IN SILICO STUDY OF Hibiscus Sabdariffa Linn. ACTIVE COMPOUNDS IN GLP-1R: POTENTIAL AS ANTIDIABETIC DRUG

GLP-1R


INTRODUCTION
Diabetes mellitus (DM) is a metabolic disorder characterized by hyperglycemia due to reduced insulin production, insulin resistance, or both.(Biessels & Reagan, 2015) In 2019, the International Diabetes Federation (IDF) estimated that there were 463 million individuals with DM, and this number is projected to increase to 578 million in 2030 and 700 million in 2045.(Daniels & Mietlicki-Baase, 2019) Indonesia is included in the IDF Asia-Pacific region, with 19.5 million DM cases found in Indonesia in 2021.(International Diabetes Federation, 2021) DM causes various complications, often associated with long-term damage, dysfunction, and failure of various organs.(Kodl & Seaquist, 2008) The most common complications included persistent kidney failure (nephropathy), nerve injury (neuropathy), which increases the likelihood of ulcers and is the main cause of amputation of the lower extremities, eye disorders (retinopathy) which causes blindness, coronary heart failure, diseases involving peripheral arteries, cognitive impairment, and cerebrovascular disorders.(Saeedi et al., 2019) Some efforts are needed to treat patients appropriately in order to reduce the morbidity and mortality due to its complications.
Management of DM requires multidisciplinary action, which includes pharmacological and non-pharmacological therapy.The high incidence and prevalence of DM encourage researchers to improve and develop better therapy for DM patients that can reduce the risk of complications and increase the quality of life.Incretin-based therapy for DM is currently being developed.(Soelistijo, 2021) The effect of incretin is a response to release more insulin due to high postprandial blood glucose levels, which is responsible for 50-70% of total insulin secretion after oral glucose administration.The incretin effect is played by two hormones, namely GLP-1 and GIP.(Baggio & Drucker, 2007) Glucagon Like Peptide-1 (GLP-1) is one of the incretin hormones produced from the preproglucagon (PPG) gene and secreted by enteroendocrine L cells in response to the presence of glucose in the intestine.(Müller et al., 2019) GLP-1 shows various positive effects, such as increasing insulin secretion and sensitivity and lowering blood glucose levels.(Cho et al., 2014) Other beneficial effects of DM conditions include delayed gastric emptying, maintenance of pancreatic β cells (proliferation, neogenesis, antiapoptosis), inhibition of food intake and energy consumption, and function in increasing glucose uptake by muscle cells.(Fujiwara et al., 2019) Interestingly, GLP-1 also has antiinflammation and antioxidant activity, and also has a positive impact on other organs such as cardiovascular and brain, and organs affected by DM complications.GLP-1 mediates its effects on various organs by binding to the GLP-1 receptor (GLP-1R) which is expressed in its related organs.GLP-1 and GLP-1R bonds will activate this receptor and produce certain cellular signaling and corresponding physiological responses.(Yildirim Simsir et al., 2018) Glucagon Like Peptide-1 receptor agonist is a new class of therapy for DM.This type of drug is represented by Exenatide, Liraglutide, Exenatide LAR, Albiglutide, and Tasphoglutide.Despite its superiority that can naturally mimic human GLP-1, of course there are side effects that patients can experience due to its consumption, including causing nausea, vomiting, constipation, diarrhea, bloating, and other digestive disorders.(Trujillo et al., 2021) (Wharton et al., 2022) Besides that, these drugs are still rare and sold at relatively high prices.(Reid, 2012) Currently, many studies have discussed the potential of new active compounds from natural ingredients, including those derived from plants to be developed as new drug.Indonesia is known as a country with high biodiversity, marked by the many plants that can be used in medicine, one of which is Hibiscus sabdariffa Linn.or Rosella which is known to have the potential to prevent DM and its complications.(Guardiola & Mach, 2014) Hibiscus sabdariffa Linn. is rich in bioactive compounds such as anthocyanins, quercetin, other flavonoids, organic acids, and polysaccharides, which have several beneficial physiological effects, such as antioxidant, antibacterial, anti-inflammatory, anticholesterol and anti-diabetic effects.(Jabeur et al., 2017) The active compounds of herbal medicines are believed to have lower side effects, high bioavailability, and low cytotoxic effects.(Umadevi et al., 2022) Candidates for a new drug in diabetes mellitus in the botanical sector must start with an in silico study.In silico studies have been widely used to search for and design new potential drugs from natural materials and are also expected to optimize the research process and prevent losses in vitro and in vivo research steps in a precise, accurate, fast, and effective manner.The mechanism of antidiabetic effect of Hibiscus sabdariffa Linn.could be through GLP-1 receptor activation.Therefore, this study aims to see how the interactions of the active compounds in Hibiscus sabdariffa Linn.against GLP-1R in silico are.

Materials and Tools
The materials used consisted of the three-dimensional structure of GLP-1R obtained through the Protein Data Bank website (PDB ID = 6ORV) and 39 compounds derived from the Hibiscus sabdariffa Linn.plant.This research used MarvinSketch, AutoDockTools, Ligplot+, and PyMOL software.The Software was operated on Lenovo ThinkPad with i7-4600U processor, 8GB RAM, Windows 13, and 64-bit operating system.

Ligands Preparation.
Ligands are the phytochemical constituents of Hibiscus sabdariffa Linn.from a study by (Herranz-López et al., 2017) as many as 39 compounds were added with N2V as the control positive ligand.Two and threedimensional ligand structures were obtained from the Pubchem® database in .sdfformat.Structures that were not found in the Pubchem® database were drawn manually using MarvinSketch.All structures were optimized in three dimensions and changed the format to .pdb using MarvinSketch.All these ligands were then given charge, set torsion and written as pdbqt format using AutoDockTools-1.5.6.

Protein Preparation
The three-dimensional structure of the GLP-1R was downloaded from the Protein Data Bank (PDB ID: 6ORV).Furthermore, the unused part including water molecules was removed, hydrogen nonpolar was added, gasteiger charged was given, and Grid box size and grid center coordinate were arranged.A grid box of 40 x 50 x 40 Å was generated around the center of the active site residues of receptor.This structure was then converted to pdbqt format.All these steps were conducted using AutoDockTools-1.5.6.

Validation of Molecular Docking Protocol
The main objective of molecular docking is to identify energetically favorable binding modes of ligands into the target receptor's binding site.(Ece, 2018) Validity is determined by redocking the native ligand to a prepared target protein.The method is said to be valid if the Root Mean Square Deviation (RMSD) value obtained is ≤ 2Å.(Puratchikody et al., 2016) To ensure that the tested ligand binds in the ideal position for each ligand, the binding site orientation is carried out using the blind docking method, and the results of all tested ligands show cavity with the highest affinity equal to the comparative ligands.Docking search parameter is used by the Lamarckian genetic algorithm with the number of genetic algorithms being 10 runs, population size is 150, the maximum number of energy evaluating is medium which is 2.500.000, the maximum number of generations is 27.000, spacing default is 0.375 Å, and the default docking parameter is used for running options.
The primary parameters used in the docking process are the free energy of binding (∆G), the inhibition constant (Ki), amino acid residues, and the number of hydrogen bonds.(Kim et al., 2016) ∆G and Ki scores determine ligand affinity to the receptor.The more negative the ∆G and the lower the Ki scores indicate higher ligand affinity toward the active site of the receptor.All tested ligands are then compared with the result of control positive ligand to determine the potency of both tested ligands.(Natesan et al., 2012) The amino acid residues of ligands are then compared with amino acid residues of control positive ligand to assess the similarity of interaction between ligands and control positive ligand.More similar amino acid residues indicate a higher probability that the ligand will have a similar activity to the control positive ligand.(Pena Neshich et al., 2015) Visualization of twodimensional and three-dimensional ligandreceptor interactions was performed using Ligplot+ and PyMOL software.

RESULTS AND DISCUSSION
Molecular docking is a computational research method that aims to estimate the interaction and affinity of a ligand with a macromolecule (usually an enzyme or protein).A ligand and protein molecule are predicted by placement techniques in a specific area (an active site) to provide optimal results (Motiejunas & Wade, 2006).The molecular docking method uses the AutoDock 4.2.6 software application.Docking validation is used successively for the x center (125.194),y center (109.599), and z center (81.738) with an RMSD value obtained from the method validation results of 2.14Å.Molecular docking results show an interaction between the active compounds Hibiscus sabdariffa Linn.which interact with GLP-1R and have an activating function (as an activator).The active compounds Hibiscus sabdariffa Linn.
interacting with GLP-1R are described in the table below.Bond-free energy is a vital aspect for assessing the interaction between the ligand and the receptor.This energy indicates the bond strength (affinity) between the tested ligand and the target protein.The more negative the value of the bond energy obtained, the more stable the complex formed is between GLP-1R (6ORV) and the active compound derived from Hibiscus sabdariffa Linn.Increasingly negative values indicate that the compound has an interaction in the form of increasing attraction between atoms.In contrast, the repulsive forces between particles become minimal so that the conformation of the compound obtained is more stable (Manna et al., 2017).The low bond energy value also indicates that less energy is required to carry out the binding or interaction of a ligand with a receptor.Meanwhile, the inhibition constant is directly proportional to the number of doses needed to cause pharmacological activity.The smaller the inhibition constant, the smaller the required dose is (Vargas et al., 2018).Several factors affect the binding affinity between the ligand and the receptor.Electrostatic, van der Waals, and hydrophobic interactions are important factors besides other factors such as entropy, desolvation, flexibility of the receptor structure, and the structure of the water molecule at the bond site (Haider, 2010).
In this study, three active compounds were found and had the best docking results, namely ethyl chlorogenate acid with ∆G (-7.73 kcal/mol) and Ki (2.16 uM), myricetin-3arabinogalactoside with ∆G (-10.64 kcal/mol) and Ki (15.82 nM), and tetra-Omethyljeediflavanone with ∆G (-9.95 kcal/mol) and Ki (50.48 nM).These three compounds have the closest free bond energy to the positive control ligand value and have the highest binding affinity among the other ligands.The analysis results also obtained amino acids which are thought to play a role in the affinity of the active compounds of Hibiscus sabdariffa Linn. to GLP-1R, as seen in Table 2.
The target binding sites of this protein are located at Trp 297,Trp 203,Met 204,Ala 200,Leu 201,Leu 217,Phe 230,Try145,Try148,Try220,Lys 197.The essential amino acid compounds contained in the positive control ligand, namely Myricetin-3arabinogalactoside, Tetra-Omethyljeediflavanone, and Ethyl chlorogenate acid, which are the three active compounds with the best docking, and they are Trp 297,Trp 203,Met 204,Phe 230,Try 220,Lys 197.Even though it differs from the position of the positive control ligand hydrogen bond (N2V), the presence of hydrogen bonds in the active compound with the best docking value has a vital role in the affinity between the compound and the target protein because it forms electrostatic interactions (donor and acceptor hydrogen) (Muttaqin, 2019).The amino acid residue plays a role in activating GLP-1R to carry out its function.The determined distance between the bonds indicates that the active compound has bonded with amino acid residues.In general, bonds occur at a distance of about 5 Ǻ and not below 1 Ǻ because there will be repulsions between atoms so that it does not meet the general bonding requirements (Faridah et al., 2021).
In Silico Study of Hibiscus Sabdariffa Linn.Active Compounds In Glp-1r: Potential as Antidiabetic Drug (T.Andriani, N. Mawaddah, L. Erlina, R. K. Anggraeni, N. Ibrahim, M. Siagian) 123 The visualization of interactions between the top three ligands and GLP-1R used Ligplot and PyMOL.The comparison between the molecular interactions of the top three ligands and the interactions of the positive control ligand molecules towards the same receptor is shown in Figures 1 and 2  The compounds with the top three docking results were then examined for their physicochemical properties to see the potential of the compounds when developed into medicinal preparations.The physicochemical parameters of the tested compounds were adjusted according to the Lipinski rule.There are five rules: (1) if the test compound has a molecular weight of more than 500 Da, it will be difficult for the compound to penetrate the cell membrane; (2) a log P value greater than 5 indicates that the compound is increasingly lipophilic and binds very tightly to the membrane, making it challenging to identify targets and is also toxic; (3) However, if the log P value is too small or negative, it is also not very good because it will be difficult for the compound to penetrate the lipid bilayer membrane; (4) Hydrogen bond donors and acceptors are the magnitudes of the hydrogen bond capacity; (5) If the hydrogen capacity is higher, the energy required in the absorption process is also higher.In general, the Lipinski rule of five (RO5) indicates the solubility of certain compounds in penetrating the membrane by passive diffusion (Brito, 2011).The test compounds are said to meet the requirements for the formation of oral preparations if there is no more than one violation of the Lipinski rules (Lipinski, 2004).Based on Table 3, of all the top active compounds analyzed, there was one compound that met the Lipinski RO5 requirements and met the requirements for making oral preparations.Further research is needed to determine the pharmacokinetic profile of these active compounds.Then the active compound must have a good ADME profile and be non-toxic, and the top three test compounds will be evaluated for their toxicity through the AdmetSAR website (Cheng et al., 2012).According to the results of the analysis, the top three active compounds are not carcinogenic, non-toxic, and included in the AOT III category, which means they are slightly toxic if ingested orally.Then the three observed ligands show that these ligands have low gastrointestinal absorption.Gastrointestinal absorption can be an indicator of whether oral administration of the compound will be effective or not.Interactions of the compounds with cytochrome P450 (CYP1A2, CYP2C19, CYP2C9, CYP2D6, CYP3A4) were identified.These iso-enzymes play a major role in drug elimination through metabolic biotransformation.If this enzyme is inhibited, the drug or its metabolites may accumulate in the body, causing toxic effects (Yang et al., 2017).Myricetin-3-arabinogalactoside and Ethyl chlorogenate acid are predicted to be noninhibitors for one of the isoenzymes.However, Tetra-O-methyljeediflavanone turned out to be a CYP2C9 inhibitor.
This study shows the potential of the active compound Hibiscus sabdariffa Linn. to interact with GLP-1R.This interaction might cause activation of the GLP-1R and initiate signal transduction and produce associated physiological responses.For example, pancreatic β cells activates several signals that have an impact on insulin secretion, increasing proliferation, and preventing apoptosis of pancreatic β cells (Ma et al., 2014) (Kartinah et al., 2019).Hibiscus sabdariffa Linn.has ability to maintain circulating GLP-1 levels to reach its target in the pancreas (Domínguez Avila et al., 2017).This in silico study showed an additional beneficial effect of Hibiscus sabdariffa Linn.whose active compounds can also act as a ligand independent variable which is predicted to have antidiabetic activity by binding to GLP-1R.

CONCLUSION
Based on the docking results of 39 test compounds on Hibiscus sabdariffa Linn.three candidate compounds with the best binding affinity with GLP-1R were obtained, namely myricetin-3-arabinogalactoside, tetra-Omethyljeediflavanone, and ethyl chlorogenic acid.Based on visualization of the interaction of active compounds in GLP-1R, it can be concluded that amino acids play a role in the active site of the receptor, namely Trp 297,Trp 203,Met 204,Phe 230,Try 220,and Lys 197.However, the results of this study must be validated further through both in vitro and in vivo studies to elucidate the potential activity through GLP-1 and GLP-1R which produce the expected responses in diabetes mellitus management. .

Table 1 .
The Results Of The Comparison Between ΔG And Ki Values From The Docking Of The Active Compounds Hibiscus sabdariffa Linn.With The GLP-1R

Table 2 .
Comparison Between Amino Acid Residues And The Number Of Hydrogen Bonds

Table 3 .
Results Of The Lipinski Rule Of Five

Table 4 .
Toxicity Test Results