In this research, the selected protein target is p53, which is known as the guardian of the genome and plays a crucial role in cellular senescence processes associated with aging. The protein structure of p53 used for molecular docking validation is a complex that contains both p53 and the native ligand. This structure serves for validation purposes before conducting molecular docking with compounds from the Plectranthus amboinicus plant. The protein structure with PDBID 5G4N can be accessed via the Protein Data Bank (PDB) website at http://www.rscb.org. This structure was chosen because it has a resolution smaller than 2 Å, specifically 1.35 Å. Protein structures with a resolution smaller than or equal to 2 Å are considered high-resolution structures where the positions of atoms can be accurately validated.20
The first step before performing molecular docking between the p53 protein target receptor and the ligands from the P. amboinicus plant is the pose validation process. This step is carried out to determine the interaction positions between the p53 protein target and the native ligand. In the validation process, an RMSD (Root Mean Square Deviation) result of 1.384 Å was obtained for the pose in the 71th run out of 100 runs using a 40 x 40 x 40 grid box with the grid position at x = 124.875, y = 105.487, z = -43.667. A pose in molecular docking is considered good if the RMSD is smaller than or equal to 2 Å. Furthermore, binding affinity results were obtained in the docking of the O83 native ligand with p53, with a value of -8.82 kcal/mol and an inhibition concentration of 343.71 nM. These figures can serve as positive controls and references when conducting an analysis of the docking of the test ligands. Therefore, it can be concluded that the protein structure of p53 used in this molecular docking process is valid and can be utilized to observe interactions with ligands from the P. amboinicus plant. Furthermore, the grid box and grid position from the validation result, which represents the best pose, will be used as the grid box and grid position for the molecular docking of the seven P. amboinicus compound ligands.
The first molecular docking is conducted between the thymol compound ligand and the p53 protein receptor. From 100 molecular docking experiments predicting the thymol pose on the p53 binding site using Autodock Tools 1.5.6, 5 poses were obtained, with the pose in the 53rd run being the best pose repeated 55 times. The Gibbs free energy (∆G) produced in the 53rd run was − 6.17 kcal/mol, with an average Gibbs free energy (∆G) of -6.14 kcal/mol, indicating moderate stability of the p53 receptor bond with thymol. This indicates that the binding formed by thymol with p53 is not as favorable as the binding formed by the O83 native ligand, which serves as the positive control with p53. In addition, the inhibition concentration (IC) obtained was 30.05 µM, signifying that only around 30.05 µM of thymol concentration in our bloodstream is required to enhance the functionality of the p53 protein. Information about the coordinates where thymol binds was also provided: x = 124.705, y = 102.458, z = -43.366. Various bonds occur when the p53 protein receptor interacts with thymol, including hydrogen bonds, Van der Waals interactions, Pi-Sulfur interactions, and Pi-Alkyl interactions, all of which strengthen and support stability. Thus, it can be stated that the compound ligand thymol can bind to the p53 protein, forming a moderately stable bond due to the Gibbs free energy (∆G) ranging from − 5 to -7 kcal/mol (-6.17 kcal/mol). The resulting inhibition concentration is also moderately good, falling within the micromolar (µM) range (30.05 µM).
The second molecular docking involves the carvacrol compound ligand and the p53 protein receptor. Out of 100 molecular docking trials predicting the carvacrol pose on the p53 binding site using Autodock Tools 1.5.6, 3 poses were obtained, with the pose in the 42nd run being the best pose repeated 67 times. The Gibbs free energy (∆G) produced in the 42nd run was − 6.55 kcal/mol, with an average Gibbs free energy (∆G) of -6.53 kcal/mol, indicating moderate stability of the p53 receptor bond with carvacrol. This indicates that the binding formed by carvacrol with p53 is not as favorable as the binding formed by the O83 native ligand, which serves as the positive control with p53. The inhibition concentration (IC) obtained was 15.68 µM, suggesting that only around 15.68 µM of carvacrol concentration in our bloodstream is needed to enhance the functionality of the p53 protein. The binding coordinates for carvacrol were provided as follows: x = 125.281, y = 104.814, z = -41.232. Similar to thymol, various bonds contribute to the stability of the interaction between the p53 protein receptor and carvacrol, including hydrogen bonds, Van der Waals interactions, hydrocarbon bonds, Pi-Sigma interactions, alkyl bonds, and Pi-Alkyl interactions. Therefore, the compound ligand carvacrol can bind to the p53 protein, forming a moderately stable bond due to the Gibbs free energy (∆G) ranging from − 5 to -7 kcal/mol (-6.55 kcal/mol). The resulting inhibition concentration is also moderately good, within the micromolar (µM) range (15.68 µM).
The third molecular docking focuses on the flavonoid compound quercetin and the p53 protein receptor. Among 100 molecular docking trials predicting the quercetin pose on the p53 binding site using Autodock Tools 1.5.6, 2 poses were obtained, with the pose in the 76th run being the best pose repeated 85 times. The Gibbs free energy (∆G) produced in the 76th run was − 9.22 kcal/mol, with an average Gibbs free energy (∆G) of -9.04 kcal/mol, indicating highly stable bonding of the p53 receptor with quercetin. Furthermore, this indicates that the binding formed by the flavonoid quercetin with p53 is more stable compared to the binding formed by the O83 native ligand, which serves as the positive control with p53. The inhibition concentration (IC) obtained was 174.91 nM, signifying that only around 174.91 nM of quercetin concentration in our bloodstream is necessary to enhance the functionality of the p53 protein. The binding coordinates for quercetin were provided as follows: x = 124.581, y = 102.961, z = -45.144. Similar to previous cases, multiple bonds contribute to the stability of the interaction between the p53 protein receptor and quercetin, including hydrogen bonds, Van der Waals interactions, hydrocarbon bonds, unfavorable donor-donor bonds, Pi-Sigma interactions, Pi-Sulfur interactions, Amide-Pi Stacked interactions, and Pi-Alkyl interactions. Consequently, the compound ligand quercetin can bind to the p53 protein, forming a highly stable bond due to the Gibbs free energy (∆G) less than or equal to -7 kcal/mol (-9.22 kcal/mol). The resulting inhibition concentration is also highly favorable, within the nanomolar (nM) range (174.91 nM).
The fourth molecular docking was performed between the flavonoid compound luteolin and the p53 protein receptor. Out of 100 molecular docking experiments aimed at predicting the luteolin pose on the p53 binding site using Autodock Tools 1.5.6, only 1 pose was obtained, with the pose in the 76th run being the best pose repeated 100 times. The Gibbs free energy (∆G) generated in the 76th run was − 9.25 kcal/mol, with an average Gibbs free energy (∆G) of -9.11 kcal/mol. This indicates that the stability of the bond between the p53 receptor and the flavonoid luteolin is highly stable. Furthermore, this indicates that the binding formed by the flavonoid luteolin with p53 is more stable compared to the binding formed by the O83 native ligand, which serves as the positive control with p53. Moreover, the inhibition concentration (IC) obtained was 167.17 nM, signifying that only approximately 167.17 nM of luteolin concentration in our bloodstream is required to enhance the functionality of the p53 protein. The binding coordinates for luteolin were provided as follows: x = 124.630, y = 102.623, z = -45.150. Similar to the previous cases, several bonds contribute to the strengthening and stabilizing of the interaction between the p53 protein receptor and the flavonoid luteolin, including hydrogen bonds, Van der Waals interactions, unfavorable donor-donor bonds, Pi-Sigma interactions, Pi-Sulfur interactions, and Pi-Alkyl interactions. Therefore, it can be concluded that the compound ligand flavonoid luteolin can bind to the p53 protein, forming an exceptionally stable bond due to the Gibbs free energy (∆G) value being less than or equal to -7 kcal/mol (-9.25 kcal/mol). Additionally, the resulting inhibition concentration is highly favorable, within the nanomolar (nM) range (167.17 nM).
The fifth molecular docking was carried out between the flavonoid compound apigenin and the p53 protein receptor. From a total of 100 molecular docking experiments aimed at predicting the apigenin pose on the p53 binding site using Autodock Tools 1.5.6, there were 2 poses, with the pose in the 72nd run being the best pose repeated 99 times. The Gibbs free energy (∆G) generated in the 72nd run was − 9.13 kcal/mol, with an average Gibbs free energy (∆G) of -9.09 kcal/mol. This indicates that the stability of the bond between the p53 receptor and the flavonoid compound apigenin is highly stable. Moreover, this indicates that the binding formed by the flavonoid apigenin with p53 is more stable compared to the binding formed by the O83 native ligand, which serves as the positive control with p53. Furthermore, the inhibition concentration (IC) obtained was 203.86 nM, indicating that only around 203.86 nM of apigenin concentration in our bloodstream is needed to enhance the functionality of the p53 protein. The binding coordinates for apigenin were provided as follows: x = 124.610, y = 102.680, z = -45.102. As observed in previous cases, multiple bonds contribute to the interaction between the p53 protein receptor and the flavonoid apigenin, reinforcing and supporting stability. These bonds include hydrogen bonds, Van der Waals interactions, Pi-Sigma interactions, Pi-Sulfur interactions, and Pi-Alkyl interactions. Consequently, it can be concluded that the flavonoid compound apigenin can bind to the p53 protein, forming an exceptionally stable bond due to the Gibbs free energy (∆G) value falling within the range of ≤ -7 kcal/mol (-9.13 kcal/mol). Additionally, it's noted that the resulting inhibition concentration is highly favorable, within the nanomolar (nM) range (203.86 nM).
The sixth molecular docking was conducted between the flavonoid compound rutin and the p53 protein receptor. From a series of 100 molecular docking experiments aimed at predicting the rutin pose on the p53 binding site using Autodock Tools 1.5.6, there were 23 poses, with the pose in the 54th run being the best pose repeated 19 times. The Gibbs free energy (∆G) generated in the 54th run was − 9.14 kcal/mol, with an average Gibbs free energy (∆G) of -7.81 kcal/mol. This indicates that the stability of the bond between the p53 receptor and the flavonoid compound rutin is highly stable. Furthermore, this indicates that the binding formed by the flavonoid rutin with p53 is more stable compared to the binding formed by the O83 native ligand, which serves as the positive control with p53. Additionally, the inhibition concentration (IC) obtained was 199.98 nM, indicating that only around 199.98 nM of rutin concentration in our bloodstream is needed to enhance the functionality of the p53 protein. The binding coordinates for rutin were provided as follows: x = 124.776, y = 105.472, z = -45.424. Similar to previous cases, multiple types of bonds are involved in the interaction between the p53 protein receptor and the flavonoid rutin, further reinforcing and supporting stability. These bonds include hydrogen bonds, Van der Waals interactions, hydrocarbon bonds, Pi-Sigma interactions, Pi-Sulfur interactions, alkyl bonds, and Pi-Alkyl bonds. Consequently, it can be concluded that the flavonoid compound rutin can bind to the p53 protein, forming an exceptionally stable bond due to the Gibbs free energy (∆G) value falling within the range of ≤ -7 kcal/mol (-9.14 kcal/mol). Furthermore, it is stated that the resulting inhibition concentration is highly favorable, within the nanomolar (nM) range (199.98 nM).
The final molecular docking was performed between the flavonoid compound eriodictyol and the p53 protein receptor. Through 100 molecular docking experiments aimed at predicting the eriodictyol pose on the p53 binding site using Autodock Tools 1.5.6, only one pose was obtained, with the pose in the 79th run being the best pose repeated 100 times. The Gibbs free energy (∆G) generated in the 79th run was − 9.34 kcal/mol, with an average Gibbs free energy (∆G) of -8.98 kcal/mol. This indicates that the stability of the bond between the p53 receptor and the flavonoid compound eriodictyol is highly stable. Furthermore, this indicates that the binding formed by the flavonoid eriodictyol with p53 is more stable compared to the binding formed by the O83 native ligand, which serves as the positive control with p53. Additionally, the inhibition concentration (IC) obtained was 142.00 nM, indicating that only around 142.00 nM of eriodictyol concentration in our bloodstream is needed to enhance the functionality of the p53 protein. The binding coordinates for eriodictyol were provided as follows: x = 124.401, y = 102.296, z = -45.330. Similar to the previous cases, various types of bonds are involved in the interaction between the p53 protein receptor and the flavonoid eriodictyol, further enhancing stability. These bonds include hydrogen bonds, Van der Waals interactions, unfavorable donor-donor bonds, Pi-Sigma interactions, Pi-Sulfur interactions, and Pi-Alkyl bonds. As a result, it can be concluded that the flavonoid compound eriodictyol can bind to the p53 protein, forming an exceptionally stable bond due to the Gibbs free energy (∆G) value falling within the range of ≤ -7 kcal/mol (-9.34 kcal/mol). Furthermore, it is stated that the resulting inhibition concentration is highly favorable, within the nanomolar (nM) range (142.00 nM).
In the results of the molecular docking between the seven ligands of P. amboinicus plant compounds and the target protein receptor p53, it was determined that the ligand sequence with the highest binding affinity is eriodictyol, followed by luteolin, quercetin, rutin, apigenin, carvacrol, and thymol. This outcome was obtained based on considerations that refer to two out of the four molecular docking parameters, namely Gibbs free energy (∆G) and inhibition constant (Ki), as observed from the histogram results in Autodock Tools v.1.5.6. The selection of these two parameters itself is founded on the theory that smaller ∆G values correspond to more negative values of the forming bond's ∆G, signifying increased stability. Furthermore, the second parameter, the smaller the inhibition concentration (IC) value, also indicates increased stability of the bond.17–19 Both of these parameters are directly proportional, and when both are optimized, the binding affinity formed between the ligand and protein improves as well.17,19 Based on the results of the molecular docking between the p53 target protein receptor and the seven ligands of P. amboinicus plant compounds, the sequence of compounds with the best binding affinity to protein p53 is as follows:
1. Eriodictyol
\(\varDelta\) G = -9.34 (kcal/mol) dan IC = 142.00 nM
2. Luteolin
\(\varDelta\) G = -9.25 (kcal/mol) dan IC = 167.17 nM
3. Quercetin
\(\varDelta\) G = -9.22 (kcal/mol) dan IC = 174.91 nM
4. Rutin
\(\varDelta\) G = -9.14 (kcal/mol) dan IC = 199.98 nM
5. Apigenin
\(\varDelta\) G = -9.13 (kcal/mol) dan IC = 203.86 nM
6. Carvacrol
\(\varDelta\) G = -6.55 (kcal/mol) dan IC = 15.68 µM
7. Thymol
\(\varDelta\) G = -6.17 (kcal/mol) dan IC = 30.05 µM
From the data above, it can be observed that the flavonoid compounds eriodictyol, luteolin, quercetin, rutin, and apigenin in the P. amboinicus plant have better binding affinities or Gibbs free energy (∆G) values compared to the compounds carvacrol and thymol. This is evident from the Gibbs free energy (∆G) values of the flavonoid compounds eriodictyol, luteolin, quercetin, rutin, and apigenin, which have values ≤ -7 kcal/mol. This implies that these five compounds have a high binding affinity, indicating stable bonds. The five flavonoid compounds also exhibit more stable binding compared to the binding between the O83 native ligand, serving as the positive control, and p53. Moreover, they have inhibition consentration (IC) with nM (nanomolar) values, signifying excellent inhibitory concentrations. Moving on to the compounds carvacrol and thymol, the Gibbs free energy (∆G) values fall within the range of -5 to -7 kcal/mol. This suggests that the binding affinity of carvacrol and thymol falls within the moderate or intermediate binding affinity category. Additionally, both compounds have inhibition concentration (IC) with µM (micromolar) values, indicating a moderate inhibitory concentration. Therefore, carvacrol and thymol are predicted to still have the potential to bind to the target protein p53, although their bonds formed might not be as strong as those formed by luteolin, apigenin, quercetin, eriodictyol, and rutin compounds. This analysis result further supports the theory that Gibbs free energy (∆G) values are directly proportional to inhibition constant (Ki) values. Thus, as Gibbs free energy (∆G) values decrease, inhibition constant (IC) values also decrease. Furthermore, as Gibbs free energy (∆G) values become more negative, the bonds formed between the receptor and ligand become more stable.
Despite the molecular docking results between the P. amboinicus plant compound ligands and the target protein receptor p53 predicting and demonstrating interactions, it cannot be denied that this study still has some limitations. These limitations are notably related to the unconfirmed predicted interaction results between the P. amboinicus plant and p53 protein, as well as the toxicity of P. amboincius to the human body, which is not yet known.