Hrev_master Healthcare in Low-resource Settings 2024; volume 12:12047 Antiretroviral activity from elderberry (Sambucus nigra L.) flowers against HIV-2 infection via reverse transcriptase inhibition: a viroinformatics study Rahadian Zainul,1,2 Viol Dhea Kharisma,3 Pauline Ciuputri,3 Arif Nur Muhammad Ansori,3-6 Mochammad Aqilah Herdiansyah,3 Sukma Sahadewa,7 Fara Disa Durry8 1Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Negeri Padang, Indonesia; 2Center for Advanced Material Processing, Artificial Intelligence, and Biophysic Informatics (CAMPBIOTICS), Universitas Negeri Padang, Indonesia; 3Division of Research of Development, Jalan Tengah, Surabaya, Indonesia; 4Uttaranchal Institute of Pharmaceutical Sciences, Uttaranchal University, Dehradun, India; 5European Virus Bioinformatics Center, Jena, Germany; 6Virtual Research Center for Bioinformatics and Biotechnology, Surabaya, Indonesia; 7Faculty of Medicine, Universitas Wijaya Kusuma Surabaya, Indonesia; 8Faculty of Medicine, Universitas Pembangunan Nasional “Veteran” Jawa Timur, Surabaya, Indonesia Abstract HIV-2 infection is a unique concern with fewer cases than HIV-1, but it poses a high mortality rate due to its resistance to all HIV-1 antiretroviral treatments. This study focuses on one type of antiretroviral, reverse transcriptase (RT) inhibitors, as they play an important role in HIV-2 replication. The screening of potential HIV-2 antiretroviral candidates was carried out using compounds from elderberry (Sambucus nigra L.) flower extract. There is a lack of research on the antiviral potential of elderberry flower extracts, particularly in HIV-2; therefore, this study is important to explain the molecular mechanism underlying the potential of elderberry (Sambucus nigra L.) flower extracts to inhibit RT activ- ity in HIV-2 through bioinformatics simulations. This study uses the in silico method, involving sample preparation in the database, drug-like molecular prediction through the server, molecular docking simulation, chemical bond interaction analysis, and three- dimensional structure visualization. Isorhamnetin has the most negative binding affinity of -9.9 kcal/mol compared to other com- pounds. It interacts with the HIV-2 RT domain at residues Trp4(B), Pro25(B), Asn137(B), Pro133(B), Gln23(B), Pro140(B), Leu21(B), Ile90(A), Thr131(B), Asn57(B), Arg22(B), and Glu89(A) with hydrophobic bond interactions. Hydrogen bond interactions are formed at the positions of Ser134(B), Gly141(B), and Thr88(A). Isorhamnetin from elderberry (Sambucus nigra L.) flower extract could be a potential HIV-2 antiretroviral candidate because it has the most negative binding affinity and the formation of hydrophobic hydrogen bond interactions on the RT domain. Introduction HIV is an RNA virus that infects/attacks lymphocyte cells in the body, causing a decrease in the body’s system of recognition.1 HIV-2, sharing similarities with HIV-1 in its envelope, is predom- inantly found in West Africa but carries the potential to become a global epidemic.2 Approximately 1 to 2 million people are living with minimally treated HIV-2, resulting in high mortality and mor- bidity. The virus, initially identified in 2018, has affected an esti- mated 38 million people worldwide.3 In 2020, globally, an esti- mated 37.7 million people were living with HIV and 1.5 million became newly infected with HIV.4 The Ministry of Health of Indonesia in 2020 reported that only 14% were known to be viral- ly suppressed after 6 months of ART.5 HIV-2 is endemic in several West African countries, namely Guinea-Bissau, Senegal, Cape Verde, Gambia, Mali, Sierra Leone, Cote d’Ivoire, and Nigeria, with a prevalence rate of 1%. Beyond West Africa, cases have also surfaced in South America, Europe, Asia, and the United States.6 In Indonesia, the death rate due to HIV/AIDS is still high.7 The Ministry of Health of the Republic of Indonesia stated that in Indonesia, in the last 12 years (data for 2010-2022, for those aged over 15 years), projections of new infections have shown a posi- tive trend, namely from 56,187 new cases to 25,740.8 Correspondence: Rahadian Zainul, Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Negeri Padang, Padang, Indonesia. E-mail: rahadianzmsiphd@fmipa.unp.ac.id Key words: antiretroviral, HIV-2, medicine, reverse transcriptase, Sambucus nigra. Contributions: RZ, VDK, PC, ANMA, conceptualization, data analysis, methodology, validation, visualization, writing - original draft, and review and editing; RZ, VDK, ANMA, MAH, SS, FDD, methodology, validation, and writing - review and editing. Conflict of interest: the authors declare no conflict of interest. Ethics approval and consent to participate: not applicable. Funding: none. Availability of data and materials: all data generated or analyzed during this study are included in this published article. Acknowledgments: the authors would like to thank Jalan Tengah (jalantengah.site) for editing the manuscript. Received: 2 November 2023. Accepted: 7 May 2024. Early access: 13 June 2024. This work is licensed under a Creative Commons Attribution 4.0 License (by-nc 4.0). ©Copyright: the Author(s), 2024 Licensee PAGEPress, Italy Healthcare in Low-resource Settings 2024; 12:12047 doi:10.4081/hls.2024.12047 Publisher's note: all claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organi- zations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. [Healthcare in Low-resource Settings 2024;12:12047] [page 525] Patients with HIV/AIDS experience various problems, both physical and psychological, one of which is health-related quality of life.9 People living with HIV require a focus on their quality of life to prevent deterioration.10 Treatment for patients diagnosed with HIV-2 involves antiretrovirals, comprising a combination of nucleoside reverse transcriptase (RT) inhibitors, integrase strand transfer inhibitors, and protease inhibitors.11 However, the virus poses a challenge by demonstrating resistance to all three types of antiretrovirals commonly used for HIV-1 treatment. While RT inhibitors have proven effective against HIV-1, their impact on HIV-2 replication is negligible. Consequently, there is a pressing need to screen new inhibitor candidates to develop treatments for HIV-2. Despite exhibiting a lower viral load compared to HIV-1, HIV-2 has developed resistance to commonly used antiretrovirals for HIV-1 treatment.11 As an enveloped virus with double-stranded RNA (dsRNA) genetic material, HIV-2 encodes several enzymes such as RT, protease, and integrase.12 This study specifically focus- es on RT due to its pivotal role in HIV-2 replication.13 The RT enzyme in HIV-2 serves a similar function to HIV-1, facilitating the formation of viral complementary DNA (cDNA) that integrates into the host cell genome.2 Reported mutations of RT in HIV-2 result in resistance to specific antiretroviral types, notably non- nucleoside RT inhibitors (NNRTIs).13 The resistance pattern posi- tions the RT enzyme as the ideal target for designing antiretrovirals to combat HIV-2 by inhibiting its enzymatic activity. Elderberry (Sambucus nigra L.) has been utilized in alternative medicine due to its potential as an antiviral, antidiabetic, antibac- terial, antitumor, and antioxidant agent.14 Polyphenolic compounds in the plant are also predicted to inhibit virus replication.15 The flower of elderberry (Sambucus nigra L.) is rarely used as an alter- native medicine; however, it contains several chemical com- pounds. These include quinic acid, caffeoylquinic acid, 1-caf- feoylquinic acid, coumaroylquinic acid, feruloylquinic acid, isorhamnetin, quercetin-3-rutinoside, quercetin-acetyl glucoside, kaempferol rutinoside, and isorhamnetin acetylhexoside.16 Previous studies have shown that flower extracts from elderberry (Sambucus nigra L.) can inhibit gram-positive bacteria such as Staphylococcus aureus and S. epidermidis.17 Currently, there is a lack of research exploring the potential of flower extracts from elderberry (Sambucus nigra L.) as an antiviral, especially for HIV- 2. This research is important for understanding the molecular mechanism underlying the potential of elderberry (Sambucus nigra L.) flower extracts to inhibit RT activity in HIV-2 using bioinfor- matics simulations. Materials and Methods Sample preparation This study used compounds extracted from elderberry (Sambucus nigra L.) flowers. Ten compounds from Sambucus nigra L. flower were selected from the chromatographic results and their data presence in the PubChem database. These com- pounds are quinic acid, caffeoylquinic acid, 1-caffeoylquinic acid, coumaroylquinic acid, feruloylquinic acid, isorhamnetin, quercetin-3-rutinoside, quercetin-acetyl glucoside, kaempferol rutinoside, and isorhamnetin acetylhexoside, as ligands.16 Information such as compound identification, simplified molecular input line entry system canonical, formula, and structure data for- mat files were obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov/). Minimization of ligands was performed using OpenBabel v2.3.1 software to convert files into protein databank format (PDB). The target chosen in this study was RT HIV-2 from RCSB PDB (https://www.rcsb.org/) with ID 1MU2 (https://www.rcsb.org/structure/1mu2), obtained in PDB format. Subsequently, water molecules and native ligands were removed through PyMOL software v.2.5.2 (Schrödinger, Inc., USA) with an academic license.18,19 Drug-like molecule prediction The objective of drug-like molecule prediction is to identify the similarity of properties in chemical compounds from elderber- ry flower extract (Sambucus nigra L.) with those found in drug molecules. In this study, the drug-like molecule prediction method refers to the Lipinski rule of five through the SCFBio server (http://www.scfbio-iitd.res.in/software/drugdesign/lipinski.jsp). The rules include molecular mass, high lipophilicity, molar refrac- tivity, and acceptor-donor hydrogen bonds. For a compound to be categorized as a drug-like molecule, it must meet at least two of these rules.20,21 Molecular docking simulation In this study, molecular docking simulations are conducted to predict the ligand binding strength of elderberry flower extract (Sambucus nigra L.) on HIV-2 RT. Molecular docking refers to the interaction between the ligand and the target, resulting in the for- mation of a molecular complex with a stable bond, and the output of this process is the binding affinity. Binding affinity is the energy generated in the ligand-protein complex, which is characterized by a negative value. The more negative the value, the higher the prob- ability of triggering specific activities, such as inhibition.22 The PyRx 0.9.9 software (Scripps Research, USA) uses a screening docking method, covering the entire target surface with a directed grid and selecting ligands based on their more negative binding affinity energy.23 Chemical interaction Chemical bonding interactions are formed in ligand-protein complexes, consisting of both hydrophobic and hydrogen bonds. These weak bonds collectively contribute to the stability of drug binding to the target and can subsequently trigger specific activi- ties, such as inhibition. The software used to identify the position and type of chemical bond interactions in this study is LigPlot+v.2.2.24,25 Structural visualization Three-dimensional visualization of docking results was dis- played through PyMOL software v.2.5.2 (Schrödinger, Inc., USA) with an academic license. Protein structures were displayed in the form of cartoons and transparent surfaces with publication stan- dards, ligands were displayed through stick structures with color- ing based on C, H, N, O, and F atoms.26 Results In this study, we revealed that the compounds retrieved from the database include quinic acid, caffeoylquinic acid, 1-caf- feoylquinic acid, coumaroylquinic acid, feruloylquinic acid, isorhamnetin, kuersetin-3-rutinoside, kuersetin-acetyl glucoside, kaempferol rutinoside, and isorhamnetin acetylhexoside (Table 1). Next, the two-dimensional structures of all compounds of elderber- ry (Sambucus nigra L.) flower extract are shown in Figure 1. Then, the druglikeness prediction results indicated two compounds not Transforming Healthcare in Low-Resource Settings: A Multidisciplinary Approach Towards Sustainable Solutions [page 526] [Healthcare in Low-resource Settings 2024;12:12047] meeting the criteria for drug-like molecules, while eight com- pounds received positive predictions as drug-like molecules (Table 2). Ligands with the most negative binding affinity values are pre- dicted to trigger inhibitory activity on the target. The molecular docking simulation results indicated that isorhamnetin has the most negative binding affinity of -9.9 kcal/mol compared to other compounds (Table 3). Three-dimensional structures on ligands and proteins from docking results are visualized with transparent sur- faces, cartoons, and sticks using specific coloring selections (Figure 2). The analysis of the position and type of interaction in the ligand-protein complex revealed that isorhamnetin interacts with HIV-2 RT domain at residues Trp4(B), Pro25(B), Asn137(B), Pro133(B), Gln23(B), Pro140(B), Leu21(B), Ile90(A), Thr131(B), Asn57(B), Arg22(B), and Glu89(A) through hydrophobic bond interactions. Hydrogen bond interactions are formed at the posi- tions of Ser134(B), Gly141(B), and Thr88(A) (Figure 3). Discussion Elderberry (Sambucus nigra L.) extract shows potential as an antiviral for treating influenza infection.27 Recent research indi- cates that flower extracts from elderberry (Sambucus nigra L.) can be used for cold flu symptoms, such as pain, fever, cough, and con- gestion.28 The antiviral activity of elderberry (Sambucus nigra L.) was demonstrated in vitro at 400 μg/mL, inhibiting DENV-2 repli- Transforming Healthcare in Low-Resource Settings: A Multidisciplinary Approach Towards Sustainable Solutions Figure 1. Elderberry (Sambucus nigra L.) flower extract compounds from database. A) Quinic acid; B) caffeoylquinic acid; C) 1-caf- feoylquinic acid; D) coumaroylquinic acid; E) feruloylquinic acid; F) isorhamnetin; G) quercetin-3-rutinoside; H) quercetin-acetyl gluco- side; I) kaempferol rutinoside; J) isorhamnetin acetylhexoside. Table 1. Ligand retrieval from PubChem. No Compounds CID SMILE Canonical Formula 1. Quinic acid 6508 C1C(C(C(CC1(C(=O)O)O)O)O)O C7H12O6 2. Caffeoylquinic acid 1794427 C1C(C(C(CC1(C(=O)O)O)OC(=O)C=CC2=CC(=C(C=C2)O)O)O)O C16H18O9 3. 1-Caffeoylquinic acid 10155076 C1C(C(C(CC1(C(=O)O)OC(=O)C=CC2=CC(=C(C=C2)O)O)O)O)O C16H18O9 4. Coumaroylquinic acid 9945785 C1C(C(C(CC1(C(=O)O)O)OC(=O)C=CC2=CC=C(C=C2)O)O)O C16H18O8 5. Feruloylquinic acid 9799386 COC1=C(C=CC(=C1)C=CC(=O)OC2CC(CC(C2O)O)(C(=O)O)O)O C17H20O9 6. Isorhamnetin 5281654 COC1=C(C=CC(=C1)C2=C(C(=O)C3=C(C=C(C=C3O2)O)O)O)O C16H12O7 7. Quercetin-3-rutinoside 5280805 CC1C(C(C(C(O1)OCC2C(C(C(C(O2)OC3=C(OC4=CC(=CC(=C4C3=O) C27H30O16 O)O)C5=CC(=C(C=C5)O)O)O)O)O)O)O)O 8. Quercetin-acetyl glucoside 10006384 CC(=O)OCC1C(C(C(C(O1)OC2=C(OC3=CC(=CC(=C3C2= C23H22O13 O)O)O)C4=CC(=C(C=C4)O)O)O)O)O 9. Kaempferol rutinoside 5318767 CC1C(C(C(C(O1)OCC2C(C(C(C(O2)OC3=C(OC4=CC C27H30O15 (=CC(=C4C3=O)O)O)C5=CC=C(C=C5)O)O)O)O)O)O)O 10. Isorhamnetin acetylhexoside 44259375 CC(=O)OC1C(C(C(OC1OC2=C(OC3=CC(=CC(=C3C2=O) C24H24O13 O)O)C4=CC(=C(C=C4)O)OC)CO)O)O CID, compound identification; SMILE, simplified molecular input line entry. [Healthcare in Low-resource Settings 2024;12:12047] [page 527] cation.29 The plant’s polyphenolic compounds are also predicted to have inhibitory effects on virus replication.15 This study is impor- tant to reveal the potential of elderberry (Sambucus nigra L.) flower extract as an antiviral for HIV-2. The compounds retrieved from the database include quinic acid, caffeoylquinic acid, 1-caf- feoylquinic acid, coumaroylquinic acid, feruloylquinic acid, isorhamnetin, kuersetin-3-rutinoside, kuersetin-acetyl glucoside, kaempferol rutinoside, and isorhamnetin acetylhexoside. The two- dimensional structures of all compounds of elderberry (Sambucus nigra L.) flower extract are shown. Transforming Healthcare in Low-Resource Settings: A Multidisciplinary Approach Towards Sustainable Solutions Table 3. The binding affinity score. Compounds CID Target PDB ID Binding affinity (kcal/mol) Quinic acid 6508 1MU2 -6.8 Caffeoylquinic acid 1794427 1MU2 -8.6 3-p-Coumaroylquinic acid 10155076 1MU2 -7.9 Coumaroylquinic acid 9945785 1MU2 -8.5 Feruloylquinic acid 9799386 1MU2 -7.3 Isorhamnetin 5281654 1MU2 -9.9 Quercetin-acetyl glucoside 10006384 1MU2 -8.0 Isorhamnetin acetylhexoside 44259375 1MU2 -9.3 CID, compound identification; PDB, protein databank format. Table 2. Drug-like molecule properties. No Compounds Molecular mass LogP HBD HBA Molar refractivity Probable 1. Quinic Acid 192.000 -2.321 5 6 39.839 Drug-like Molecule 2. Caffeoylquinic Acid 354.000 -0.645 6 9 82.518 Drug-like Molecule 3. 3-p-Coumaroylquinic acid 354.000 -0.645 6 9 82.518 Drug-like Molecule 4. Coumaroylquinic Acid 338.000 -0.351 5 8 80.853 Drug-like Molecule 5. Feruloylquinic Acid 368.000 -0.342 5 9 87.405 Drug-like Molecule 6. Isorhamnetin 316.000 2.313 4 7 78.937 Drug-like Molecule 7. Quercetin-3-rutinoside 610.000 -1.878 10 16 137.495 Non drug-like Molecule 8. Quercetin-acetyl glucoside 506.000 -0.159 7 13 115.821 Drug-like Molecule 9. Kaempferol rutinoside 594.000 -1.584 9 15 135.830 Non drug-like Molecule 10. Isorhamnetin acetylhexoside 520.000 0.143 6 13 120.708 Drug-like Molecule HBD, hydrogen bond donor; HBA, hydrogen bond acceptor. Figure 2. Structural visualization from the molecular docking simulation. A) Quinic acid_RT HIV-2; B) caffeoylquinic acid_RT HIV-2; C) 3-p-coumaroylquinic acid_RT HIV-2; D) coumaroylquinic acid_RT HIV-2; E) feruloylquinic acid_RT HIV-2; F) isorhamnetin_RT HIV-2; G) quercetin-acetyl glucoside_RT HIV-2; H) isorhamnetin acetylhexoside_RT HIV-2. RT, reverse transcriptase. [page 528] [Healthcare in Low-resource Settings 2024;12:12047] The prediction of druglikeness in compounds from elderberry (Sambucus nigra L.) flower extract aimed to identify the physico- chemical characteristics of a drug-like molecule. The prediction uses Lipinski’s rules of five, which stipulate that compound with drug-like molecule characteristics must fulfill at least two rules.30 The druglikeness prediction results indicated two compounds not meeting the criteria for drug-like molecules, while eight com- pounds received positive predictions as drug-like molecules. Compounds exhibiting drug-like molecule characteristics are pre- dicted to induce biological activity and possess the ability to pen- etrate the cell membrane, reaching targets within the cytoplasmic environment.31 Molecular docking aims to identify how ligands interact with the target domain.32 In this study, grid docking is used with the position set at Center (Å) X: 2.746, Y: -25.031, Z: 16.706, Dimensions (Å) X: 105.798, Y: 83.933, Z: 106.561 to orient the ligand on the target. The ligand consists of compounds from elder- berry (Sambucus nigra L.) flower extract characterized as a drug- like molecule, and the target is RT HIV-2 (PDB ID: 1MU2). A screening docking method is employed to identify or screen the ligand activity on the target with the most negative binding affinity.33 Ligands with the most negative binding affinity values are predicted to trigger inhibitory activity on the target. The molec- ular docking simulation results indicated that isorhamnetin has the most negative binding affinity of -9.9 kcal/mol compared to other compounds. A more negative binding affinity value signifies a stronger influence produced by ligand binding on the target.34 Isohamnetin from elderberry (Sambucus nigra L.) flower extract is predicted to be a good antiretroviral candidate through inhibition of HIV-2 RT activity. Three-dimensional structures on ligands and proteins from docking results are visualized with transparent sur- faces, cartoons, and sticks using specific coloring selection. The molecular interactions formed in the docking result com- plex consist of hydrogen and hydrophobic bonds.35 Both bonds contribute to the strength of the interaction and the stability of the drug molecule.36 The analysis of the position and type of interac- tion in the ligand-protein complex revealed that isorhamnetin interacts with the HIV-2 RT domain at residues Trp4(B), Pro25(B), Asn137(B), Pro133(B), Gln23(B), Pro140(B), Leu21(B), Ile90(A), Thr131(B), Asn57(B), Arg22(B), and Glu89(A) through hydrophobic bond interactions. Hydrogen bond interactions are formed at the positions of Ser134(B), Gly141(B), and Thr88(A). Isorhamnetin is predicted to interact through hydrogen bonding and hydrophobicity on the RT domain of HIV-2, potentially trig- gering inhibitory activity on the target. The predicted mechanism of HIV-2 RT inhibitors in this study refers to NNRTIs in HIV-1 by targeting a hydrophobic pocket for inhibition of polymerization reactions.37 In contrast to HIV-1, HIV-2 has mutations in RT with positions K65R, K70E, L74V, Q151M, M184I/V; RT mutations in HIV-2 trigger significant antiretroviral resistance compared to HIV-1.11 The position of amino acid residues in the pocket binding domain of isorhamnetin from elderberry (Sambucus nigra L.) has a type of hydrophobic bond interaction at the position of amino acid residues that do not include mutation regions, allowing isorhamnetin to be predicted as a potential HIV-2 RT inhibitor. Conclusions Isorhamnetin from elderberry (Sambucus nigra L.) flower extract shows promise as a potential HIV-2 antiretroviral candidate because it has the most negative binding affinity and the formation of hydrophobic hydrogen bond interactions on the RT domain. 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