Submitted : 29-06-2020 Revised : 10-12-2020 Accepted : 24-03-2021 Trad. Med. January-April 2021 Vol. , p 71-78 ISSN-p : 1410-5918 ISSN-e : 2406-9086 In Silico Study on the Effect of Heliannuol A. E Compounds of Sunflower (Helianthus annuus L. ) on Dual PI3K/mTOR . OQ. Enzyme Roihatul Mutiah*. Yen Yen Ari Indrawijaya. Tanaya Jati Dharma. Jamilah Damaiyanti Pharmacy Study Program. Faculty of Medicine and Health Sciences. Maulana Malik Ibrahim State Islamic University of Malang. Indonesia ABSTRACT Heliannuol is a sesquiterpene that has a benzoxepine ring, oxepin. Many derivatives of benzoxepine compounds show anticancer activity by inhibiting the phosphoinositide 3-kinase (PI3K) enzyme. These enzymes play a role in cell proliferation and growth. The study aims to predict the physicochemical properties using LipinskiAos Rule of Five parameters on phosphoinositide 3- kinase (PI3K/Mtor. PDB 5OQ. enzyme and the toxicity of Heliannuol A. E compounds. The process uses the pkCSM online tool. The validation of receptor 5OQ4 is done using the value parameter RMSD < 2 . I). Protox online tool dan pkCSM online tool is employed to predict the toxicity using parameter LD50, skin sensitization. Ames toxicity, hepatotoxicity, and toxicity class. The interaction of ligan and enzyme is tested using Molegro Virtual Docker Heliannoul A. E compounds fulfill LipinskiAos Rule of Five. The receptor 5OQ4 is known valid using the value of RMSD 0,923 . I). Heliannuol A. E compounds inhibit Dual PI3K / mTOR enzyme less than Bimiralisib. As a result of the toxicity test of compounds Helliannouls A. E, and Bimiralisib compounds are included in class 4, while Helliannouls D compounds are included in class 5. Keywords: in-silico. Heliannuols. PI3K/mTOR. INTRODUCTION Brain cancer covers about 85-90% of all central nervous system cancers. The incidence rate for malignant brain cancer worldwide based on world standard population rates is 3. 4 per 100,000 The mortality rate is 4. 25 per 100,000 population per year. Understanding the molecular principles and signaling pathways involved in glioblastoma is essential for the development of more effective therapies. (AnZhenyi et al. , 2018. (Pedoman Nasional Pelayanan Kedokteran Tumor Otak, 2. The phosphoinositide 3-kinase signaling pathway have a central role in the regulation of signal transduction, mediating various biological processes, including cell proliferation, apoptosis, metabolism, motility, and angiogenesis in Excessive activation of the PI3K or Akt pathway provides rapid growth, tumor development, and multi-drug resistance to glioblastoma cells (Zhao et al. , 2. Heliannuols is a new group of phenolic sesquiterpenes isolated from sunflower cultivars (Helianthus annuu. The chemical structure of Heliannuols has a benzoxepine ring in which various benzoxepine derivatives have shown activity towards various types of cancer. *Corresponding author: Roihatul Mutiah Email: roiha@farmasi. uin-malang. The benzoxepine derivative mechanism inhibits the enzyme phosphoinositide 3- kinase (PI3K) (Hefron et al. , 2011. Kuntala et al. , 2. The insilico test is a method through simulation carried out with computer media. silico test was conducted by tethering the molecule . olecular dockin. Selected drug candidates to the receptor. In Silico testing has the advantage of efficiently finding new drugs using a modeling method that is a simulation with the help of computational technology (Pelkonen et al. , 2. Prediction of drug activity at the receptors can be performed using in silico test. This research was carried out because, in the development of cancer drugs, it was needed to predict anticancer activity, toxicity, and Heliannuol A. E compounds contained in sunflower plants (Helianthus annus L. ) towards phosphatidylinositol 3-kinases receptors. METHODOLOGY Materials Tools: Chem Bio Draw Ultra Version 12 (CambridgeSof. Avogadro. Molegro Virtual Docker 6. 0, protox online tool, pkCSM online tool. Ingredients: Dual PI3K / mTOR enzyme . OQ. , protein subtype: gamma isoform subunit. Native Ligand: 5- . ,6-dimorpholin-4-yl-1,3,5-triazine-2y. - 4- . pyridin-2-amine or Traditional Medicine Journal, 26. , 2021 | DOI : 10. 22146/mot. Roihatul Mutiah bimiralisib (PQR. , resolution: 2. 70 yI. 2dimensional and 3-dimensional structures of Heliannuol A. E, and Bimiralisib compounds along with the SMILE code. Methods Ligand Preparation and Receptors Receptor preparation was done through downloading on the protein data bank website https://w. org/structure/5OQ4. Ligand preparation was done by drawing ligands in 2D and obtained the SMILE code used Chem Bio Draw Ultra version 12. Then 3D was changed using Avogadro. Geometry optimization using Avogadro with the MMFF94 method. Physicochemical Test The Physicochemical prediction was carried out using Lipinski parameter 5 was molecular weight, the logarithm of octanol/water partition coefficient, number of bonds between rotating atoms. Hydrogen Bond Acceptors. Hydrogen Bond Donors, and Polar Surface Activity by entering SMILE code into the online tool pkCSM. Determination of Cavity The determination of the cavity was used to detect the location of the ligand with 5OQ4 The selection was made by looking at the native sites of the ligands that interact with the 5OQ4 receptor. A cavity at the receptor is shown in Method Validation The validation parameter seen was the value of RMSD (Root Mean Square Deviatio. through backing natives with receptors. Validation was done by re-docking between native ligands and 5OQ4 receptors. Docking Heliannuols A. E, and Bimiralisibat the 5OQ4 receptor Docking Heliannuols A. E, and Bimiralisib were performed used Molegro Virtual Docker 6. RS value . erank scor. was the bond energy needed to form bonds between ligands and receptors so that the activity of a compound can be predicted (Zaidan et al. , 2. Toxicity Test Prediction of compound toxicity based on skin sensitization. Ames toxicity. Hepatotoxicity. LD50 using the online Protox tool, and online The toxicity class is based on the Globally Harmonized System. RESULT AND DISCUSSION This physicochemical properties with the legal parameters of five Lipinski, activity towards the phosphoinositide 3-kinase enzyme (PI3K/ mTOR. PDB 5OQ. , and the toxicity of the compounds Heliannuols A. Ligand Preparation and Receptors The Dual PI3K / mTOR . OQ. enzyme was used as a receptor protein and the ligand used is the compound Heliannuol A. E with the comparative compound Bimiralisib. The results of two-dimensional images are shown in figure 1. The process of geometry optimization aims to minimize energy to obtain the most stable structure (Susanti et al. , 2. The results of the geometry optimization of Heliannuol A. E, and Bimiralisib compounds are shown in table I. Physicochemical Test The physicochemical test aims to predict the absorption properties of the compounds Heliannuols A. Bimiralisib. Compounds have low absorption if there are more than five donor H-bonds . xpressed as the number of OH and NH), molecular weights more than 500. Log P is more than 5, and there are more than 10 H-bond acceptors . xpressed as the number of Ns and O. (Lipinski et al. , 2. Prediction results of physicochemical properties can be seen in table II. The results of the analysis in table II that the compounds Heliannuols A. E, comparative compounds (Bimiralisi. meet the five Lipinski legal requirements, the compounds are predicted to be easily absorbed. They have good permeability, so it requires further testing related to distribution, metabolism, and excretion in a manner in silico. Determination of Cavity Native sites of binding ligands have potential as active holes so that they can be used for docking ligands from the compound Heliannoul B. Bimiralisib. The cavity at the receptor is shown in green. figure 2 A (A) shows the five cavities that may interact with the 5OQ4 receptor. The five cavities were selected by looking at the places of native ligands that interact with receptors 5OQ4. The results of cavity determination can be seen in figure 2. The results of the selection obtained one cavity, namely volume 452. 608, with a surface area of 1149. Traditional Medicine Journal, 26. , 2021 In Silico Study on the Effect of Heliannuol A. E Compounds of Sunflower (A) (B) (C) (D) (E) (F) Figure 1: Two-dimensional structure of Heliannuols A (A). Two-dimensional structure of Heliannuols B (B). Two-dimensional structure of Heliannuols C (C). Two-dimensional structure of Heliannuols D (D). Twodimensional structure of Heliannuols E (E). Two-dimensional structure Bimiralisib dimension (F). Table I. Results of determining the minimum energy of Heliannuols compounds with MMFF94 in Avogadro Compound Heliannuols A Heliannuols B Heliannuols C Heliannuols D Heliannuols E Bimiralisib Minimum Energy (Kcal/mo. Replication I Replication II Replication i 75,835 75,835 75,834 76,461 76,462 76,460 67,480 67,480 67,480 63,585 63,585 63,585 54,188 54,188 54,188 67,265 67,265 67,265 Average (Kcal/mo. A SD 75,835 A 0,0003 76,461 A 0,0010 67,480A 0,0001 63,585A 0,0000 54,188 A 0,0000 67,265 A 0,0000 Table II. Results of the determination of physicochemical properties and prediction of toxicity to the compounds Heliannuols A. Bimiralisib Parameters of the Five Lipinski Laws Compound Heliannuols A Heliannuols B Heliannuols C Heliannuols D Heliannuols E Bimiralisib BM . /mo. Log P Torsion HBA HBD PSA (A. Application of the Five Lipinski Laws Yes Yes Yes Yes Yes Yes Note: BM (Molecular weigh. <500. Log P (The logarithm of octanol/water partition coefficient <5. Torsion (H-bonding that can rotat. HBD . onor H-bond. <5. HBA (H-bond acceptor. <10. PSA (Polar Surface Activit. Traditional Medicine Journal, 26. , 2021 Roihatul Mutiah (A) (B) Figure 2. The results of cavity detection at the 5OQ4 receptor (A). Cavity that binds to the native ligand 5OQ4 receptor . avity 2 (B)) Note Figure 2A : Cavity 1 Vol = 1339. 39 Surface = 2714. Cavity 2 Vol = 452. 608 Surface = 1149. Cavity 3 Vol = 285. 696 Surface = 697. Cavity 4 Vol = 143. 872 Surface = 559. Cavity 5 Vol = 73. Surface = 243. Note Figure 2B : Red: Heliannuols A. Yellow: Heliannuols B. Green: Heliannuols C. Blue: Heliannuols D. Purple: Heliannuols E. Pink: Bimiralisib (A) (B) (C) (D) (E) (F) Figure 3. The results of two-dimensional interaction of hydrogen bonds . lue lin. and steric bonds . ed line. Heliannuols A (A). Heliannuols B (B). Heliannuols C (C). Heliannuols D (D). Heliannuols E (E). Bimiralisib(F) compounds with the 5OQ4 receptor amino acid. Table i. Method validation results Ligand Nilai RMSD Rerank Score Moldock Score Replication I 0,99 3AW_1201 Replication II Replication i 0,98 Average. ASD 0,923 . A 0,1069 -100 . A 1,4930 -113 . A 5,1411 Note : RMSD : A deviation value that represents the ratio between the ligand receptor conformation in the simulation process and the initial ligand-receptor conformation (Dermawan et al. , 2. Traditional Medicine Journal, 26. , 2021 In Silico Study on the Effect of Heliannuol A. E Compounds of Sunflower (A) (B) (C) (D) (E) Figure 4. Three-dimensional form of interaction between Heliannuols A (A). Heliannuols B (B). Heliannuols C (C). Heliannuols D (D). Heliannuols E (E). and Bimiralisib compounds with the amino acid receptor 5OQ4. Note : Red: Heliannuols A. Yellow: Heliannuols B. Green: Heliannuols C. Blue: Heliannuols D. Purple: Heliannuols E. Pink: Bimiralisib. Method Validation The validation of the method was replicated three times by re-docking the native ligand with the hole . of the 5OQ4 target protein Based on the results of re-docking, the average RMSD value is 0. 923 yI, which indicates that the docking method meets the validity criteria, so that the parameter can be used for docking the test compound (Muttaqin et al. , 2. The results of research validation can be seen in Table i. Traditional Medicine Journal, 26. , 2021 Molecular Docking and Amino Acid Interaction The 5OQ4 receptor docking analysis . n enzyme of Dual PI3K /mTOR) of Heliannuols A. E compounds are obtained. The lowest average rerank score was -69,000 Kcal/mol in Heliannuol E test compound so that it has the highest activity compared to Heliannuols compounds A. Bond energy shows the amount of energy needed to form bonds between ligands and receptors. The smaller the bond energy Roihatul Mutiah Table V. Results of Interactions with Amino Acids Hydrogen Bonding Steric Bonding Amino Acid (Distance . I)) Ligand Group Amino Acid (Distance . I)) Ligand Group Heliannuols A Glu 880 . Ile 963 . Ile 831 . Met 953 . Heliannuols B Val 882 . Met 953 . Glu 880 . Ile 831. Ala 885. Ile 831. Val 882. Heliannuols C Asp 964 . Asp 841. Ile 831. Ile 831. Ile 963. Heliannuols D Tyr 867 . Glu 880. Asp 964. Ile 963. Ile 963 . Met 953. Trp 812. Ile 831. Ile 831. Heliannuols E Glu 880 . Glu 880 . Val 882 . Val 882. Ile 879. Ile 879. Bimiralisib Asp 836 . NH2 Ile 963 . Val 882 . Glu 880. Asp 841 . NH2 Glu 880. Asp 964 . NH2 Asp 964. Asp 841. Compound Table VI. Toxicity results of Heliannuols and Bimiralisib compounds Toxicity Compounds Heliannuols A Heliannuols B Heliannuols C Heliannuols D Heliannuols E Bimiralisib LD50 g/k. * Test AMES** Hepatotoxic ** Yes Skin Sensitivity ** Toxicity Class * Note : * Using Protox II Online Tool. ** Using pkCSM Online Tool means, the more stable the bond is. The more stable the ligand binds with the receptor, it can be predicted that the activity will also be more significant (Hardjono, 2. The bonds formed between the groups in the ligand with the receptors are essential in determining the affinity formed. The OH group bond with the receptor causes the rerank score of Heliannuols A. E to be more excellent, and the bonding groups F. N, and O on the Bimiralisib causes the value of the score to be smaller, so it has a greater affinity. It is because in Bimiralisib there are more bonds with groups that have large electronegativity so that they have a stronger hydrogen bond compared to Heliannuols A. The results of ligand interactions with amino acids in the 5OQ4 receptor are shown in and in table V and figure 2. Figure 3 shows the interaction of hydrogen bonds in Aspartic 964 amino acid residues in Traditional Medicine Journal, 26. , 2021 In Silico Study on the Effect of Heliannuol A. E Compounds of Sunflower Bimiralsib which binds to NH2 groups also occur in Heliannuol C. D compounds that bind to OH ligand groups, but do not occur in Heliannuol A and E. Compounds. At the same time, hydrogen bonds with acid residues Amino Valin 882 in Bimiralisib also occur in Heliannuols B and E. The hydrogen bond occurs when a bond between an H atom has a partial positive charge and another electronegative atom and has a pair of free electrons with complete octets such as O. N, and F (F. Siswandono, 2. Whereas steric interactions in Bimiralisib occur in amino acids Isoleucine 963. Glutamate 880. Aspartate 841 which also binds to Heliannuol A. Compounds Steric bonds are involved in the interaction of benzene of the receptor plane and in the interaction of the hydrocarbon chain with macromolecules or receptors (Siswandono, 2. Research Bianchi et al. , 2004 explained that glioblastoma growth is associated with increased concentrations of choline. GABA, isoleucine, leucine, lysine, phenylalanine, taurine, tyrosine, and valine. In grade i and grade IV tumors in concentrations of extracellular phenylalanine, isoleucine, tyrosine, valine, and lysine, all are increased compared to normal tissue. Toxicity Test The next In Silico research was to predict the compounds Heliannuol A. Bimiralisib. Toxicity prediction results are shown in Table VI, obtained LD50 values of Heliannuol D compound . oxicity class 5 . 0