In Silico Study of Anticancer Activity of Red Betel Leaves Bioactive Compounds against Colon Cancer Marker Proteins

Colon cancer or colorectal cancer is one of the major health problems in the world. Previous research has proven that red betel leaves extract has anticancer activity against breast cancer cells. The study aimed to look at the potential of compounds contained in red betel ethyl acetate fraction as anticancer substances for colon cancer by conducting in silico tests through the docking of three colon cancer biomarker receptors against eight red betel leaves compounds. The parameters of binding affinity energy and inhibition constant used in the molecular docking analysis showed that there are several compounds that have the potential as inhibitors against colon cancer marker proteins to inhibit the development of colorectal cancer and have high bioavalibility as oral drugs. Based on in silico test it is known that the compound N-1,N-9-Bis [E-(2-nitophenyl) methylene] non anedihydrazide has the highest inhibition of human leukotriene A4 hydrolase receptor with binding affinity energy of 11.3160 Kcal/mol. The next compound is 4-({4,6-Bis[3R,5S)-3,5-diamino-1-piperydinyl]-1,3,5-traizin-2-yl}amino) benzenosulfon amide which has the highest inhibition in Chek1 with binding affinity energy of 9.7110 Kcal/mol, and the compound 4-(4-methoxy-phenylamino)-2,3-dihydro-1H-4a, 9-diaza-cyclopenta (b) fluorine-10-carbonitrile has the highest inhibition in the Bcl 2 navitoclax analog receptor with binding affinity energy of 8.4470 Kcal/mol.


Introduction
Colon or colorectal cancer is a malignant tumor that arises from the epithelial tissue of the colon or rectum. There are several treatments that can be done to treat colorectal cancer, including surgery, radiotherapy, and adjuvant chemotherapy (Sayuti and Nouva 2019). Some chemotherapy drugs that have been discovered today have a mechanism of action by inducing cytotoxicity, some of which are by influencing DNA replication transcription, activation of death receptor signals, as well as by activation of mitochondrial pathways. Those mechanisms can cause apoptosis of cancer cells and prevent metastases. However, chemotherapy drugs also have side effects, one of which is that it can interfere with body performance such as renal toxicity or cartilage suppression (Florea and Busselber 2011).
The side effects caused by chemotherapy encourage researchers to develop herbal medicine that can replace the role of synthetic chemotherapy drugs that are relatively more effective in increasing the immunity of patients with cancer (Rizki et al. 2015).
One of the plants that has the potential to be efficacious in reducing the malignant effects of colon cancer is red betel. This is because the water extract of red betel leaves contains phytochemical compounds in the form of flavonoids, tannins, and alkaloids (Safithri et al. 2016). Ethyl acetate which is a semipolar solvent is able to attract compounds with a wide polarity range from polar to nonpolar, so that it is able to attract such compounds (Putri et al. 2013). The use of active compounds from fraction with ethyl acetate solvent is expected to obtain high antioxidant activity. Several studies have proven that semipolar solvent such as ethyl acetate have a stronger ability as an antidote to free radicals than methanol, ethanol, chloroform, petroleum ether, and hexane solvent (Suryanto and Momuat 2017).
In addition, the in vitro method shows that red betel water extract has the potential to be an activator of the SOD enzyme with the highest SOD activity of 3.41 U/ml (Safithri 2012). Related to anticancer research, also the results of the in vitro study related to cancer, methanol extract of red betel leaves can inhibit the growth of breast cancer (T47D) through inhibition of phosphorylation p44/p42, which is a protein tyrosine kinase that plays a very important role in regulating the growth, differentiation, and survival of a cell (Wicaksono et al. 2009). Further study using the in vivo method based on the Brine Shrimp Lethality Test (BSLT) showed that the greater the concentration of red betel methanol extract, the greater the mortality of Artemia salina. The extract is said to be active or has anticancer activity according to the BSLT method if it has LC 50 <1,000 μg ml -1 , the results obtained in this study have an average value of 70,013 μg ml -1 , so it is concluded that the methanol extract of red betel leaves has anticancer activity (Rosyadi et al. 2020).
Based on the previous research about the potentials of red betel leaves, it is necessary to conduct research related to its potential as a colon anticancer by in silico method. This study aims to see the potential of compounds contained in red betel ethyl acetate fraction which has the potential to be an anticancer for colon cancer with in silico test through the docking of three receptors of colonic cancer biomarkers against compounds of the ethyl acetate fraction of red betel leaves.

Protein Structure Quality Analysis
Analysis of the structure quality of each receptor, consisting of Human Leukotriene A4 hydrolase, Chek1, and Bcl 2 navitoclax analog was carried out on the PROCHECK website (https://servicesn.mbi. ucla.edu/PROCHECK/). The receptors structures from modeling results that have been downloaded in PDB format were previously uploaded on the website. In the next stage, the quality of the structure from modeling result was analyzed to identify stable amino acid residues, where these residues were divided into four quadrants. The four quadrants include most favoured regions, additional allowed regions, generously allowed regions, and disallowed regions (Kurnia and Ruswanto 2019).
The next step was receptors preparation with YASARA Structure software. The receptors used were target proteins from 3D modeling results of human Leukotriene A4 hydrolase, Chek1, and Bcl 2 navitoclax analog. Unneeded parts in the docking protocol were removed, such as water molecules, residues, and native ligands. After that, hydrogen atoms were added while aliphatic hydrogens were not displayed (Agistia et al. 2013).

Molecular Docking Validation
Validation of molecular docking is carried out by directed molecular docking using YASARA Structure by the targeted docking method. The ligand docking zone was bounded by a gridbox around the active side on all three 3D proteins. Gridbox validation began with the stage of re-docking the natural ligands of the preparation results as well as receptors were being re-docked 999 times by YASARA Structure. The gridbox validation was then continued by determining the best gridbox size, also to obtain the RMSD (root mean square distance) value. The gridbox with the best size which had RMSD score less than 2 was then selected so that it can be used in molecular docking simulations (Agistia et al. 2013). The gridbox size tested was from size 0, 0.5, 1.0, 1.5 to size 5 with YASARA structure.

Physicochemical Predictions of Ligands
Lipinski's prediction of test ligands should be done first before molecular docking simulation. The physicochemical properties were tested on Lipinski SCFBio website (http:/Avww.scfbioiitd.res.in/ software/dr ugdesign/lipinski.jsp) which is based on the five rules of Lipinski (Adriani 2018). There should not be more than two rules violation in this step.

Molecular Docking Simulation
Molecular docking of test ligand was carried out with the optimum gridbox size obtained from the consisting of analysis of hydrogen, hydrophobic interactions, and distances performed with Discovery Studio software.

Protein Structure Quality
The results of the protein structure stability quality of 4LXD receptor with a crystallography resolution of 1.90 Å showed that the protein was the most stable, where as many as 121 residues (96.0%) were in the most favored region and as many as 5 residues (4.0%) in additional allowed region ( Figure 1). Followed by 3U9W receptor that got 501 residues in total (92.4%) in the most favored region and 39 residues (7.2%) in additional allowed region (Figure 1), in addition to which this receptor has a crystallography resolution of 1.25 Å. The last resptor tested for stability was 2R0U with 1.90 Å resolution, at this receptor there were 204 residues (87.6%) in the most favored region, previous gridbox validation. The receptor preparation file was opened in yasara structure software. The file was then prepared by forming a ligand-receptor complex based on the optimum gridbox. The docking results that have been obtained were then read with notepad software. Docking results such as binding energy values and non-covalent interactions of ligand-receptor complexes were obtained from this step (Agistia et al. 2013).

Data Analysis
Molecular docking results were analyzed by looking at the value of the Gibbs free energy (∆G*/ binding affinity), the inhibition constant (Ki) and the interaction of amino acid residues. The value of Gibbs free energy was used in the analysis of the affinity that occurs between the ligand and the receptor, further analysis of the results of molecular docking 3D visualization of the complex formed between the test ligand and the receptor. The visualization and as many as 26 residues (11.2%) were in additional allowed region (Table 1).

Molecular Docking Validation
The result of the molecular docking validation of each receptor showed that the 4LXD receptor best RMSD score was found at a gridbox size of 3 Å with 3D visualization shown in Figure 2, with an RMSD score of 1.6731 Å, the best binding affinity energy obtained was 6.3880 Kcal/mol. The 3U9W receptor got the best RMSD score of 0.0664 Å at a gridbox size of 3.5 Å with a binding affinity energy of 13.4720 Kcal/mol, and the 2R0U receptor had the best RMSD score which was 0.6895 Å at gridbox with a size of 4 Å, also with a binding affinity energy of 8.1350 Kcal/ mol. These gridbox optimum size, binding affinity enrgy score, dissociation constant, and RMSD score of each receptor can be seen in Table 2.

Physicochemical Predictions of Ligands
Analysis with the Lipinski Rule of Five of eight test ligands of compounds isolated from the ethyl acetate fraction of red betel leaves passed all test ligands used. Even though ligand with CID code of 2724185 has one violation with molar refractivity rule that has score less than 40 (Table 3) and ligand 44418672 has two violations with hydrogen donor more than 5 bounds, and the hydrogen acceptor more than 10 bounds.
The remaining ligands passed this stage without any rule breakage at all. Specifically in this selection step the ligand is still acceptable if it did not break more than two rules. Therefore these two ligands are still acceptable for next analysis.

Ligand Docking to BCL 2 Navitoclax Analog Receptor
The interactions that occurred at the Bcl 2 navitoclax analog receptor (PDB: 4LXD) resulted in the best binding affinity energy, which was 8.4470 Kcal/mol with a compound of 4-(4-methoxyphenylamino) -2.3-dihydro-1H-4a,9-diazacyclopenta (b)fluorine-10-carbon itrile with CID number 1077234, shown by Figure 3 with a value of Ki 6.33 x 10 -7 μM. The results from Table 4 show that there are 12 residues involved in the interaction of ligand and this receptor, of which 7 residues are active site residues, namely Tyr105, Met112, Glu133, Leu134, Gly142, Arg143, and Ala146. There were no hydrogen bonds from ligand and receptor, but there were hydrophobic interactions with 7 residues (Table  5).

Ligand Docking to Human Leukotriene A4 Hydrolase Receptor
Molecular docking on Human Leukotriene A4 Hydrolase receptor (PDB: 3U9W) showed the best interaction with compound N-1,N-9-Bis [E-(2nitrophenyl) methylene] non anedihydrazide (CID: 9595915) with a binding affinity energy of 11.3160 Kcal/mol, the best Ki (Inhibition constant) value of 7.48 x 10 -8 4.97 x 10 -9 μM shown in Table 4. Software analysis showed that the ligand interacted with 28 residues in total, 14 of which are receptor active site residues consisting of Gln1136, Tyr1267, Gly1269, Met1270, His1295, Glu1296, Trp131, Phe1314, Val1367, Pro1374, Ala1377, Tyr1378, Pro1382, and Tyr1383. The interactions of hydrogen bond were found with the Gly1268, Arg1563, and Tyr1383 residues. There were also hydrophobic interactions occurred between the ligand and 18 residues from the 3U9W receptor shown in Table 5. The 3D visualization of this protein and ligand interaction shows that the catalytic area is wide enough to cover ligand's surface, and wider than 4LXD and 2R0U receptor, this is due to the large number of residues involved in molecular docking (Figure 3).

Ligand Docking to Human Leukotriene A4 Hydrolase Receptor
The results of 3D visualization between the receptor and the ligand that have the best binding affinity energy are shown by Figure 3. The Parameters observed for the determination of the energy of the ligand affinity to the receptor in this study ware the binding affinity energy, the value of the inhibition constant, and the amino acid residue. When hydrophobic bonds increase from a drug compound, the activeness of the compound will increase (Patil et al. 2010). The statement corresponds to the 3U9W receptor where as the hydrophobic bond increases, the better the affinity score, and the smaller the value of Ki or its inhibition constant. The value is the concentration of inhibitors needed to decrease by half the activity of the enzyme. The smaller the value of Ki, the stronger the inhibitor (Pannindriya et al. 2021).
Previous studies have shown that active pocket located in the area of the N-terminal catalytic domain which is hydrophobic. The docking results showed that the interaction of short length bonds and the number of hydrogen bonds formed was found more in the interaction of protein Leukotriene A4 Hydrolase-Bullatalisin than the protein Leukotriene A4 Hydrolase-standard, the compound bullatasin itself is an acetogenin derivative compound that has the potential to become an inhibitor against Leukotriene A4 hydrolase. Bullatalicin ligands, along with standard ligands can occupy protein active pocket alternative of leukotriene A4 Hydrolase receptors (Noviardi and Fachrurrazie 2015).

Ligand Docking to Chek1 Receptor
The Chek1 protein is necessary for effective cell division in the phases of the normal cell cycle, so that both during cancer development and progression and under stressful environments, the amount of Chek1 protein is strictly controlled (Zhang and Hunter 2013). These findings suggest that inhibiting Chek1 can be an effective treatment for cancers that overexpress the protein. The study on kaempferitrin docking with Serine/threonineprotein kinase Chek1 (PDBID: 2R0U), showed 8 HAYATI J Biosci 119 Vol. 30 No. 1, January 2023 4. Discussion

Protein Structure Quality
Diffraction resolution chategory is divided into four categories namely: small (<3.00 Å), medium (2.70-2.00 Å), high (2.00-1.50 Å), and very high (< 1.5 Å). The smaller the crystallographic value, the more specific to image generated visualization (Made and Pathni 2018). this statement indicates that all three receptors used from Figure 1 have high resolution. In addition, the results of the Ramachandran diagram of a structure with ˃90% residue in favoured regions indicate an accurate structure (Rai and Rieder 2012), it is hereby known that all three receptors also have accurate structure to proceed at the molecular docking stage.

Molecular Docking Validation
The validation stage aims to ensure that the size of the grid box obtained is appropriate and already includes the active side of the tested receptor. The validation stage will produce an RMSD value that will show the accuracy of the docking conformation that has been carried out. Docking results are considered good if it has an RMSD value of less than 2 Å (Purnomo 2013). Therefore, from this statement, it is concluded that the three receptors are accurate and good for use in the molecular docking stage (Table 2)

Physicochemical Predictions of Ligands
Lipinski's rules are used to evaluate the drug likeness of a chemical compound. The rules consist of a molecular weight of <500 Da, a LogP value of <5, the number of hydrogen bond donors <5, the number of acceptors of hidrogen bond <10 and a molar refractivity between 40-130 (Dwi and Abdul 2011). Results from Table 3 shows that all eight test ligands have very high bioavailability because the highest violations are only two rules. Thus it can be concluded that the compounds have good compatibility because they pass these rules and shows that these compounds have the potential to have similarities in orally active drugs (Tumilaar et al. 2021). In addition, these things indicate that each of the compounds easily binds to receptors and how well the permeability of the compounds are in crossing the cell membrane (Singh et al. 2013).
The conformation of docking is determined from the result of the highest affinity in the form of bond energy (∆G) with YASARA structure. Analysis of molecular interactions can show specific interactions between ligands and receptors. The interactions between ligands and receptors are stabilized by hydrogen bonds and hydrophobic reactions (Perdana and Permana 2021). The binding affinity energy score will affect the intermolecular activity of the compound and have an impact on the strength of the bond. The best binding affinity energy score will provide greater strength of protein and ligand interaction (Hanif et al. 2020), therefore the 3U9W and 2R0U receptors interact most strongly with the ligands with the best binding affinity energy.
In conclusion of this research there are several compounds that have the potential as inhibitors against colon cancer marker proteins by in silico method to inhibit the development of colorectal cancer and have high bioavalibility as oral drugs. The compound N-1,N-9-Bis [E-(2-nitophenyl) methylene] non anedihydrazide has the highest inhibition of human leukotriene A4 hydrolase. The next compound is 4-({4,6-Bis[3R,5S)-3,5d i a m i n o -1 -p i p e r y d i nyl ] -1, 3 , 5 -t ra i z i n -2 -yl } amino) benzenosulfonamide which has the highest inhibition in Chek1 and the compound 4-(4-methoxy-phenylamino)-2,3-dihydro-1H-4a, 9-diaza-cyclopenta (b) fluorine-10-carbonitrile has the highest inhibition in the Bcl 2 navitoclax analog receptor.