2.1. Pharmacokinetic and Toxicological Properties
The pharmacokinetic properties are described in Table 1
for 14 molecules selected from virtual screening using the Rapid Overlay of Chemical Structures method implemented in the ROCS software followed by electrostatic similarity calculation using the EON software. The “star” parameter means the number of property values or descriptors that are outside the 95% range of similar values for known drugs. A large number of “stars” suggest that a molecule is less drug-like than molecules containing few stars.
After thorough evaluation of Absorption, Distribution, Metabolism, and Excretion (ADME) parameters, such as MW (molecular weight), QP logKp (predictable permeability of the skin), HB donor (estimated number of hydrogen bonds that would be donated by the solute to water molecules in aqueous solution), HB acceptor (estimated number of hydrogen bonds that would be accepted by solute molecules of water in aqueous solution); according to rule of five, all the compounds satisfied the conditions [22
]. All molecules tested in the present study exhibit hydrogen bonding and also display hydrophobic interactions with corresponding amino acids, according to the molecular docking simulations performed here (see Section 2.3
). All the molecules investigated here did not present any violation, except for the template compound (pyriproxyfen), but they are in accordance with Lipinski’s rule of five, and only five molecules violated Jorgensen’s rule of three, which is in accordance with studies carried out by Gaddaguti et al. (2016) [25
]. Compounds with less, or preferably no violations, of these rules are more likely to be administered/available by the oral route [25
The toxicological properties of the molecules with toxicity alarms are shown in Table 2
. It was noted that the pyriproxyfen in the toxicity analysis did not indicate any alert, a fact that can be justified considering the low concentration in which it acts in the active site [25
Analysis of the toxicological properties allowed the observation that of the 14 molecules, nine presented toxicity alerts characterized as plausible or acceptable and five (ZINC13537284, ZINC00001021, ZINC01530718, ZINC00000257, and ZINC00001624) did not present any type of alert.
Hepatotoxicity of steroid hormones describes the pathological conditions associated with the administration of these compounds and includes intrahepatic cholestasis, vascular disorders, and neoplasms [27
]. The mechanism of cholestasis induced by steroids is believed to include a measure of intrinsic toxicity. The structural similarity with endogenous bile acids led to the suggestion that competition with bile acid transport could contribute to the observed effect [28
The skin sensitizing activity results from the phenyl acylation of skin protecting esters, following the nucleophilic attack of skin proteins on the carbonyl of the ester group [30
]. Activity for such compounds has been demonstrated in various skin sensitization assays, including the guinea pig maximization test [32
] and adjuvant single injection tests. Skin sensitization in humans has also been described [33
Related evidence has been extensively reviewed for the binding of 17-β-estradiol to the estrogen receptor [34
]. The receptor seems to involve the ligand and, as a result, all four rings of the steroid nucleus contribute significantly to the binding. In addition, the hydrogen bond between the receptor and the phenolic and β-hydroxyl groups also plays a role. The binding is generally reduced by the introduction of polar substituents into the structure, while hydrophobic groups are tolerated at various positions subject to steric constraints. In vitro receptor binding assays suggest that the phenolic hydroxyl group is more important than the 17-β-hydroxyl group in terms of estrogen receptor binding affinity [35
The alert describes the teratogenicity of 17-β-estradiol and its analogs. These compounds are potential ligands for the estrogen receptor (ER) and may cause birth defects as a result of their interaction with that receptor. In the present study, 17beta-estradiol [36
], ethinyl estradiol [38
], and dipropionate of estradiol [36
] produced malformation in the reproductive organs of both male and female offspring when administered orally or subcutaneously to the mother during the second half of gestation (including the period of sexual differentiation).
The alert describes the genotoxicity of alkylating agents wherein the carbon containing the functional group is a primary or secondary alkyl carbon atom. In addition to the alkyl halides, it also includes the alkyl, sulphonated, and sulfonated sulfonates which lack a hydroxyl group directly attached to the sulfur [39
]. Alkyl halides are electrophilic species capable of directly alkylating the DNA. Therefore, many compounds are mutagenic in the Ames test in the presence and absence of the S9 mixture, particularly Salmonella typhimurium
in strains TA100 and TA1535 [40
The in vitro prediction of inhibition of hERG channels is one of the toxicological factors that are related to side effects that new drug candidates may present. The hERG channels belong to the Shaker family, a subtype of gene that belongs to a subunit of potassium channels that are voltage controlled. Its inhibition or alteration causes prolongation of the ventricular repolarization phase of the heart and may still cause cardiac arrhythmias [43
2.3. Molecular Docking Study
In order to validate the molecular docking method used here, the compounds with the crystallographic information were subjected to the development of docking until the spatial conformation was found using AutoDock 4.2/Vina 1.1.2 software, via graphical interface PyRx by comparison with the original crystallographic structure of the acetylcholinesterase (AChE) inhibitors (PDB IDs 1QON and 4EY6) and the juvenile hormone III structure (PDB ID 5V13).
Retrieving the pose of each AChE inhibitor (I40, GNT, and JHIII), it was possible to perform validation of the molecular docking protocols used here, calculating root-mean-square deviations (RMSD) of 0.82, 0.37, and 1.27 Å, respectively. According to Gowtham et al. (2008) [46
] and Hevener et al. (2009) [47
], the binding mode predicted using docking indicates that when the RMSD is less than 2.0 Å regarding the crystallographic pose of a respective ligand, validation is considered satisfactory. The best results can be seen in Figure 3
The molecular docking method used here identified a conformation that allows the ligand to also bind the residues of the I40-active sites (PDB ID 1QON) around the α-helix between amino acid residues Tyr370–Tyr374 and around the β-sheet between amino acid residues Ile82–Thr85 and Val478–His480. For the ligand, it is possible to see hydrogen bonds in common with residues Tyr370 and His480. There was also a hydrophobic interaction with residues Tyr71, Trp83, Tyr370, Phe371, and Leu479, corroborating the studies of Harel et al. (2000) [48
Residues of the GNT-active sites (PDB 4EY6) were located around the α-helix between amino acid residues 336–338 and on β-sheet between the amino acid residues 85–87, 121–124, and 202–203. For the ligand it was possible to see hydrogen bonds in common with the residues Tyr124, Glu202, and Ser203. There were also hydrophobic interactions with residues Trp86, Gly121, and Tyr337, as identified in studies conducted by Gaddaguti et al. (2016) [25
Interactions with the JHIII site (PDB 5V13) were located around the α-helix between the amino acid residues Ser30-Ala38, Arg45-Glu51, Val60-Gln71, Phe123-Leu130, Val132-Arg136, Leu138-Arg143, and Val280-Trp286 for the β-sheet between the amino acid residues Pro52-Pro55, Tyr72-Val73, Thr144-Val145, and Arg276-Gln279. For the ligand, it was possible to observe hydrophobic type interactions with all amino acid residues, according to studies carried out by Olmstead et al. (2003) [20
In order to evaluate if the interactions had a higher binding affinity than the specific ligand (I40, GNT, and JHIII) for acetylcholinesterase from different organisms (Drosophila melanogaster
and Homo sapiens
organism) and mosquito juvenile hormone (Aedes aegypti
organism), it was observed that of the five compounds submitted to docking, only two presented values higher than or equal to the negative controls used here. Compound ZINC00001624 has a binding affinity of −10.5 kcal/mol, followed by ZINC00001021 with −9.2 kcal/mol compared to the controls I40 and pyriproxyfen (PDB ID = 1QON, Drosophila melanogaster
organism), according to Figure 4
The I40 exhibited a binding affinity of −13.1 kcal/mol higher than pyriproxyfen of −8.9 kcal/mol. However, the compound ZINC0001624 showed a binding affinity value of −10.5 kcal/mol higher than the controls used in molecular docking. Thus, by comparing the compound ZINC0001624 to the I40 control, a difference of ±2.6 kcal/mol was observed, whereas a variation of ±1.6 to ±1.3 kcal/mol was observed for the others, as shown in Figure 4
In the human h
AChE, the inhibitors showed higher binding affinity and free energy values compared to the pyriproxyfen used in the molecular docking procedure performed here. These values corroborate the obtained similarity in the amino acid residue sequence in which the compound ZINC00001624 showed a high affinity value of −10.3 kcal/mol, followed by ZINC00001021 with −9.9 kcal/mol, according to Figure 5
GNT exhibited a binding affinity of −9.9 kcal/mol and pyriproxyfen, of −9.1 kcal/mol. However, compound ZINC0001624 has a binding affinity value of −10.3 kcal/mol, hence higher than the obtained for the controls docked here. Thus, by comparing the compound ZINC0001624 to the GNT control, a difference of ±0.4 kcal/mol was observed, whereas for the others, a variation of ±0.6 to ±0.4 kcal/mol was observed, as shown in Figure 5
We observed that of the five compounds submitted to the molecular docking studies, only two show values higher than or equal to the controls used. Regarding the JHIII complex (Aedes aegypti
organism), the compound ZINC0001021 and ZINC00001624 presented higher value than the controls used (JHIII and pyriproxyfen), with values of −11.4 and −10.4 Kcal/mol, respectively. Results of the affinity values can be observed according to Figure 6
. JHIII showed binding affinity of −8.9 kcal/mol, i.e., lower than pyriproxyfen (of −10.3 Kcal/mol). However, the compound ZINC0001021 shows a binding affinity value of −11.4 kcal/mol, i.e., higher than the controls used in molecular docking. Therefore, by comparing the ZINC0001021 compound to the JHIII control, a difference of ±2.5 Kcal/mol was observed, and the others a variation of ±1.0 to ±1.1 Kcal/mol.
With these data, we propose that the compounds are capable of binding to active sites. However, the compound ZINC0001624 has higher affinity to the active site of the human acetylcholinesterase, whereas ZINC0001021 has higher binding affinity to the active site of the mosquito juvenile hormone-binding protein. Compound ZINC00001021 shows similar interactions to the active site of acetylcholinesterase for I40 around the α-helix between amino acid residues Tyr370–Tyr374 and β-sheet with amino acid residues Trp83 and Leu479. The binding affinity value similar to the observed for the control shows that such a compound is a potential insecticide.
The interactions individually observed after docking of compound ZINC0001021 and ZINC0001624 had similar interactions with I40 regarding the acetylcholinesterase active site, located around the α-helix between amino acid residues Tyr370 and β-sheet with amino acid residues Trp83, Leu479, and Gly481, as can be seen in Figure 7
Compounds with potential insecticidal activity may irreversibly inhibit the production of acetylcholinesterase; this enzyme is responsible for the hydrolysis of acetylcholine (ACh) that terminates the nerve impulse. Inhibition of the acetylcholinesterase enzyme is particularly the initial mechanism for a substance to be considered to have potential insecticide in the larval phase, considering the knowledge cited by several authors [49
]. It is essential to observe interactions formed inside the active site of the acetylcholinesterase, in which three important characteristics are present in order to know the mechanism of elucidation of biological action of the enzyme production.
The interactions obtained after molecular docking of the compounds with the amino acid residues Trp71, Trp83, Tyr370, Phe371, and His480 of acetylcholinesterase are similar to those reported in the literature [53
]. In the compound ZINC0001624, the most significant contributions of the interactions were observed with the docking study performed here, where the contribution of the residues Trp83, Tyr370, and Gly481 to the increase of the binding affinity could thus inactivate the enzyme acetylcholinesterase by competition with the I40 for the AChE active site.
In considering the active site of acetylcholinesterase that binds GNT around the α-helix between amino acid residues Tyr337 and β-sheet with amino acid residues Trp86 and Tyr124, the compound ZINC00001021 had similar interactions. The binding affinity value obtained was similar to the observed for galantamine, pointing out that such a compound is a potential insecticide.
Compounds ZINC00001021 and ZINC00001624 had similar interactions to the observed between the acetylcholinesterase active site and GNT, i.e., around the α-helix between amino acid residues Tyr337 and β-sheet with amino acid residues Trp86, Tyr124, Ser125, Ser203, Tyr341, and His447, such as can be seen in Figure 8
According to Meriç [55
], in the AChE active site the catalytic triad (Ser203, Glu334, and His447) is located in the lower portion of the active site, surrounded by three important aspects for the catalytic activity: the acyl pocket (residues Phe295, Phe297, and Phe338), the oxy-anion channel (main residue nitrogen Gly121, Gly122, and Ala204) and the choline binding site (Trp86 and Tyr337). For the compound ZINC00001624, the most significant contributions of the interactions were observed in the docking study, where the contribution of the catalytic triad (represented by Ser203 and His447) and choline binding (Trp86 and Tyr337) for the increase of the binding affinity was observed, thus inactivating the enzyme acetylcholinesterase by competition with the active site with the GNT.
The individual interactions observed performing docking of molecules ZINC00001021 and ZINC00001624 were similar to the observed for JHIII inside the active site of the juvenile hormone, i.e., around the α-helix between amino acid residues Val68, Typ129, and Phe144 and in β-sheet with Val51, Trp53, and Pro55, as shown in Figure 9
The growth, development, metamorphosis, and reproduction of insects are under control of juvenile and ecdysteroid hormones, or molting hormones, secreted by specific endocrine glands, corpora allata, and prothoracic glands. The receptors of these two large groups of insect hormones have become targets for neurotoxic pesticides and insecticides. The development of these “biorational” insecticides, such as methoprene and tebufenozide, are based on classical bioassays that measure the agonist activity of these hormones [56
The crystal structure of the mosquito hormone binding protein (mJHB) bound to JHIII was determined by molecular substitution using an N-terminal polyalanine model of the N-terminal domain of a D7 (salivary odoriferous proteins) [57
]. The N-terminal domain of the long D7s binds to vertebrate eicosanoid mediators in Aedes
. In Aedes
sp., the C-terminal domain binds biogenic amines including serotonin and histamine, while the C-terminal domain of Anopheles
does not appear to have a small molecule binding site [19
Proteins of this type would bind to small hydrophobic molecules that act on essential physiological processes. In studies, a D7-like protein in the hemolymph of Ae. aegypti
is a ligand-specific JH binding protein. The crystallographic structure of the JHIII-protein complex reveals a single binding site, and this causes the protein to undergo a conformational change in the hormone load that stabilizes the ligand in the binding pocket [19
A single well-ordered molecule of JHIII (refined occupations 0.91–0.96) was present in the N-terminal domain binding pocket of mJHB. Three molecules of the complex were present in the asymmetric unit of the crystal, and the conformation of the linker was essentially identical in all three [19
]. The epoxy of JHIII was located in the center of the domain, while the methyl ester group of the hormone was oriented towards its surface. The epoxy group forms a hydrogen bond with the phenolic hydroxyl of Tyr-129, and the remainder of the isoprenoid chain was surrounded by hydrophobic side chains, including Phe144, Tyr64, Trp53, Val65, Val68, Leu72, Leu74, Val51, and Tyr33.
Molecule ZINC00001021 (Z21) showed significant contributions to the binding affinities (−11.4 kcal/mol) calculated using molecular docking, because it conforms to the active site, in which they are represented by amino acids surrounded by hydrophobic side chains including Try33, Val51, Trp53, Val65, and Val68.
Quantitative data on residues, distances, type, and free binding energies (ΔG) between the promising compounds and the insect/human acetylcholinesterase and juvenile hormone enzyme can be seen in Tables S1–S3 (see Supplementary Materials)
. It is possible to verify that the reference molecules (I40, GNT, and JHIII ligand) and the other ligands had an increase in the number of interactions that resulted in the decrease of the free binding energy, indicating a higher degree of spontaneity of the interactions. We have observed that interactions with the residues Trp83, Tyr370, Tyr374, and Leu479 are common and most recurrent for all the compounds used here for molecular docking. Interaction with the Trp83 and Tyr370 residue is also common in most molecules, however less recurrent than Phe371, Gly481, and His480, as shown in Table S1, Supplementary Materials
. These results make it possible to infer that these two residues play a key role in the potential insecticidal activity.
Regarding to the molecule ZINC0001624 (Z24), with higher free energy (−12.00 kcal/mol), hydrogen bonding interactions occur with His480, as well as pi-alkyl with Trp83, and they are similar to those that occur with I40, indicating that such a compound has a potential insecticidal action, as well as it could interact more effectively with the enzyme active site. The most promising molecule ZINC0001624 shows good results by the docking analysis, because it has a high value of free energy, which contributes to its greater stability when interacting with the active site of the insect acetylcholinesterase.
Interactions with residues Trp86, Tyr124, Tyr337, and His447 are common and most recurrent in all compounds investigated here. The interaction with the Ser125 residue is also common in most molecules, however, less recurrent than Tyr133 and Ser203.
Molecule ZINC0001624 (Z24
) shows free energy of −10.40 kcal/mol, hydrogen bonding interactions with Ser203, as well as pi-alkyl interactions with Trp86 in a similar way to those observed for GNT, indicating that such a molecule has a potential insecticidal action, for inhibiting the production of the enzyme acetylcholinesterase, as shown in Table S2, Supplementary Materials
. The most promising molecule is ZINC0001624 because it shows a free energy value (ΔG) higher than the observed for GNT, molecules complexed with acetylcholinesterase, and used here in the molecular docking.
Binding affinity and ΔG values for the insect and human acetylcholinesterase enzymes were matched, since there was homology and high sequence similarity shared among Ae. aegypti
, D. melanogaster
, and H. sapiens
. Inhibitor selectivity was due to Tyr71, Tyr73, Glu80, and Asp375, which in vertebrates are Asp, Gln, Ser, and Gly, according to studies of Harel et al. [48
Significant differences in binding affinity values and ΔG were due to a 50% reduction in the active site cleft of the insect acetylcholinesterase, compared to the human enzyme, according to the literature [9
], thus corroborating the values obtained with molecular docking.
Molecule ZINC00001624 (Z24
) also had significant affinities calculated for residues Phe144, Tyr64, Trp53, Val65, Val68, Leu72, Leu74, Val51, and Ty33. However, comparing the distances of the key interactions that occur inside the pocket (including Try33, Val51, Trp53, Val65, and Val68) the interaction forces were more intense for ZINC00001021 (Z21
), with higher affinity values and ΔG value of −8.13 kcal/mol, as shown in Table S3, Supplementary Materials
The most common interactions observed for compounds docked here with juvenile hormone were with the amino acid residues Val51, Trp53, Pro55, and Val68. Interactions of the residues Tyr129 and Phe144 occurred for compound ZINC00001624 and control JH, and the less common interactions occurred with Leu74.