ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. IN SILICO STUDIES OF COMPOUNDS PRESENT IN AZADIRACHTA INDICA (NEEM) AND THEIR ABILITY TO BIND HIV INTEGRASE PROTEIN ZHONGRUI ZHANG, YIN HEI LAU SONIA ARORA (FACULTY ADVISOR) ✵ ABSTRACT Azadirachta indica (Neem) is an evergreen tree that belongs to the Meliaceae family. It is native to the Indian subcontinent and grows worldwide. It is also known as the "village pharmacy" in India for its wide range of therapeutic and pharmacological properties. An in vitro study indicated that A. indica showed anti-HIV properties. However, the exact mechanism for the supposed anti-HIV properties re- mains unknown. This study aimed to construct an in- silico database of the compounds present in A. in- dica and propose a computational analysis of these compounds against HIV integrase. We performed a thorough literature search to gather relevant infor- mation on the plant compounds, including chemical structure, location within the plant, extraction method, and percent yield of each compound found in the plant. We took a comprehensive ap- proach to closely study the binding pockets of HIV integrase and performed molecular docking on A. indica compounds using Molecular Operating Envi- ronment. A deductive analysis of the docking ener- gies of these compounds revealed thirty potential binders against HIV integrase proteins. We further validated these binders by comparing the ligand in- teractions to known inhibitors using Ligplot+, which identified the presence of numerous hydrogen bonds and hydrophobic interactions at the protein binding pocket. In conclusion, we propose an un- derlying binding potential for several A. indica com- pounds with HIV integrase, yielding a potential mechanism for the anti-HIV activity of A. indica. KEY TERMS: Neem, anti-HIV, medicinal plant, molecular docking 1 INTRODUCTION Azadirachta indica (family: Meliaceae), com- monly known as neem, is a large, fast-growing, trop- ical evergreen tree that has been widely used in tra- ditional medicine since prehistoric times (Abdel- hady et al., 2015; Paul, Prasad, & Sah, 2011; Sadeghian & Mortazaienezhad, 2007). A. indica is indigenous to the Indian subcontinent and is culti- vated in at least 30 countries worldwide (Abdelhady et al., 2015). In India, A. indica is also known as "the village pharmacy," "the wonder tree," "nature's drug store," and "the life-giving tree" (Hossain et al., 2013; Patel et al., 2016; Paul et al., 2011). All parts of the tree can be used in disease treatments due to the presence of various phytochemicals. Medicinal plants play an important role in the health of human society. A. indica is a medicinal plant with a broad spectrum of therapeutic applica- tions. Bioactive phytochemicals, such as flavonoids, terpenoids, tannins, carbohydrates, and proteins, provide A. indica with its healing properties. Many unique compounds have been identified and iso- lated from all parts of A. indica (Sarah et al., 2019). Azadirachtin, nimbin, gedunin, and quercetin are some of the most studied compounds in A. indica. These compounds carry various biological and pharmacological properties such as antimicrobial, antiviral, antifungal, antimalarial, anti-inflammatory, antiulcer, and anticancer properties (Jerobin et al., 2015 and Paul et al., 2011). Clinical trials have af- firmed various therapeutic properties of neem. For example, neem bark extract was found to control gastric hypersecretion as well as gastroesophageal and gastroduodenal ulcers (Bandyopadhyay et al., 2004). A recent randomized controlled trial also found the therapeutic potential of neem in prevent- ing COVID-19 infection (Nesari et al., 2021). There- fore, A. indica has been used in the treatment of fe- ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV ver, malaria, intestinal infections, inflammation, ar- thritis, and skin diseases (Abdelhady et al., 2015; Anyaehie, 2009). Because of its medicinal proper- ties in disease prevention and treatment, the United Nations proclaimed A. indica as the "Tree of the 21st Century” (Hossain et al., 2013). The human immunodeficiency virus (HIV) is a retrovirus that attacks the human immune system. The genetic material of the retrovirus is inserted into the host genome by the retroviral integrase during the process of integration (Komal et al., 2020 and Smith & Daniel, 2006). As a result of a weakened im- mune system, various symptoms — such as fever, cough, swollen lymph nodes, mouth ulcers, and muscle aches — can develop. If HIV is not treated properly, it can lead to severe diseases such as tu- berculosis, cryptococcal meningitis, cancers, and acquired immunodeficiency syndrome (AIDS) (World Health Organization [WHO], 2021; Centers for Disease Control and Prevention [CDC], 2021). Hence, urgent treatment is necessary for HIV pa- tients. According to the WHO, there were approxi- mately 37.7 million HIV cases in 2020. However, due to the development of resistance to current medica- tion targeting integrase, the investigation of new in- tegrase inhibitors is needed (Mesplède et al., 2012). In a previous in vitro study conducted by Udeinya et al. (2004), a fractionated acetone-water extract pre- pared from A. indica showed anti-cytoadhesion ac- tivity, which protects lymphocytes against invasion by HIV and suggests the anti-retroviral property of this plant. In-silico screening, or computer-aided drug design (CADD), has become a crucial part of the modern drug discovery process. It uses a variety of bioinformatics applications and algorithms to effi- ciently screen for potential drug candidates and sig- nificantly reduce the time and resources needed in the traditional lab-bench-based drug delivery pro- cess (Rodrigues and Schneider, 2015). In addition, the aforementioned algorithms can be used to pre- dict the pharmacological properties and interac- tions of molecules. Molecular docking is a type of CADD that predicts the protein-ligand interaction between the drug target and the drug candidate. It runs computer simulations of the potential drug candidates (ligands) with different 3D postures in- teracting with the drug targets (proteins) and measures the favorableness of such interactions in terms of binding energy. Such predictions could therefore be used as the first step of drug candidate screening and can eliminate unlikely candidates within a relatively short time frame using fewer re- sources. Despite recent findings on A. indica’s anti- HIV potential, the exact mechanism of action is still unknown. Previous studies from our lab have fo- cused on several anti-HIV targets such as HIV prote- ase and reverse transcriptase. However, none of these studies have led to conclusive data (un- published observations). Therefore, this study aimed to construct an in-silico database of the com- pounds present in A. indica and propose a compu- tational analysis of these compounds against HIV in- tegrase — one of the important proteins in the HIV life cycle — to investigate the potential inhibitory ac- tivity in reducing viral load. 2 METHODS LITERATURE REVIEW AND DATABASE BUILDING A literature search on A. indica was first con- ducted to collect common compounds present in this plant with readily available structures. For each of these compounds, the percent yield and the lo- cation within the plant containing the highest abun- dance of compounds were also collected. The three-dimensional (3D) structures of the com- pounds were collected on PubChem (Kim et al., 2020) (SDF format) or ChemSpider (MOL format). For compounds without readily available 3D struc- tures on these public sources, the 2D structures were collected and converted to 3D models using Discovery Studio (D.S.) Visualizer (BIOVIA & Das- sault Systèmes, 2017). Hydrogen atoms were added to all 3D compound structures. The geometry of each compound was cleaned using the built-in Min- imize Structure tool in UCSF Chimera (Pettersen et al., 2004) to reduce the internal energies. All opti- mized A. indica compounds were saved in a MOL2 format and ready for molecular docking. ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV PROTEIN VISUALIZATION AND OPTIMIZATION The 3D protein structures of HIV integrase were retrieved from the RCSB Protein Data Bank to serve as the target model of investigation (Berman et al., 2000). The human protein structures with higher resolution and known bounded ligands were prioritized during the collection process. Minor pro- tein processing was performed to optimize the pro- tein model and minimize errors. All selected pro- teins underwent energy optimization and geometry cleaning with the Structural Preparation and Proto- nate 3D tool in Molecular Operating Environment (MOE) ("Molecular Operating Environment (MOE), 2019.01," 2022), and the processed proteins were saved in MOE format. A pre-docking binding pocket analysis was also performed using Lig- plot+(Laskowski & Swindells, 2011) to collect base- line protein-ligand interactions between the known inhibitor compounds and the HIV integrase pro- teins. DOCKING PARAMETER OPTIMIZATION AND BASELINE BUILDING The minimized HIV integrase protein files were re-docked using MOE Docking. Re-docking was performed as a suitability control experiment, which involved taking out the originally bounded ligands in each protein file and docking them back into the protein binding pocket with various binding parameters to identify the most optimal conditions for the experiment. The default placement method of Triangle Matcher with London dG scoring system and the refinement method of Rigid Receptor with GBVI/WSA dG scoring system were used as the docking methods. These methods and scoring sys- tems are known to give reliable results, and the re- sults were estimated in terms of free energy re- ported in kcal/mol (Corbeil et al., 2012; Galli et al., 2014). However, other parameters such as receptor region, docking site, and number of docked poses were tested for the most optimized parameter com- binations for each protein target, which was meas- ured by the RMSD values between the original lig- and and the re-docked models. The docking ener- gies for the most suitable docking parameters (often resulted in the lowest RMSD values) were also rec- orded for baseline purposes. A. INDICA COMPOUND DOCKING A MOE database file in MDB format was cre- ated with the name and the structure in MOL2 for- mat of each optimized A. indica compound. The op- timized docking parameters were used albeit the MOE compound database file, which was used as the docking ligands. The docked poses with the best docking energies (most negative) for each compound were recorded. For each A. indica com- pound, the average docking energies against all protein targets were compared with the average docking energies in optimization. Since the docking energy was measured in free energy, any A. indica compounds with more negative average docking energies than optimization was more thermody- namically favorable to bind and therefore identified as potential HIV integrase binders. POST-DOCKING ANALYSIS Ligplot+ was used to perform a post-dock- ing analysis on all binders in complex with their pro- tein binding pockets. Two of the most common and relatively strong protein-ligand interactions — hy- drogen bonds and hydrophobic interactions — were examined to help explain and verify the favor- able docking energies obtained by the binder com- pounds. 3 RESULTS COMPOUND DATABASE BUILDING A comprehensive database comprised of 50 compounds present in A. indica was created. FIG- URE 1 shows the structures of a few compounds pre- sent in the database. In addition to the 3D structures of each compound, the database also collected the locations where these compounds are found on the plant, a brief categorization of each compound, the extraction methods, and the corresponding percent yield found in the plant (TABLE 1). ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV Azadirachtin Nimbin Gedunin Nimbolide Nimbolin Salannin Cycloeucalenol Nimbosterol Nimbolicin FIGURE 1. The 3D structures of nine representative A. indica compounds collected in the database. Grey: carbon; white: hydrogen; red: oxygen. PROTEIN DATA COLLECTION AND BINDING POCKET ANALYSIS OF HIV INTEGRASE Four HIV integrase proteins (PDB IDs: 1QS4, 3NF6, 3NF7, 6WC8) were selected (Goldgur et al. 1999; Gorman et al. 2020; Peats et al. 2010). These PDB files had the highest res- olution, were derived from human targets, and contained at least one known HIV integrase in- hibitor. Clustal Omega multiple protein se- quence alignment revealed that these selected integrase proteins were at least 95% identical between any two proteins (Sievers et al. 2011). The difference in protein sequence was due to the presence of 2-4 unique mutation sites in each protein. The number of files used in this study was a balance between accuracy and resource, as previous studies from our lab had demonstrated that using four target files was sufficient to generate reliable results. Bind- ing pocket analysis of each of the four proteins revealed the presence of numerous hydrogen bonds and hydrophobic interactions between the known inhibitor ligands and the integrase proteins, as shown in TABLE 2. ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV TABLE 1: In-silico compound database for A. indica. All data was collected from publicly available journal sources as indicated in Reference column. The percent yields corresponded to the extraction method as listed. N/A: Data not available. Compound Source Extraction method Percent Yield Reference 1 Azadirachtin Flower, fruit, leaf, and seed Aqueous 0.1-0.3% in seed (Biswas et al, 2002; Kaushik, 2021; Morgan, 2009; Paul et al., 2011; Ponnusamy et al., 2015; Sadeghian & Mortazaienezhad, 2007; Singh et al., 2017) 2 Isomargolonone Bark N/A N/A (Biswas et al., 2002; Singh et al., 2017) 3 Azadiradione Fruit, leaf, seed Aqueous 0.3% in leaf (Paul et al., 2011; Ponnusamy et al., 2015; Sadeghian & Mor- tazaienezhad, 2007) 4 Epicatechin Bark N/A N/A (Biswas et al., 2002; Singh et al., 2017) 5 Mahmoodin Seed oil N/A N/A (Biswas et al., 2002) 6 Azadirone Fruit, leaf, seed oil Aqueous 2.46% in leaf (Paul et al., 2011; Ponnusamy et al., 2015; Sadeghian & Mor- tazaienezhad, 2007) 7 Flavanone Flower N/A N/A (Nakahara et al., 2003) 8 Margolone Bark N/A N/A (Biswas et al., 2002; Singh et al., 2017) 9 Catechin Bark N/A N/A (Biswas et al., 2002; Singh et al., 2017) 10 Nimbin Leaf, seed oil, trunk and root bark Aqueous 2.6% in leaf (Biswas et al., 2002; Kaushik et al., 2021; Paul et al., 2011; Ponnusamy et al., 2015; Sadeghian & Mor- tazaienezhad, 2007; Singh et al., 2017) 11 Gedunin Leaf, Seed oil N/A N/A (Anand, 2017; Biswas et al., 2002; Paul et al., 2011; Ponnusamy et al., 2015; Sadeghian & Mor- tazaienezhad, 2007; Singh et al., 2017) 12 Nimbinin Leaf, seed oil, trunk and root bark N/A N/A (Koul, Isman, & Ketkar, 1990; Paul et al., 2011) 13 Nimbolide Leaf, Seed oil Aqueous 2.20% leaf (Biswas et al., 2002; Kaushik et al., 2021; Sadeghian & Mor- tazaienezhad, 2007; Singh et al., 2017) 14 Nimbidin Leaf, Seed N/A N/A (Biswas et al., 2002; Koul et al., 1990; Singh et al., 2017) 15 Nimbolin A Trunk wood N/A N/A (Paul et al., 2011) 16 Nimbolin B Trunk wood N/A N/A (Paul et al., 2011) 17 Quercetin Flower and leaf N/A N/A (Kaushik et al., 2021; Paul et al., 2011) 18 Salannin Leaf, seed oil Aqueous 5.6% in leaf (Paul et al., 2011; Ponnusamy et al., 2015; Sadeghian & Mor- tazaienezhad, 2007) 19 Nimbidol Leaf N/A N/A (Anand, 2017) 20 Cycloeucalenol Wood oil N/A N/A (Paul et al., 2011) ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV 21 Nimbosterol (beta-si- tosterol) Leaf, wood oil N/A N/A (Kaushik et al., 2021) 22 Nimbinone Bark N/A N/A (Ara, Siddiqui, Faizi, & Siddiqui, 1988) 23 Nimbolicin Bark N/A N/A (Read & French, 1993) 24 Margocin Root bark N/A N/A (Ara et al., 1990) 25 Gallic acid Bark N/A N/A (Biswas et al., 2002; Singh et al., 2017) 26 2-methyl-5-ethylfuran Leaf Butanol 4.8273% (Hossain et al., 2013) 27 Arabinose Bark N/A N/A (Kumar et al., 2017) 28 m-toluylaldehyde Leaf Methanol 22.7669% (Hossain et al., 2013) 29 2-methyl-benzalde- hyde Leaf Butanol 11.8674% (Hossain et al., 2013) 30 Levoglucosenone Leaf Butanol 7.1217% (Hossain et al., 2013) 31 Methyl isoheptade- canoate Leaf Hexane Chloroform Methanol 2.1921% 11.6299% 12.2749% (Hossain et al., 2013) 32 Methyl petroselinate Leaf Hexane 11.2380% (Hossain et al., 2013) 33 Phytol Leaf Hexane Ethyl Acetate Chloroform 2.6170% 61.2401% 10.0515% (Hossain et al., 2013) 34 Butyl palmitate Leaf Hexane 6.6981% (Hossain et al., 2013) 35 Isobutyl stearate Leaf Hexane 4.2521 % (Hossain et al., 2013) 36 Oxalic acid Leaf Hexane 13.7094% (Hossain et al., 2013) 37 Methyl 14-methylpen- tadecanoate Leaf Methanol Ethyl Acetate Chloroform Butanol 38.1251% 6.4278% 31.8674% 13.4471% (Hossain et al., 2013) 38 Hexahydrofarnesyl ac- etone Leaf Ethyl Acetate 2.5888% (Hossain et al., 2013) 39 Lineoleoyl chloride Leaf Methanol Chloroform Butanol 26.8329% 11.3587% 13.6057% (Hossain et al., 2013) 40 Nonacosane Leaf Chloroform Butanol 20.6575% 12.8752% (Hossain et al., 2013) 41 Stearic acid Kernel Oil N/A 18% (Do et al., 2022 ) 42 Palmitic acid Kernel Oil N/A 16.9% Do et al., 2022 ) 43 Oleic acid Kernel Oil N/A 45.9% (Do et al., 2022 ) 44 Linoleic acid Kernel Oil N/A 15.69% (Do et al., 2022 ) 45 Pyroligneous acid Heartwood N/A 38.4% (Kumar et al., 2017) 46 Hentriacontane Leaf Butanol 13.9887 (Hossain et al., 2013) 47 Heptacosane Leaf Hexane 8.1010% (Hossain et al., 2013) 48 Octacosane Leaf Hexane 7.0926 (Hossain et al., 2013) 49 Eicosane Leaf Hexane 10.0136 (Hossain et al., 2013) 50 Nonadecane Leaf Hexane 3.7587% (Hossain et al., 2013) ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV TABLE 2: Binding pocket analysis of the four selected HIV integrase proteins. The hydrogen bonds and hydrophobic interactions were identified using Ligplot+. All ligands present in these proteins had shown existing binding activity toward HIV integrase. 100: 1-(5-chloroindol-3-yl)-3-hydroxy-3-(2h-tetrazol-5-yl)-propenone IMV: 5-[(2-oxo-2,3-dihydro-1H-indol-1-yl)methyl]-1,3-benzodioxole-4-carboxylic acid CIW: 5-[(5-chloro-2-oxo-2,3-dihydro-1H-indol-1-yl)methyl]-1,3-benzodioxole-4-carboxylic acid TQM: {5-(3-fluorophenyl)-2-[(thiophen-2-yl)ethynyl]-1-benzofuran-3-yl}acetic acid PDB # Ligand Hydrogen Bond Hydrophobic 1QS4 100 (5ClTEP) Thr66 Asp64 Asn155 Lys159 Gln148 Lys156 Ile151 Glu152 3NF6 IMV Glu170 Gln95 Ala128 Lys173 His171 Tyr99 Ala129 Met178 Thr174 Leu102 Trp132 Thr125 Ala169 3NF7 CIW Val77 Val150 Leu158 Val79 Ser153 His183 Gly82 Met154 Ile84 Glu157 6WC8 TQM Gln95 Leu102 Glu96 Ala128 Ala98 Ala129 Tyr99 Trp132 DOCKING PARAMETER OPTIMIZATION AND BASELINE BUILDING Docking optimization was performed on each of the four HIV integrase targets. The re- docked models were compared with the origi- nal ligands to identify the best docking parame- ters to use for A. indica compounds. After the optimization process, the protein atoms without the surrounding solvent were set as the docking receptor. The protein residues within the 5Å space of the original ligand were defined as the protein active sites for docking. Thirty place- ment poses and five refinement poses were deemed the best parameters for later studies. For each of the four protein targets, these pa- rameters yielded RMSD values of 1.99, 0.35, 0.73, 1.63, respectively, which were low enough to generate accurate docked results (FIGURE 2). The average docking energy of redocked lig- ands was -5.6358 kcal/mol. MOLECULAR DOCKING OF A. INDICA COMPOUNDS INTO HIV INTEGRASE BINDING POCKET Molecular docking was performed on all 50 A. indica compounds against each of the four HIV integrase proteins using the optimized docking parameters. After a comparison be- tween the average docking energy for the plant compounds and the redocked ligands, 30 A. in- dica compounds were predicted to have a more favorable binding energy and were identified as potential HIV integrase binders (TABLE 3). ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV FIGURE 2: Ligand-bounded HIV integrase binding pocket (PDB# 3NF7). (A) Docking optimization with original ligand (green) and redocked ligand (pink). (B) A. indica compound salannin (blue) and gedunin (orange). TABLE 3: The docking energy for each of the A. indica compounds and the redocked ligands. Each of these energy measures was the average docking energy of each compound against all four HIV integrase proteins (PDB# 1QS4, 3NF6, 3NF7, 6WC8). Highlighted compounds were identified as binders. * Redocked ligands from docking optimization. Compound Average Docking Energy (kcal/mol) Compound Average Docking Energy (kcal/mol) 0 Redocked* -5.6358 26 2-methyl-5-ethylfuran -4.1258 1 Azadirachtin -6.3591 27 Arabinose -4.1509 2 Isomargolonone -5.3900 28 m-toluylaldehyde -4.1068 3 Azadiradione -5.4921 29 2-methyl-benzaldehyde -3.9820 4 Epicatechin -5.2782 30 Levoglucosenone -3.5953 5 Mahmoodin -5.9337 31 Methyl isoheptadecanoate -6.2995 6 Azadirone -5.6070 32 Methyl petroselinate -6.4152 7 Flavanone -5.0024 33 Phytol -6.4347 8 Margolone -5.3343 34 Butyl palmitate -6.5009 9 Catechin -5.2375 35 Isobutyl stearate -6.7462 10 Nimbin -6.0113 36 Oxalic acid -3.0358 11 Gedunin -5.8379 37 Methyl 14-methylpentadecanoate -6.3077 12 Nimbinin -5.6339 38 Hexahydrofarnesyl acetone -6.0264 13 Nimbolide -5.6445 39 Lineoleoyl Chloride -6.2090 14 Nimbidin -5.4625 40 Nonacosane -7.4238 15 Nimbolin A -6.6793 41 Stearic acid -6.3773 16 Nimbolin B -6.3832 42 Palmitic acid -6.1378 17 Quercetin -4.9746 43 Oleic acid -6.2499 18 Salannin -5.9750 44 Linoleic acid -6.3096 19 Nimbidol -5.1154 45 Pyroligneous acid -3.3098 20 Cycloeucalenol -6.0074 46 Hentriacontane -7.3965 21 Nimbosterol -6.3417 47 Heptacosane -7.0865 22 Nimbinone -5.1792 48 Octacosane -7.3098 23 Nimbolicin -6.4237 49 Eicosane -6.4206 24 Margocin -5.4672 50 Nonadecane -6.1945 25 Gallic acid -4.2497 FIGURE 2A FIGURE 2B ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV POST-DOCKING ANALYSIS A post-docking analysis was performed on all thirty A. indica binders against each of the four protein targets. The Ligplot+ images re- vealed the intermolecular interactions of these binders in complex with the HIV integrase bind- ing pockets (FIGURE 3). Most of the binders were shown to be surrounded by large, hydrophobic clusters. Some hydrogen bonds were also ob- served with some A. indica compounds. The common interacting residues of a few repre- sentative compounds are shown in TABLE 4. FIGURE 3: Representative figure of the post-docking analysis on A. indica compound bounded HIV integrase proteins. Semi-circles indicate protein residue involved in hydrophobic interactions. Arrow-pointed orange compounds indi- cate protein residue involved in hydrogen bonds. The purple compounds are A. indica compounds of interest. ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV TABLE 4: The common interacting HIV integrase residues of a few representative A. indica binders and the original redocked ligand via either hydrophobic interactions or hydrogen bonds. *Residues involved in hydrogen bonds. Compound Average Docking Energy (kcal/mol) Common Interacting Residues Re-docked -5.6358 Val77, Val79, Gly82, Ile84, Val150, Ser153, Met154, Glu157, Leu158 Azadirachitin -6.3591 Val79, Gly82, Val150, Ser153, Met154, Glu157, His183, Lys188*, Arg199* Isobutyl Stearate -6.7462 Val77, Val79, Gly82, Val150, Met154, Glu157, His183, Lys186, Lys188 Nimbolicin -6.4237 Val77, Val79, Ala80*, Gly82, Val150, Met154, Glu157, His183, Lys186, Lys188, Arg199 Nimbolide -5.6445 Ser81, Gly82, Val150, Ser153, Met154, Glu157, His183, Lys188*, Arg199* Nimbolin A -6.6793 Val77, Val79, Ala80, Gly82, Val150, Ser153, Met154, Glu157, His183, Lys188, Arg199 Nimbosterol -6.3417 Val77, Val79, Gly82, Val150, Met154, Glu157, His183, Lys186, Lys188 Octacosane -7.3098 Val77, Gly82, Val150, Ser153, Met154, Glu157, Lys188 Salannin -5.975 Ser153, Met154, Glu157, His183, Lys186*, Lys188*, Arg199 4 DISCUSSION & CONCLUSION A comprehensive in-silico database of A. indica compounds was created, providing detailed information on many compounds re- garding their sources, extraction methods, and percent yields extracted from the plant. Alt- hough the exact percent yield of these com- pounds varies greatly depending on the extrac- tion method, this information could provide val- uable insights for later drug discovery stages. The 3D structures collected for each of these compounds were also extensively used in the molecular docking studies against HIV inte- grase. In the docking optimization process, the original ligands in each protein file were docked back into the protein binding pockets; the re- sulting model was referred to as re-docked lig- ands. The relative position and identity of the re- docked ligands were visually compared with the original ligands to determine the reliability of the docking methods. The re-docked ligands were shown to occupy a highly similar 3D space with the original ligand. This was also quantified via the RMSD values, which measured the aver- age distance between the atoms of the original ligand and the re-docked ligand. Therefore, the low RMSD values also reflected highly similar postures between the predicted model and the original ligand. Both verification methods indi- cated that the optimized docking parameters and algorithm were highly accurate in predict- ing the binding affinity of the A. indica com- pounds. ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV Meanwhile, the average docking en- ergy in optimization was also an important base- line for identifying the potential A. indica bind- ers. A closer look at the 30 identified A. indica binders revealed that they were bound at the same binding pocket as the original ligands, suggesting a potentially similar allosteric effect. Interestingly, the average docking energies of six compounds (nonacosane, hentriacontane, octacosane, heptacosane, isobutyl stearate, and nimbolin A) were one standard deviation more favorable than the redocked ligands, indicating more efficient binding activity than the original ligands. Post-docking analysis was performed to explain and validate the favorable docking en- ergies predicted by the docking algorithm. The large hydrophobic clusters surrounding most A. indica binders and the presence of hydrogen bonds with some binders were both excellent indicators of strong intermolecular interactions. Some interacting residues in the original lig- ands, such as Val77, Val79, Gly82, Val150, Ser153, Met154, Glu157, and His183, were commonly retained across many A. indica bind- ers. In addition to the retained interactions, most binders also gained new hydrophobic in- teractions; some of these binders, such as aza- dirachitin and nimbolide, also gained new hy- drogen bonds. The A. indica binders that gained new interactions suggest a more potent binding ability to the target proteins compared to the corresponding binding ability of the orig- inal ligands. Therefore, the intermolecular inter- actions present in these new protein-ligand complexes validated the favorable binding en- ergies predicted by the docking algorithms, which supported the identified A. indica binders against HIV integrase. Overall, we have identified 30 out of 50 A. indica compounds as binders of HIV inte- grase proteins. The large proportion of the binder compounds present in this plant pro- vides a feasible explanation of this plant's HIV viral reducing potential. Therefore, we propose a potential mechanism for the anti-HIV activity for A. indica which could offer insights into a novel HIV treatment candidate. However, the fact that the docking energies of the existing known binders were used as the cut-off point in A. indica binder identification may be a poten- tial limitation this study. The possibility of the A. indica compounds with less-than-ideal docking energies binding to the targets still exists. This study only provides a computational screening of the potential HIV drug candidates; further bench testing on promising candidates is still necessary to validate the results. Therefore, fu- ture goals include further testing these com- pounds in a wet lab setting to validate the po- tential inhibitory potential against HIV integrase. The database created in this study may play an important role in future studies of this plant compared to other biological targets, which in turn enables exploration of other therapeutic targets∎ 5 ACKNOWLEDGEMENTS We would like to express our sincere gratitude to our mentor and research advisor Dr. Sonia Arora for her continuous support throughout the duration of the project. This re- search work would not be possible without her enthusiasm and knowledge to the topic. Her guidance was always inspiring and this project was a great learning opportunity on in-silico ap- proaches, which could have endless applica- tions in drug discovery works. 6 REFERENCES [1] Abdelhady, M. I. S., Bader, A., Shaheen, U., El- Malah, Y., Abourehab, M. A. S., & Barghash, M. F. (2015). Azadirachta indica as a Source for Antiox- idant and Cytotoxic Polyphenolic Compounds. Biosciences Biotechnology Research Asia, 12(2), 1209-1222. doi:10.13005/bbra/1774 [2] Alzohairy, M. A. (2016). Therapeutics Role of Aza- dirachta indica (Neem) and Their Active Constit- uents in Diseases Prevention and Treatment. Evid Based Complement Alternat Med., 2016. doi:10.1155/2016/7382506 [3] Anand, N. (2017). Antifungal, Contraceptive, Anti-Cancer, Mosquito Repellent Properties of ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV Azadirachta Indica: A review. Texila International Journal of Basic Medical Sciences, 2(1). doi:10.21522/TIJBMS.2016.02.01.Art006 [4] Anyaehie, U. B. (2009). Medicinal properties of fractionated acetone/water neem [Azadirachta indica] leaf extract from Nigeria: a review. Niger J Physiol Sci., 24(2). doi:10.4314/njps.v24i2.52926 [5] Ara, I., Siddiqui, B. S., Faizi, S., & Siddiqui, S. (1988). Terpenoids from the stem bark of Aza- dirachta indica. Phytochemistry, 27(6), 1801- 1804. doi:10.1016/0031-9422(88)80447-3 [6] Ara, I., Siddiqui, B. S., Faizi, S., & Siddiqui, S. (1990). Tricyclic diterpenoids from root bark of Azadirachta indica. Phytochemistry, 29(3), 911- 914. doi:10.1016/0031-9422(90)80044-H [7] Bandyopadhyay, U., Biswas K., Sengupta, A., Moitra, P., Dutta, P., Sarkar, D., Debnath, P., Gan- guly, C. K. & Banerjee, R. K. (2004). Clinical stud- ies on the effect of Neem (Azadirachta indica) bark extract on gastric secretion and gastroduo- denal ulcer. Life Sci, 75(24):2867-78. [8] Berman, H. M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T. N., Weissig, H., . . . Bourne, P. E. (2000). The Protein Data Bank. Nucleic Acids Re- search, 28, 235-242. [9] BIOVIA, & Dassault Systèmes. (2017). Discovery Studio Visualizer. San Diego: Dassault Systèmes. [10] Biswas, K., Chattopadhyay, I., Banerjee, R. K., & Bandyopadhyay, U. (2002). Biological activities and medicinal properties of Neem (Azadirachta indica). Current Science, 82(11), 1336-1345. [11] Centers for Disease Control and Prevention [CDC]. (2021). About HIV. Retrieved from HTTPS://WWW.CDC.GOV/HIV/BASICS/WHATISHIV.HTML [12] Corbeil, C. R., Williams, C. I., & Labute, P. (2012). Variability in docking success rates due to da- taset preparation. Journal of computer-aided molecular design, 26(6), 775–786. HTTPS://DOI.ORG/10.1007/S10822-012-9570-1 [13] Council, N. R. (1992). Neem: A Tree For Solving Global Problems. Washington, DC: The National Academies Press. [14] Do, D. N., Pham, H. D., Le, X. T., Le, M. T., Ngu- yen, N. P., Bui, Q. M. & Luu, X. C. (2022).Mechan- ical Extraction of Neem Seed Kernel (Azadirachta indica A. Juss.) Harvesting in Ninh Thuan, Viet Nam by Using Hydraulic Pressing: Effect of Pro- cessing Parameters. Materials Science Forum, 1048, 437-444. [15] Galli CL, Sensi C, Fumagalli A, Parravicini C, Mari- novich M, Eberini I. A computational approach to evaluate the androgenic affinity of iprodione, procymidone, vinclozolin and their metabolites. PLoS One. 2014 Aug 11;9(8):e104822. doi:10.1371/journal.pone.0104822. [16] Goldgur, Y., Craigie, R., Fujiwara, T., Yoshinaga, T., Davies, D.R. (1999). Core domain of HIV-1 in- tegrase complexed with Mg++ and 1-(5- chloroindol-3-yl)-3-hydroxy-3-(2H-tetrazol-5- yl)- propenone. doi: 10.2210/pdb1QS4/pdb [17] Gorman, M.A. & Parker, M.W. (2020). HIV Inte- grase core domain in complex with inhibitor 2- (5-(3-fluorophenyl)-2-(2-(thiophen-2-yl)ethynyl)- 1- benzofuran-3-yl)ethanoic acid. doi:10.2210/pdb6WC8/pdb [18] Hossain, M. A., Al-Toubi, W. A. S., Weli, A. M., Al- Riyami, Q. A., & Al-Sabahi, J. N. (2013). Identifi- cation and characterization of chemical com- pounds indifferent crude extracts from leaves of Omani neem. Journal of Taibah University for Sci- ence, 7(4), 181-188. doi:10.1016/j.jtusci.2013.05.003 [19] Jerobin, J., Makwana, P., Kumar, R. S., Sundara- moorthy, R., Mukherjee, A., & Chandrasekaran, N. (2015). Antibacterial activity of neem nanoemulsion and its toxicity assessment on hu- man lymphocytes in vitro. Int J Nanomedicine, 10(Suppl 1), 77-86. doi:10.2147/IJN.S79983 [20] Kaushik, P., Ahlawat, P., Singh, K., & Singh, R. (2021). Chemical constituents, pharmacological activities, and uses of common ayurvedic medic- inal plants: a future source of new drugs. Ad- vances in Traditional Medicine. doi:10.1007/s13596-021-00621-3 [21] Kim, S., Chen, J., Cheng, T., Gindulyte, A., He, J., He, S., . . . Bolton, E. E. (2020). PubChem in 2021: new data content and improved web interfaces. Nucleic Acids Research, 49(D1), D1388-D1395. doi:10.1093/nar/gkaa971 [22] Komal, D., Khushboo, J., Aaftaab, S., Lakshmi, S., & Mallika, A. (2020). Targeting Integrase En- zyme: A Therapeutic Approach to Combat HIV Resistance. Mini-Reviews in Medicinal Chemistry, 20(3). doi:10.2174/1389557519666191015124932 [23] Koul, O., Isman, M. B., & Ketkar, C. M. (1990). Properties and uses of neem, Azadirachta indica. Can. J. Bot., 68(1), 1-11. doi:10.1139/b90-001 [24] Kumar, B., Bhaskar, D., Rajadurai, M., & Sathy- amurthy, B. (2017). In vitro studies on the effect of Azadirachta indica Linn. in lung cancer A549 https://www.cdc.gov/hiv/basics/whatishiv.html https://doi.org/10.1007/s10822-012-9570-1 ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV cell lines. World J Pharm Pharm Sci, 6(9), 1627- 1640. [25] Laskowski, R. A., & Swindells, M. B. (2011). Lig- Plot+: multiple ligand-protein interaction dia- grams for drug discovery. Journal of Chemical In- formation and Modeling, 51, 2778-2786. [26] Mesplède, T., Quashie, P. K. & Wainberg, M. A. (2012). Resistance to HIV integrase inhibitors. Curr Opin HIV AIDS, 7(5), 401-408. doi:10.1097/COH.0b013e328356db89 [27] Molecular Operating Environment (MOE), 2019.01. (2022). Chemical Computing Group ULC, 1010 Sherbooke St. West, Suite #910, Mon- treal, QC, Canada, H3A 2R7. [28] Morgan, E. D. (2009). Azadirachtin, a scientific gold mine. Bioorganic & Medicinal Chemistry, 17(12), 4096-4105. doi:10.1016/j.bmc.2008.11.081 [29] Nakahara, K., Roy, M. K., Ono, H., Maeda, I., Ohnishi-Kameyama, M., Yoshida, M., & Trakoontivakorn, G. (2003). Prenylated fla- vanones isolated from flowers of Azadirachta in- dica (the neem tree) as antimutagenic constitu- ents against heterocyclic amines. J Agric Food Chem., 51(22), 6456-6460. doi:10.1021/jf034666z [30] Nesari, T. M., Bhardwaj, A., ShriKrishna, R., Ruknuddin, G., Ghildiyal, S., Das, A., Pandey, A. K., Chaudhary, N., Soman, G. & Barde, M. (2021). Neem (Azadirachta Indica A. Juss) Capsules for Prophylaxis of COVID-19 Infection: A Pilot, Dou- ble-Blind, Randomized Controlled Trial. Altern Ther Health Med, 27(S1):196-203. [31] Patel, S. M., Venkata, K. C. N., Bhattacharyya, P., Sethi, G., & Bishayee, A. (2016). Potential of neem (Azadirachta indica L.) for prevention and treatment of oncologic diseases. Semin in Can- cer Biol., 40-41, 100-115. doi:10.1016/j.semcancer.2016.03.002 [32] Paul, R., Prasad, M., & Sah, N. K. (2011). Anti- cancer biology of Azadirachta indica L (neem): a mini review. Cancer Biol Ther., 12(6), 467-476. doi:10.4161/cbt.12.6.16850 [33] Peat, T.S., Newman, J., Deadman, J.J., Rhodes, D. (2010). Structural basis for a new mechanism of inhibition of HIV integrase identified by frag- ment screening and structure based design. doi:10.2210/pdb3NF7/pdb [34] Pettersen, E., Goddard, T., Huang, C., Couch, G., Greenblatt, D., Meng, E., & Ferrin, T. (2004). UCSF Chimera--a visualization system for explor- atory research and analysis. Journal of Computa- tional Chemistry, 25(13), 1605-1612. [35] Ponnusamy, S., Haldar, S., Mulani, F., Zinjarde, S., Thulasiram, H., & RaviKumar, A. (2015). Gedunin and Azadiradione: Human Pancreatic Alpha-Am- ylase Inhibiting Limonoids from Neem (Aza- dirachta indica) as Anti-Diabetic Agents. PLoS One., 10(10). doi:10.1371/journal.pone.0140113 [36] Read, Michael D. & French, H. James H., eds. (1993). Genetic Improvement of Neem: Strate- gies for the Future. Proc. of the International Con- sultation on Neem Improvement held at Kasetsart University, Bangkok, Thailand, 18 - 22 January 1993. Bangkok, Thailand: Winrock International. 194 + x pp. [37] Rodrigues, T., Schneider, G. (2015). Chapter 6 - In Silico Screening: Hit Finding from Database Mining. The Practice of Medicinal Chemistry, 4, 141-160. HTTPS://DOI.ORG/10.1016/B978-0-12-417205-0.00006-7 [38] Sadeghian, M. M., & Mortazaienezhad, F. (2007). Investigation of Compounds from Azadirachta indica (Neem). Asian Journal of Plant Sciences, 6, 444-445. doi:10.3923/ajps.2007.444.445 [39] Sarah, R., Tabassum, B., Idrees, N., & Hussain, M. K. (2019). Bioactive Compounds Isolated from Neem Tree and Their Applications. In Natural Bio-active Compounds: Springer. [40] Sievers, F., Wilm, A., Dineen, D., Gibson, T. J, Kar- plus, K., Li, W., Lopez, R., McWilliam, H., Rem- mert, M., Söding, J., Thompson, J. D, Higgins, D. G, (2011) Fast, scalable generation of high-qual- ity protein multiple sequence alignments using Clustal Omega. Molecular Systems Biology, 7. 539. doi: accession:10.1038/msb.2011.75 [41] Singh, H., Kaur, M., Dhillon, J. S., Batra, M., & Khurana, J. (2017). Neem: a magical herb in en- dodontics. Stomatological Dis Sci, 1, 50-54. doi:10.20517/2573-0002.2016.10 [42] Smith, J. A., & Daniel, R. (2006). Following the Path of the Virus: The Exploitation of Host DNA Repair Mechanisms by Retroviruses. ACS Chem. Biol., 1(4), 217-226. doi:10.1021/cb600131q [43] Udeinya, I. J., Mbah, A. U., Chijioke, C. P., & Shu, E. N. (2004). An antimalarial extract from neem leaves is antiretroviral. Transactions of The Royal Society of Tropical Medicine and Hygiene, 98(7), 435-437. doi:10.1016/j.trstmh.2003.10.01 https://doi.org/10.1016/B978-0-12-417205-0.00006-7 ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV [44] World Health Organization [WHO]. (2021). HIV/AIDS. Retrieved from HTTPS://WWW.WHO.INT/NEWS-ROOM/FACT-SHEETS/DE- TAIL/HIV-AIDS Zhongrui Zhang is a recent graduate from Rutgers University-New Brunswick. He has a B.S. in Biotechnology, bioinformatics from the School of Environmental and Biological Science. He had been con- ducting research in Dr. Sonia Arora’s lab on two projects over the course of two years. One was to utilize in-silico techniques to study the compounds present in Ocimum sanctum against inflammatory pathways. The other was to investigate the potential anti-HIV activity of Azadirachta indica in reducing viral loads. He also assisted PhD stu- dents in Dr. James Simon's lab in conducting synthetic and analytical organic chemistry work, where he gained hands-on experience work- ing with plant compounds. These research experiences sparked his interest in drug discovery, and he is currently working at Bristol Myers Squibb within the Biologics Department. In the future, Zhongrui would like to pursue graduate studies in the drug development-re- lated field. Zhongrui can be reached at zhongrui.zhang@rutgers.edu. Yin Hei Lau is a graduate of Rutgers University. She has a B.S. degree in Biotechnology – Bioinformatics from the School of Environmental and Biological Science. She has a broad interest in health and medi- cine and would like to conduct further research in the medical field. Her research, under the guidance of Dr. Sonia Arora, investigated the anti-HIV properties of compounds in A. indica through in-silico ap- proach. She also worked in Dr. Judith Storch's research lab for more than two years. She assisted a PhD student in functional analysis of enterocyte fatty acid binding proteins (FABP). For independent pro- jects, she studied the hepatic lipid metabolism in the intestine-spe- cific liver FABP (LFABP) knockout mice and the intestinal lipid metab- olism in the liver-specific LFABP knockout mice. Yin Hei can be contacted at: yinhei.lau@rutgers.edu. https://www.who.int/news-room/fact-sheets/detail/hiv-aids https://www.who.int/news-room/fact-sheets/detail/hiv-aids mailto:zhongrui.zhang@rutgers.edu mailto:yinhei.lau@rutgers.edu