ACCEPTED MANUSCRIPT This is an early electronic version of an as-received manuscript that has been accepted for publication in the Journal of the Serbian Chemical Society but has not yet been subjected to the editing process and publishing procedure applied by the JSCS Editorial Office. Please cite this article as P. J. P. Tjitda, F. O. Nitbani, D. Mbunga and T. D. Wahyuningsih, J. Serb. Chem. Soc. (2023) https://doi.org/10.2298/JSC220913045T. This “raw” version of the manuscript is being provided to the authors and readers for their technical service. It must be stressed that the manuscript still has to be subjected to copyediting, typesetting, English grammar and syntax correc- tions, professional editing and authors’ review of the galley proof before it is published in its final form. 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Soc.00(0)1-20 (2023) Original scientific paper JSCS–12064 Published DD MM, 2023 1 Natural flavonoids in Delonix regia leaf as an antimycobacterial agent: an in silico study PUTRA JIWAMURWA PAMA TJITDA1*, FEBRI ODEL NITBANI2, DOMINUS MBUNGA1 and TUTIK DWI WAHYUNINGSIH3 1Department of Pharmacy, Health Polytechnic of Kupang, Indonesia, 2Department of Chemistry, Faculty of Sains and Engineering, Nusa Cendana University, Indonesia, and 3Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Gadjah Mada, Indonesia (Received 13 September 2022; Revised 23 December 2022; Accepted 21 July 2023) Abstract: Multi-drug resistant (MDR) and extensively-drug resistant (XDR) as a result of continuous use of antibiotics encourage the development of new antimycobacterial drugs. In this study, 13 flavonoid compounds from the flamboyant leaf plant were studied for their inhibitory properties of MtKasA, MtDprE, and MtPank which are significant enzymes in Mycobacterium tuberculosis, as well as for their molecular docking, molecular dynamics, and prediction of ADMET-drug likeness. The results of molecular docking studies revealed that compound F13 (Apigenin) was the most potent compound because it was able to bind the most amino acids as indicated by the native ligand of each enzyme. Molecular dynamics studies showed that compound F13 forms a stable complex with MtKasA. The results of the ADMET-Drug Likeness analysis concluded that compound F13 was the most promising compound. Overall, compound F13 has the potential to be used as a treatment therapy against Mycobacterium tuberculosis. Keywords: tuberculosis, molecular docking; MtKasA; MtDprE; MtPank. INTRODUCTION Tuberculosis (TB) is a deadly disease caused by Mycobacterium tuberculosis. Many parties have and are paying special attention to the handling of tuberculosis. WHO, in the global tuberculosis report 2021, reported that the pandemic of COVID-19 has a negative impact on efforts to treat tuberculosis. Limited access to diagnosis and treatment of tuberculosis has increased the number of tuberculosis deaths by 1.2 million since 2019. 1 WHO also noted four countries contributed the most prominent TB cases, namely India (41%), Indonesia (14%), the Philippines (12%), and China (8%). Another report noted that cases of drug-resistant TB against first-line drug usage for TB therapy have increased by 15%. 1 This is *Corresponding authors E-mail: putrachemist_jc@yahoo.com https://doi.org/10.2298/JSC220913045T A cc ep te d m an us cr ip t mailto:putrachemist_jc@yahoo.com https://doi.org/10.2298/JSC220913045T 2 TJITDA et al. primarily due to the prolonged treatment of TB, as well as the use of antibiotic combinations with side effects that result in the development of multi-drug resistant (MDR) and extensively drug-resistant (XDR). 2 As a result of all these reports, tuberculosis treatment needs urgent attention as well as appropriate and quick solutions. There are still many drug discovery studies underway to overcome the problem of drug resistance, including tuberculosis. 3 Molecular docking is a computational method for new drug discovery based on structure-based design. Application of molecular docking methods includes virtual screening, prediction of adverse drug reactions, drug repositioning, and drug mechanism of action against the targeted protein. 4 Therefore, molecular docking is an excellent and powerful method for predicting drug activity against specific proteins. Several targeted proteins to predict drug activity against the growth of Mycobacterium tuberculosis are MtKasA, MtDprE1, and MtPank. 5 MtKasA and MtDprE1 are necessary enzymes used for cell wall biosynthesis. MtKasA is responsible for elongating the fatty acid carbon chain in the FAS-II pathway and provides a precursor for mycolic acid biosynthesis. 6 MtDprE1 performs the epimerization reaction of decaprenyl phosphoryl--D-ribose (DPR) to decaprenyl phosphoryl-- D-arabinofuranose (DPA) in the periplasmic space of the cell membrane. DPA is used for the biogenesis of arabino galactan and lipoarabino mannan. 7,8 MtPank is also an essential drug target to overcome drug-resistant TB. MtPank enzyme plays a role in the growth of Mycobacterium tuberculosis through the biosynthesis of Coenzyme-A (CoA). 9 Various studies have been conducted to study the plant’s active compounds and their biological activity against Mycobacterium tuberculosis. 10–13 The content of secondary metabolites in plants significantly contributes to certain biological activities and is always accompanied by low side effects. 14 Among the secondary metabolites, flavonoids have been reported to have inhibitory activity against the growth of Mycobacterium tuberculosis. 15–17 Chemically, flavonoids have two benzene rings, A and B, which are connected by a pyran heterocyclic ring (C ring). Flavonoid groups include flavonols, flavanones, flavones, isoflavones, flavanols, and anthocyanins. 18 Flamboyant (Delonix regia) is a plant that comes from the Caesalpiniaceae family and is widely found in various regions of Indonesia. Previous studies reported that the isolation of flamboyant leaves with ethanol as a solvent produced 13 flavonoid compounds. 19 The presence of flavonoid led to the investigation of their ability to inhibit 3 key enzymes in the growth of Mycobacterium tuberculosis. Therefore, this study aimed to identify the potential of flavonoid compounds contained in flamboyan leaves as antimycobacterial agents, using an in silico study approach. As a result, the potential compounds obtained could be used as reference compounds for laboratory testing. A cc ep te d m an us cr ip t ANTIMYCOBACTERIAL NATURAL FLAVONOIDS IN DELONIX REGIA LEAF 3 EXPERIMENTAL Materials As test ligands, natural flavonoids were obtained from previous studies investigating the HPLC profiles of Delonix regia leaves extract.19 The proteins analyzed in this study include MtKasA, MtDprE1, and MtPank taken from the RSCB database with PDB ID were 2WGE, 4FF6, and 4BFT, respectively. The software used for molecular docking was AutoDock Vina; the structures of ligand compounds were drawn with ChemOffice Professional 16 and Chem3D Professional 16, and structure optimization was performed using Gaussian 09. Molecular docking results were visualized in Discovery Studio client 2021. Validation of docking protocol Each receptor protein was prepared by separating the native ligand from the protein using AutoDock Tool (ADT) 1.5.6. Missing atoms of the receptor protein were checked and corrected through ADT. Furthermore, the water molecule on the receptor protein was cleaned and the protein was added with Kollman Charge and then stored in the pdbqt extension format. The separated native ligands were prepared by adding hydrogen atoms and Gasteiger charge. Prepared proteins and native ligands were then docked by following several parameters. The molecular docking for MtKasA protein used a grid box dimension of 25 x 25 x 25 Å with cartesian coordinates x, y, and z respectively 37.883, 0.813, and -5.898. MtDprE1 protein used a grid box size of 12 x 12 x 12 Å with cartesian coordinates x, y, and z respectively 14.697, - 21.165, and 36.955 and for MtPank protein, it was 14 x 14 x 14 Å with cartesian coordinates x, y, and z respectively -19.242, -10.217, and 12.648. An exhaustiveness of 16 was applied to the protocol docking validation process. In order to dock all proteins, the spacing value of 1 Armstrong was used. RMSD value was determined using Pymol. The docking protocol was used to dock the tested compound if it had an RMSD value below 2 Å.20 Preparation of natural flavonoid as tested ligand The structure of natural flavonoids from Delonix regia leaves extract was taken from the PubChem database. Compounds that were not found in the database were drawn with ChemDraw Professional 16, as shown in Fig. 1. Furthermore, all compounds were converted to 3-dimensional structure and geometric optimization using Gaussian 09 with the DFT method with a base set B3LYP 6-31G.21 Furthermore, the test compound was prepared using ADT, which involved the addition of hydrogen atoms, merging non-polar hydrogen atoms, and adding Gasteiger charge. All compounds that had been prepared were then stored in a pdbqt format file. Antimycobacterial profile prediction The antimycobacterial properties of 13 flavonoids contained in flamboyant leaf extract were predicted. Each SMILE code of the flavonoid compound was inputted into PASS online (http://www.way2drug.com/passonline/predict.php). Activity Predicted values (Pa) and Inactive Predictions were recorded, tabulated, and presented in graphical form. Molecular docking Natural flavonoids that had been optimized were docked to the three receptor proteins. Docking was conducted using AutoDock Vina following a validated docking protocol. The docking results generated binding energy as a value that indicated the binding energy between the ligands and the receptor protein. The 2D and 3D interactions of the tested compound with A cc ep te d m an us cr ip t http://www.way2drug.com/passonline/predict.php 4 TJITDA et al. key amino acid residues of the receptor protein were visualized using Discovery Studio client 2021. ADMET-Drug-likeness analysis Pharmacokinetic properties of natural flavonoid compounds, including adsorption, distribution, metabolism, excretion, and toxicity, were predicted using the pkCSM tool.22 The drug-likeness properties were studied using the MolSoft tool (https://www.molsoft.com/publications.html). Molecular dynamic simulation The lead compounds obtained from molecular docking simulations were then studied for the stability of their interactions with amino acid receptors, in this case MtKasA, MtDprE1, and MtPank. This was done through molecular dynamic simulations. Simulations used CAB Flex 2.0 server (http://biocomp.chem.uw.edu.pl/CABSflex2/index). The simulation setting used a number of cycles and 50 trajectory frames. Furthermore, the temperature of the simulation and the global weight were respectively 1.4 and 1.0. RESULTS AND DISCUSSION Validation of docking protocol The molecular docking method is a powerful in silico method for predicting the interaction between ligands and receptors. 23 To study the antimycobacterial properties of natural flavonoids in the ethanolic extract of Delonix Regia leaves, three significant proteins were used to inhibit Mycobacterium Tuberculosis activities. These proteins were MtKasA, MtDprE1, and MtPank. Before molecular docking of the test compounds was carried out on the three proteins, docking protocol validation was performed. As shown in Fig. 1, the native ligands are superimposed and include MtKasA complexes with thiolactomycin (TLM), MtDprE1 complexes with 3-(hydroxyamino)-N-[(1R)-1-phenylethyl]-5- (trifluoromethyl)benzamide (0T4), and MtPank with 2-chloro-N-[1-(5-[[2-(4- fluorophenoxy)sulfanyl]-4-methyl-4H-1,2,4-triazo-3-yl]benzamide (ZVT). Validation results in the form of RMSD values for MtKasA, MtDprE1, and MtPank were 0.828 Å, 1.512 Å, and 0.689 Å, respectively. Antimycobacterial profile prediction Prediction of the antimycobacterial profile of flavonoid compounds was carried out using the PASS server and gave results as shown in Fig. 2. The results of the analysis from the PASS server showed that all compounds had a Pa value >0.3, however the value was still <0.7 except for compound F5 (0.276). This means that the compound was predicted to have moderate anti-mycobacterial potential in the laboratory. 24 Furthermore, this compound could be further confirmed through molecular docking. A cc ep te d m an us cr ip t https://www.molsoft.com/publications.html http://biocomp.chem.uw.edu.pl/CABSflex2/index ANTIMYCOBACTERIAL NATURAL FLAVONOIDS IN DELONIX REGIA LEAF 5 A) B) C) Fig 1. Superimpose re-docking ligand (green) into native ligand (blue) of protein receptor: A) Complex of MtKasA (id pdb: 2wge) - TLM; B) Complex of MtDprE1 (id pdb: 4ff6) - 0T4; C) Complex of MtPank (id pdb: 4bft) - ZVT F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Compound ID Pa Value Pi Value Fig 2. Graph of antimycobacterial profile prediction by flavonoid compounds from flamboyant leaves Molecular docking The chemical structure of flavonoid compounds contained in flamboyant leaf extract that were used as tested ligands, is shown in Table I. In this study, molecular docking of flavonoids in flamboyant plants was conducted to determine potential compounds for antituberculosis drugs. The binding energy and chemical A cc ep te d m an us cr ip t 6 TJITDA et al. interactions of each flavonoid with the targeted protein were studied and compared to natural inhibitors of each enzyme. TABLE I. Natural flavonoid compounds from Flamboyant leaves (Delonix regia). Comp. ID Chemical structure PubChem ID IUPAC name (PubChem generated) F1 137832345 6-[3,4-dihydroxy-5- (hydroxymethyl)oxolan- 2-yl]-5,7-dihydroxy-2- (4-hydroxyphenyl)-8- [3,4,5-trihydroxy-6- (hydroxymethyl)oxan-2- yl]chromen-4-one F2 74977441 5,7-dihydroxy-2-(4- hydroxyphenyl)-8- [3,4,5-trihydroxy-6- (hydroxymethyl)oxan-2- yl]-6-(3,4,5-trihydroxy- 6-methyloxan-2- yl)chromen-4-one F3 5291488 2-(3,4- dihydroxyphenyl)-5- hydroxy-7-[3,4,5- trihydroxy-6- (hydroxymethyl)oxan-2- yl]oxychromen-4-one F4 3594 5-hydroxy-2-(3- hydroxy-4- methoxyphenyl)-7- [3,4,5-trihydroxy-6- [(3,4,5-trihydroxy-6- methyloxan-2- yl)oxymethyl]oxan-2- yl]oxy-2,3- dihydrochromen-4-one F5 5315615 3-(3,4- dihydroxyphenyl)-2- [(E)-3-(3,4- dihydroxyphenyl)prop- 2-enoyl]oxypropanoic acid F6 5385553 5-hydroxy-2-(4- hydroxyphenyl)-7- [3,4,5-trihydroxy-6- (hydroxymethyl)oxan-2- yl]oxychromen-4-one A cc ep te d m an us cr ip t ANTIMYCOBACTERIAL NATURAL FLAVONOIDS IN DELONIX REGIA LEAF 7 F7 5459217 7-[4,5-dihydroxy-6- (hydroxymethyl)-3- (3,4,5-trihydroxy-6- methyloxan-2- yl)oxyoxan-2-yl]oxy-5- hydroxy-2-(4- hydroxyphenyl)chromen -4-one F8 12305415 5-hydroxy-2-(4- hydroxyphenyl)-3,7- bis[(3,4,5-trihydroxy-6- methyloxan-2- yl)oxy]chromen-4-one F9 5353915 2-(3,4- dihydroxyphenyl)-5,7- dihydroxy-3-(3,4,5- trihydroxy-6- methyloxan-2- yl)oxychromen-4-one F10 131752460 [2-[5,7-dihydroxy-2-(4- hydroxyphenyl)-4- oxochromen-3-yl]oxy- 4,5-dihydroxy-6- (hydroxymethyl)oxan-3- yl] (E)-3-(4- hydroxyphenyl)prop-2- enoate F11 15559329 7-[4,5-dihydroxy-6- (hydroxymethyl)-3- (3,4,5-trihydroxy-6- methyloxan-2- yl)oxyoxan-2-yl]oxy-5- hydroxy-2-(4- methoxyphenyl)chromen -4-one F12 5280863 3,5,7-trihydroxy-2-(4- hydroxyphenyl)chromen -4-one A cc ep te d m an us cr ip t 8 TJITDA et al. F13 5280443 5,7-dihydroxy-2-(4- hydroxyphenyl)chromen -4-one Molecular docking of the MtKasA enzyme was carried out on 13 flavonoids contained in flamboyant leaves and the results are shown in Table II. For MtKasA molecular docking, TLM which acted as a natural inhibitor, produced a binding energy value of -32.22 kJ/mol, while some other test compounds also demonstrated satisfactory inhibitory activity compared to natural inhibitors. F6 compound had the highest binding energy (-38.91 kJ/mol), followed by the F5 compound with a binding energy value of -36.40 kJ/mol, while F3, F12, and F13 compounds showed the same binding energy value of -35.95 kJ/mol. TABLE II. Binding energy of natural flavonoid compound from Delonix regia leaf. Id compound Name Binding energy (kJ/mol) MtKasA MtDprE1 MtPank TLM Thiolactomycin -32.22 - - 0T4 3-(hydroxyamino)-N-[(1R)-1-phenylethyl]- 5-(trifluoromethyl)benzamide - -37.66 - ZVT 2-chloro-N-[1-(5-{[2-(4- fluorophenoxy)ethyl]sulfanyl}-4-methyl- 4H-1,2,4-triazol-3-yl)ethyl]benzamide - - -39.33 Flavonoid compounds F1 Apigenin-6-arabinose-8-galactose -28.03 -33.47 -36.82 F2 Apigenin-6-rhamnose-8-glucosa -28.87 -33.47 -34.31 F3 Luteolin-7-glucose -35.98 -35.56 -36.82 F4 Hesperidin -35.15 -34.31 -35.15 F5 Rosmarinate -36.40 -33.47 -30.96 F6 Apigenin-7-O-glucose -38.91 -37.66 -39.33 F7 Apigenin-7-O-neohespiroside -31.38 -35.98 -32.64 F8 Lespedin -30.96 -35.98 -31.38 F9 Quercetin-3-O-Rhamnoside -24.69 -33.47 -34.73 F10 2''-O-Trans-P-Coumaroylastragalin -30.54 -39.75 -39.33 F11 Fortunellin -31.80 -34.31 -37.66 F12 Kaempferol -35.98 -33.89 -34.31 F13 Apigenin -35.98 -34.31 -33.05 A cc ep te d m an us cr ip t ANTIMYCOBACTERIAL NATURAL FLAVONOIDS IN DELONIX REGIA LEAF 9 A) B) C) D) E) F) Fig 3. Chemical interaction of MtKasA enzyme with A) TLM; B) compound F6; C) compound F5; D) compound F3; E) compound F12; F) compound F13. A cc ep te d m an us cr ip t 10 TJITDA et al. An investigation of the chemical interaction of TLM in the MtKasA enzyme binding pocket has been carried out (Table SI). The oxygen atom of the thiolactone ring is linked to Cys171 by hydrogen bonds. The binding of His311 was formed by the pi-sulfur bond on the S atom of the thiolactone ring. The bond stabilization in the enzyme MtKasA binding pocket was facilitated by hydrophobic interactions with amino acids Phe237, Phe404, and Pro280. This fact was in accordance with the results of a study conducted and reported by Luckner et al. that the catalytic triad Cys171-His311-His345 was the active site of the MtKasA binding pocket (Fig. 3). 25 F5 and F6 compounds showed hydrogen bonds to the His311 amino acid. Hydrogen bonds also supported the stabilization of F6 compounds in interacting with Pro280. F5 compound has slightly lower binding energy, whereas binding of the compound to Pro280 involves hydrophobic interactions. The loss of hydrogen bonds in the catalytic triad was suspected as a reason for the lower binding energy of F3, F12, and F13 compounds. Additionally, F3, F12, and F13 compounds involving hydrophobic interactions also bind to essential amino acids like Cys171, Pro280, and Phe404. This evidence was an indication that F3, F12, and F13 compounds also interact at the MtKasA enzyme active site. Inhibition of the tested compounds against the MtDprE1 enzyme was also tudied. Molecular docking revealed that all analyzed compounds were potent inhibitors of the MtDprE1 enzyme with binding energies ranging from -33.89 to -39.75 kJ/mol. The F10 compound was the tested compound with the largest binding energy, which was -39.75 kJ/mol, while the F6 compound produced the same binding energy as the native ligand (0T4), which was -37.66 kJ/mol. In this study, we also found that the core structure of flavonoid compounds in apigenin and kaempferol had excellent binding ability. F7 and F13 compounds with an apigenin core structure had binding energies of -35.98 and -34.31 kJ/mol, respectively. F8 and F12 compounds, which had a kaempferol core structure, gave bond affinities of -35.98 and -33.89 kJ/mol, respectively. The chemical interaction visualization of the tested compound against the MtDprE1 enzyme provided data to support data binding energy. 0T4 compound as a native ligand bound several key amino acids (Table SII). Cys387 amino acid is found in the substrate-binding domain of MtDprE1 enzyme. 26 In addition, several amino acids, such as Lys134, Gly117, and Gly113, which are located in the FAD- interacting domain, 7 were also seen in Fig. 4. F10 compound was able to bind the amino acid Cys387 through hydrogen bonding. Lys134 and Gly117 amino acids were also bonded via hydrogen bonding with the F10 compound. For the F7 compound, adherence to Cys387 and Lys134 amino acids involved hydrophobic interactions, and the F13 compound only attached Lys134 via hydrogen bonding. In this case, the hydroxyl group of the rhamnoside substituent is used to bond the F8 compound to Cys387 essential amino acid. The weak binding affinity of the A cc ep te d m an us cr ip t ANTIMYCOBACTERIAL NATURAL FLAVONOIDS IN DELONIX REGIA LEAF 11 F12 compound was due to hydrophobic interactions attached to Cys387 amino acid, according to binding energy data. Molecular docking studies of the tested compounds were also carried out on pantothenate kinase (MtPank). Binding energy data showed that all the tested compounds had excellent affinity potential for the MtPank enzyme, ranging from -30.96 to -39.33 kJ/mol. This study used the native ligand ZVT as a comparison ligand. The molecular docking results showed that flavonoid core structures in apigenin and kaempferol gave the most effective binding affinity, as could be seen for F6 and F10 compounds with a binding energy value of -39.33 kJ/mol. It was also revealed that compounds derived from the apigenin core structure, such as those in F1 and F2 compounds resulted in slightly lower binding energy in molecular docking results. With the MtPank, the F11 compound also produced a fairly significant binding affinity of -37.66 kJ/mol compared to MtKasA and MtDprE1 enzymes. In accordance with the binding energy value, the F11 compound did not have the ability to bond to MtKasA or MtDprE1. It was interesting to discuss the chemical interactions between all tested compounds and the MtPank enzyme. ZVT compounds as native ligands showed interactions in the MtPank binding pocket (Table SIII). Triazole ring of ZVT compound formed hydrogen bonds with Tyr235 and Asn277 with bond lengths of 1.94 Å and 2.46 Å, respectively (Fig. 5). These two amino acids were active sites for binding to pantothenate.9,27 Bond stabilization was also formed through binding the ZVT compound with Arg238 via hydrogen bonding. In addition, the hydrophobic interaction of ZVT with Met242 in the presence of a benzene ring also played a role in stabilizing the bonding of the ZVT-MtPank complex. In general, all the tested compounds showed the same inhibitory mechanism. All tested compounds were in the binding pocket and bound to the active site of the MtPank enzyme. For the F10 compound, the hydroxyl group of the glucose substituent facilitated hydrogen bonding with Tyr235. In contrast, the Asn277 amino acid was bonded by the oxygen atom of the coumaroyl carbonyl group. F6 compound only binds Tyr235 with a bond length greater than 3.87 Å, which matches the binding energy value and is slightly lower than the F10 compound. A cc ep te d m an us cr ip t 12 TJITDA et al. A) B) C) D) E) Fig 4. Chemical interaction of MtDprE1 enzyme with A) 0T4; B) compound F10; C) compound F7; D) compound F13; E) compound F8. A cc ep te d m an us cr ip t ANTIMYCOBACTERIAL NATURAL FLAVONOIDS IN DELONIX REGIA LEAF 13 A) B) C) Fig 5. Chemical interaction of MtPank enzyme with A) ZVT; B) compound F10; C) compound F6. It was strongly suspected that the presence of apigenin and kaempferol as core structures contributed to the inhibition of the tested compound against the MtPank enzyme. This was shown by the interactions of F12 and F13 compounds. Visualization of the bond between these two compounds exhibited the presence of binding to two key amino acid residues of the MtPank enzyme. F12 and F13 compounds showed very effective binding to Tyr235 and Asn277. This means that these two compounds were in the binding pocket of MtPank and had the same inhibitory mechanism. These molecular docking results confirm that F12 and F13 compounds were potentially active as MtPank enzyme inhibitors. A cc ep te d m an us cr ip t 14 TJITDA et al. ADMET-Drug-likeness analysis The pharmacokinetic properties of the tested compounds were further analyzed using pkCSM web tools to reveal their bioavailability. The results are presented in Table II. In terms of absorption, the highest absorption ability across the intestinal membrane was shown by F12 and F13 compounds with values of >80%. Furthermore, distribution character was studied through the value of Volume of distribution (VD), blood-brain barrier (BBB) permeability, and Central Nervous System (CNS) permeability. All of the tested compounds showed moderate (-0.15-0.45 log L Kg -1 ) to low (VD < -0.15 log L Kg -1 ) VD values. F8, F9, F10, F11, and F13 compounds with low VD indicated that these compounds tended to be distributed in plasma. Furthermore, the BBB permeability value revealed that all compounds were unable to penetrate the BBB (<1.00). 22 The same case was also shown by the CNS permeability value of all tested compounds. The results of the analysis showed that all compounds had poor absorption into the CNS. Table II. ADMET properties of natural flavonoid compounds from Delonix regia leaf. Id Comp Absorption Distribution Metabolism Excretion Toxicity Intestinal absorption Volume of distribution (VDss) BBB permeability CNS permeability Substrate CYP Inhibitor Total clearance AMES toxicity 2D6 3A4 2D6 3A4 Numeric (%absorbed) Numeric (log L Kg-1) Numeric (log BB) Numeric (log PS) Categorial (Yes/No) Numeric (log mL min-1 Kg-1) Categorial (Yes/No) Flavonoid compounds F1 21.25 0.01 -2.87 -5.82 No No No No 0.32 Yes F2 30.96 0.04 -2.80 -5.79 No No No No 0.34 Yes F3 56.15 0.10 -1.82 -4.51 No No No No 0.69 No F4 38.87 0.36 -2.58 -5.18 No Yes No No 0.59 Yes F5 48.16 0.41 -1.59 -3.62 No No No No 0.42 No F6 41.10 0.11 -1.52 -4.03 No No No No 0.60 No F7 30.44 0.03 -2.33 -5.33 No No No No 0.50 No F8 26.98 -0.40 -1.81 -5.22 No No No No 0.09 No F9 60.01 -0.24 -2.11 -4.83 No No No No 0.64 No F10 61.35 -0.98 -1.80 -4.15 No No No Yes 0.14 No F11 34.94 -0.22 -2.32 -5.29 No No No No 0.60 Yes F12 84.79 0.01 -1.32 -2.44 No No No Yes 0.62 Yes F13 91.64 -0.18 -1.05 -2.25 No No No Yes 0.67 No Cytochrome P450 is an enzyme in charge of drug metabolism, and in this study, the isoforms used were 2D6 and 3A4. The analysis showed that only the F4 compound could act as a CYP3A4 substrate and F10, F12, and F13 compounds were inhibitors of CYP3A4. The next analysis was total clearance, a value describing the drug’s ability to be metabolized in the liver and excreted through the kidney. 28 The results revealed that almost all compounds had a fairly acceptable A cc ep te d m an us cr ip t ANTIMYCOBACTERIAL NATURAL FLAVONOIDS IN DELONIX REGIA LEAF 15 total clearance value ranging from 0.14-0.67, except for the F8 compound, which had a low total clearance value (0.09). Furthermore, the toxicity properties of a drug were also a critical aspect of further drug development. The results of AMES toxicity analysis found that F1, F2, F4, F11, and F12 compounds had mutagenic and carcinogenic properties, but other compounds had the opposite properties. Thus, they had the potential to be further developed as drug candidates. Table III. Drug-likeness of natural flavonoid compounds from Delonix regia leaf. Compound MW Log P Number of HBA Number of HBD PSA (A2) Violation of Lipinski rule Drug- likeness score Flavonoid compound F1 564.49 -0.18 14 10 198.92 3 0.26 F2 578.16 -0.00 14 10 196.75 3 0.36 F3 448.10 0.47 11 7 151.29 2 0.60 F4 610.19 -0.81 15 8 186.70 3 0.94 F5 360.08 1.54 8 5 114.28 - 0.37 F6 432.11 0.91 10 6 135.81 1 0.59 F7 578.16 0.30 14 8 182.25 3 0.81 F8 578.16 -0.71 14 8 179.86 3 0.73 F9 448.10 0.32 11 7 150.41 2 0.82 F10 578.14 1.71 12 7 167.32 3 0.63 F11 592.18 0.82 14 7 172.17 3 0.70 F12 286.05 1.61 6 4 87.13 - 0.50 F13 270.05 3.22 5 3 73.57 - 0.39 Drug-likeness evaluation of all tested compounds was then carried out and used as a screening stage based on the Lipinski rule. 29 Table III presents the data from the drug-likeness analysis of all tested compounds. F5, F12, and F13 compounds did not violate the Lipinski rule. The molecular weight of the compound (MW, >500) indicated that the compound had poor absorption ability. Log P value described the lipophilic character and all tested compounds had a Log P value below the cut-off value (>5). Referring to the Lipinski rule, hydrogen bonding acceptor (HBA) must be below 10, and hydrogen bonding donor (HBD) must be below 5. 30 These two values were correlated with the Polar Surface Area (PSA) value. When a compound has a violation of HBA and HBD, it tends to have low bioavailability with a PSA value greater than 140.00 A 2 . All the tested compounds had HBA >10 and HBD > 5 except compounds F5, F12, and F13. For the drug-likeness score, all compounds gave a fairly acceptable value with a range of 0.26-0.94. Compound F4 had the largest drug-likeness score, namely 0.94. Unfortunately, this compound also had toxic properties based on the results of the AMES toxicity analysis. Compared with the results of ADMET analysis and drug-likeness analysis, the F12 compound had toxic properties while the F5 compound had poor adsorption A cc ep te d m an us cr ip t 16 TJITDA et al. characteristics. As a result, compound 13 as the lead compound is further investigated for its stability against all 3 enzymes. Molecular dynamic simulation Molecular dynamics using the CABS Flex 2.0 server was performed and provided a Root Mean Square Fluctuation (RMSF) graph to evaluate the stability of F13 key important enzymes of mycobacterium tuberculosis (Fig. 6). As reported by Jamroz et al., RMSF analysis using the CABS Flex server demonstrated significant correlation with NMR spectroscopy analysis. 31 As shown in Fig. 6, compound F13 formed the most stable complex against the MtKasA enzyme. It was found that the fluctuating value of all amino acid residues was 1-3 Å. 32 The F13-MtDprE1 complex showed high fluctuations at residue numbers 17, 60, 166, 258, and 284. Meanwhile, high fluctuation level for residue numbers 85, 248, 249, 250, 251, and 252 were found in the F13-MtPank complex. The high fluctuating value means that the flexibility of amino acid residues is very considerable. This reduces the stability of the complex. Although the results of in silico studies indicated that compound F13 was the most effective compound in inhibiting mycobacterium tuberculosis by forming a stable complex with the MtKasA enzyme. There is still a need to confirm compound F13's activity in vitro and in vivo through laboratory experiments. A cc ep te d m an us cr ip t ANTIMYCOBACTERIAL NATURAL FLAVONOIDS IN DELONIX REGIA LEAF 17 A) B) C) Fig 6. Multimodel and RMSF of compound F13 against: A) MtKasA enzyme; B) MtDprE1 enzyme; and C) MtPank enzyme. A cc ep te d m an us cr ip t 18 TJITDA et al. CONCLUSION In silico study of natural flavonoid compounds from Delonix regia leaves extract showed that all tested compounds had antimycobacterial properties based on in silico analysis. ADMET and drug-likeness screening showed that among all the existing test compounds, only compound F13 had the potential to be studied further as an antimycobacterial candidate with binding energy values for MtKasA, MtDprE1, and MtPank respectively -35.98 kJ/mol, -34.31 kJ/mol, and -33.05 kJ/mol. The molecular dynamics simulation results suggested that compound F13 formed the most stable complex against the MtKasA enzyme compared to MtDprE1 and MtPank. SUPPLEMENTARY MATERIAL Supplementary Materials are available electronically from https://www.shd- pub.org.rs/index.php/JSCS/article/view/12064 , or from the corresponding authors on request. И З В О Д ПРИРОДНИ ФЛАВОНОИДИ У ЛИСТОВИМА DELONIX REGIA КАО АНТИМИКОБАКТЕРИЈСКИ АГЕНСИ: IN SILICO СТУДИЈА PUTRA JIWAMURWA PAMA TJITDA1, FEBRI ODEL NITBANI2, DOMINUS MBUNGA1 И TUTIK DWI WAHYUNINGSIH3 1Department of Pharmacy, Health Polytechnic of Kupang, Indonesia, 2Department of Chemistry, Faculty of Sains and Engineering, Nusa Cendana University, Indonesia, и 3Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Gadjah Mada, Indonesia Отпорност на многоструке лекове (MDR) и екстензивна отпорност на лекове (XDR) као резултат сталне употребе антибиотика охрабрује развијање нових антимикобактериских лекова. У овом раду је проучавано 13 флавоноидних једињења из листова биљке Delonix regia због њихових инхибиторних особина за MtKasA, MtDprE, и MtPank, који су значајни ензими код Mycobacterium tuberculosis, као и њиховог молекулског докинга, молекулске динамике, те предиђања ADMET сличности са лековима. Резултати студије молекулског докинга откривају да је једињење F13 (Apigenin) било најпотентније једињење пошто је било у стању да веже већину аминокиселина на које указује нативни лиганд сваког ензима. Студије молекулске динамике су показале да једињење F13 формира стабилан комплекс са MtKasA. Резултати ADMET-анализе сличности са лековима закључују да је једињење F13 оно које највише обећава. Све у свему, једињење F13 има потенцијал да се користи као терапија лечења Mycobacterium tuberculosis. (Примљено 13. септембра 2022, ревидирано 23. децембра 2022, прихваћено 21. јула 2023.) A cc ep te d m an us cr ip t https://www.shd-pub.org.rs/index.php/JSCS/article/view/12064 https://www.shd-pub.org.rs/index.php/JSCS/article/view/12064 ANTIMYCOBACTERIAL NATURAL FLAVONOIDS IN DELONIX REGIA LEAF 19 REFERENCES 1. 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