The L66 molecule shows a low ΔG (−3.88 Kcal/mol), however, it interacts with Ser-203, Glu-202, His-447, Tyr-124 and Tyr-337, mainly via π-alkyl bonds, with a mean distance of 3.45 A, and its Ki is 1.42 nM (see
Table 4). With increasing temperature, the hydrogen bonding fuses and each interacting -CH contributes with about −600 cal/mol to the stability (ΔG) of the complex. The Van der Waals energy type bonds show a lower value for electrostatic energy because of the detailed interactions of the atoms with each residue of the protein-activator. In addition, the increased hydrophobicity of the amino acid residues and the loss of α-helix content (from 63.57 to 51, 83%) in presence of hydrogen bond acceptors, reveals their motif [
54,
55,
56].
