2.1. Essential Oil Extracts
Untargeted metabolite profiling of the n
-hexane extract of the mabolo essential oil yielded several peaks as shown in the chromatogram (Figure 1
and Table 1
). Metabolites identified are esters, specifically butanoates (methyl, 5.6 %; ethyl, 8.5 %; butyl, 6.0 % & benzyl, 15.2 %), and benzoates (methyl, 53.4 % & butyl, 1.4 %) and benzyl alcohol (9.8 %). Metabolites detected from the sample is comparable with previous results [25
]. Butanoates were detected at low abundance, possibly due to hydrolysis reaction as suggested from previous studies [26
]. Alkyl butanoates are hydrolyzed to produce free butyric acid, which gives the olfactory signature of mabolo during steam distillation [26
]. Only the corresponding benzyl alcohol was detected among the possible alcohol hydrolysis products. Furthermore, the butanoates and benzyl alcohol were previously detected from yellow passion fruit (Passiflora edulis
(Sims. f.). Flavicarpa
Degener) and were identified to be odor active constitutents [31
]. Butanoates are decribed to have fruity, sweet and floral aroma, while benzyl alcohol has herbal, moldy and roasted seed aroma [31
Methyl benzoate is the most abundant ester present in the sample. Compared with aliphatic esters, the hydrolysis of organic esters, such as benzoates, occurs much slower, which could account for the higher percentage of benzoates versus butanoates [30
]. Methyl benzoate is one of the most abundant phenylpropanoid-derived volatile emitted from different plant parts and sources [32
]. It is documented to have attractant activity to Hylastinus obscurus
(clover root borer) [33
] but potential repellent activity to Apis mellifera
] and strong repellency to several pestiferous social wasps (yellow jackets, Vespula pennyslvanica
and paper wasps, Polistes dominulus
Previously, methyl butanoate and methyl propionate were reported to be more effective in eliciting a response in sensilla trichodea of mosquitoes than the ethyl esters of these compounds at similar stimulus intensities [37
]. These results showed that methyl esters attracted more gravid female mosquitoes than did the ethyl esters of the same compounds—propionic and butyric acids—when used in 0.1% aqueous solution. Moreover, sensilla trichodea’s response is relatively specific for chemical substances reported to be oviposition attractants by mosquitoes [37
]. In addition, yellow jackets were found to be attracted to the combination of butyl butanoate with acetic acid as well as to isobutanol and heptyl butanoate with acetic acid [38
2.2. Protein Model Verification
Due to the absence of an OAMB crystal structure, homology modeling was performed. Here, we used the web-based server, GPCR-I-TASSER, to construct a full-length protein model of OAMB. Top templates used for modeling OAMB includes the human M2 muscarinic acetylcholine receptor (PDB: 3UON, 467 residues) [39
-adrenergic receptor (PDB: 3D4S, 490 residues) [40
], human cannabinoid receptor CB1 (PDB: 5TGZ, 452 residues) [41
], rat neurotensin receptor NTS1 (PDB: 4GRV, 510 residues) [42
], human A
adenosine receptor (PDB: 3EML, 488 residues) [43
], and a human membrane protein/hydrolase (PDB: 5D6L, 500 residues) [44
The aforementioned proteins were used as templates to create five OAMB models. Among the five resulting structures, the best model was selected using C-score as the standard. This metric measures the quality of the models based on the template alignments, and it typically has values ranging from −5 to +2. The best OAMB model had a C-score value of −2.74. The reason for this low score could be attributed to the low sequence identity and a large difference in length between OAMB (645 residues) and the templates (452 to 510 residues).
The homologs used for modeling were between 18% and 22% identical with OAMB. After performing multiple sequence alignment of OAMB and the templates, it was found that most of the conserved sequences are located in the transmembrane region of the proteins, as shown in Figure 2
Comparison of the protein structures were done by performing 3D alignment of OAMB and the homologs. Heavily conserved structural domains are found in the transmembrane region, which is an evolutionary characteristic of G-protein coupled receptors. Large structural discrepancies can be observed in the loop regions, i.e., intracellular and extracellular loops. This major problem in protein structure prediction can be attributed to sequence unalignment of highly varying sequences within a given structural motif.
2.3. Ensemble Docking
A structural ensemble was obtained from the molecular dynamics simulations of the non-liganded OAMB. This was used to study the ensemble binding affinity and binding sites of various ligands at different conformations of the protein. After obtaining ligand structures and an ensemble of protein conformations, molecular docking was performed using AutoDock Vina. The putative ligand, octopamine, along with the isolated compounds (Table 1
), i.e., benzyl alcohol, butyl benzoate, benzyl butanoate, butyl butanoate, ethyl butanoate, methyl benzoate, and methyl butanoate, were docked to obtain the binding free energy of the complexes formed between the receptor and the ligands of interest. The inhibition constant
could be derived from the binding affinities using the formula
. Since there is an inverse relationship between binding affinity and
, it follows that the compound with the best binding affinity will have the lowest concentration requirement to inhibit OAMB.
Ensemble docking results (Table 2
) show that the compound with the highest mean binding affinity is benzyl butanoate with a value of −6.03 ± 0.09 kcal·mol
, followed by butyl benzoate, which has a comparable affinity of −6.02 ± 0.01 kcal·mol
. Octopamine, the putative ligand, possess an average binding affinity of −5.18 ± 0.07 kcal·mol
. Moreover, methyl benzoate, the most abundant compound isolated, had a binding affinity of −5.61 ± 0.07 kcal·mol
. Benzyl butanoate, butyl benzoate, and methyl benzoate are slightly better than octopamine. Other extracted compounds are inferior than octopamine, i.e., benzyl alcohol, butyl butanoate, ethyl butanoate, and methyl butanoate, with binding affinities of −4.93 ± 0.06, −4.88 ± 0.06, −4.41 ± 0.06, −4.06 ± 0.05, in kcal·mol
Results from the docking studies suggest that an aromatic motif induces favorable binding. Investigating the binding sites of the ligands reveal a similar set of amino acids (Figure 3
). The probability of each ligand to bind in a specific site was also analyzed. Here, the most frequent binding region includes the residues Val69, Val99, Met103, Cys104, Ser107, Trp532, Gly559, Trp560, and Asn562. Based on the ensemble docking, these residues have >50% probability of ligand interactions. Furthermore, these residues belong to the transmembrane helices 2, 3, 6, and 7. Other residues that interact with the ligands were Ala65, Asp66, Trp80, Trp96, Leu97, Asp100, Val101, Trp159, Trp285, Cys287, Glu288, Phe304, Phe535, Phe536, Arg542, and Phe556 of the extracellular loops 2, 3, and 4 and transmembrane helices 2, 3, 4, 5, 6, and 7 with ligand interaction probabilities of 10–50%. Aromatic residues in the binding regions, i.e., Trp80, Trp96, Trp159, Trp285, Phe304, Trp532, Phe535, Trp560, Phe536, and Phe556 interacting with the phenyl ring of the aromatic ligands (
stacking interactions) seems to be the origin for the observed binding affinity.
Further analysis shows that there are two possible binding pockets. The site with the higher probability of ligand binding (>50%) consists of Ala65, Ala66, Val69, Trp96, Leu97, Val99, Asp100, Val101, Met103, Cys 104, Ser107, Trp532, Phe535, Phe536, Gly559, Phe556, Tyr560, and Asn562. The second binding pocket with a lower ligand interaction probability (<50%) consists of Trp80, Trp159, Trp285, Cys287, Glu288, Phe304, and Arg542 of the extracellular loops 2, 3, 4, and transmembrane helices 4 and 5. The aforementioned residues in the first binding pocket is located at the core of the transmembrane domain in helices 2, 3, 6, and 7.
The fruit of mabolo is a good source of aromatic compounds (Table 1
). The most abundant compound methyl benzoate, with a 53.4% percent abundance in the fruit, has a better binding affinity than octopamine. Interestingly, benzyl butanoate, the best binding ligand is also the second most abundant compound in the fruit with a percent abundance of 15.23%. This indicates that the mabolo fruit is an excellent source of pest control compounds.
There are very few available studies regarding OAMB. Various works show that OAMB is known to be crucial for olfactory learning and motor control [21
]. Moreover, OAMB affects female fruit fly ovulation [20
], which could possibly be impaired by antagonistic activity against OAMB. Hindering the function of this receptor could potentially cause a decline in fruit fly reproduction [20
] and erratic behavior, leading to a decrease in crop produce damage. The antagonistic effect of the mabolo fruit extracts will need to be tested via live insect assay to empirically determine the effects of the compounds, i.e., attractants, repellents, and/or sterilizer (inhibit egg fertilization) and obtain a feedback for the modeling experiments. Thus, this study may well serve as a guide for the synthesis of potent biopesticides by rational design of functional derivatives that possess better binding affinities than the isolated compounds.