Assessment of phytochemicals from Syzygium aqueum as inhibitors of ATP-dependent 6-phosphofructokinase: in silico and in vitro studies

Suwendar Suwendar; Sani Ega Priani; Dina Mulyanti; Taufik Muhammad Fakih; Ibrahim Jantan

doi:10.3897/pharmacia.71.e132917

Abstract

Helminth infections can be effectively treated with various anthelmintics. However, the potential for drug resistance and the need for repeated doses to ensure successful treatment are some limitations to the treatment. This study aimed to evaluate the ability of phytochemicals from Syzygium aqueum to inhibit ATP-dependent 6-phosphofructokinase (6-PFK) from Ascaris lumbricoides and Ascaris suum using the in silico method (molecular docking, molecular dynamics simulation, and ADMET analysis) and in vitro evaluation using mortality, paralysis, and ovicidal activity testing. Molecular docking outcomes indicated that out of 76 compounds from Syzygium aqueum, 18 molecules exhibited strong inhibition against the target enzyme, based on binding free energy and interaction patterns. Molecular dynamics simulation demonstrated that samarangenin A and butyrospermol complexes remained stable for up to 200 ns within a range below 1 nm. The most promising compounds also exhibited good ADMET properties. In the in vitro egg hatch test, a 10% concentration of Syzygium aqueum extract showed potent ovicidal properties by significantly reducing the number of fertile eggs but was less effective than 0.12% albendazole. However, ferulic acid at various concentrations exhibited minimal effect on egg fertility but showed increased mortality and incidence of paralysis at higher doses against male worms. These findings yielded valuable insights into the underlying mechanisms, providing essential clues for the development of highly efficient inhibitors for parasitic worm infections through structure-based design.

Keywords

Syzygium aqueum, Ascaris species, ATP-dependent 6-phosphofructokinase, in silico study, in vitro approach

Introduction

Helminth infections, caused by parasitic worms, pose a significant human health challenge, affecting millions of people globally. Worldwide, an estimated one-third of the world’s population currently suffers from neglected tropical parasitic diseases (Dixit et al. 2017; Rennie et al. 2021). These infections affect communities in the developing world and are recognized as neglected tropical diseases (Loukas et al. 2021). Many suffer from multiple worm species or co-infections with pathogens like HIV, malaria, or bacteria. Ascaris lumbricoides and Ascaris suum are prevalent nematodes affecting humans and pigs, respectively (Liu et al. 2012). Recent data indicates around 1.2 billion people are infected. These infections are particularly common in tropical and subtropical regions, notably in China, East Asia, Latin America, and Sub-Saharan Africa. Additionally, many of these parasitic infections are zoonotic, transmitted from animals to humans (Fitzsimmons et al. 2014; Wiedemann and Voehringer 2020). Helminth infections can lead to various health problems, such as anemia, malnutrition, and impaired cognitive development, and if left untreated, they can be life-threatening. There are several anthelmintic drugs that are generally effective in treating the diseases and can help eliminate the parasites from the body. Although anthelmintics like albendazole, ivermectin, and mebendazole are effective against infections like Ascaris, they often face issues of resistance and side effects that limit their long-term use (Djune-Yemeli et al. 2020). These limitations have spurred research into developing new treatment alternatives, including the use of natural compounds. Natural compounds offer high potential efficacy with a lower risk of resistance and side effects, making them promising candidates in anti-parasitic therapy (Williams et al. 2014).

ATP-dependent 6-phosphofructokinase (6-PFK) can be a promising target for the parasites Ascaris lumbricoides and Ascaris suum. This enzyme is a homotetramer, with each subunit having a molecular weight of 34 kDa, resulting in a total molecular weight of approximately 140 kDa (Hansen and Schönheit 2001). It is ubiquitous across all life domains, spanning bacteria, archaea, and eukaryotes. Among its shared traits in bacterial and eukaryotic domains is the homotetrameric architecture and the allosteric control over activity by intermediary metabolic compounds (Zhang et al. 2022; Jin et al. 2023). Typically, bacterial variants of this enzyme are allosterically spurred by adenosine diphosphate (ADP) and curbed by phosphoenolpyruvate (PEP). Consequently, this enzyme serves as a pivotal regulatory hub in sugar degradation through the Embden-Meyerhof pathway (Hansen et al. 2002). Understanding the intricate regulation and structural features of this enzyme provides valuable insights for devising targeted therapeutic strategies against parasitic infections. Given its involvement in regulating parasite metabolism, this enzyme stands as an optimal target for antiparasitic intervention. Consequently, in theory, if small molecules can bind to ATP-dependent 6-PFK, they could impede parasitic infections within host cells. Multiple in vitro analyses have validated the potential of ATP-dependent 6-PFK as a target for impeding the entry of parasitic worms.

Syzygium aqueum, locally known as watery rose apple, is native to Indonesia and Malaysia, particularly Java Island, and is prevalent in tropical regions, Africa, and parts of Southern Asia, notably India and Thailand (Sonawane 2018; Yumita et al. 2023; Longevity 2024). Diverse studies have investigated and reported various biological properties of different components of Syzygium aqueum, including anti-diabetic, antioxidant, anti-inflammatory, hepatoprotective, lipolytic, anticellulite, and anticancer effects (Sirisha and Shreeja 2019). Earlier studies suggested that polyphenols, which were the most abundant constituent in various parts of Syzygium aqueum, possessed anthelmintic properties (Gayen et al. 2016; Charitha et al. 2017). These compounds not only offer nutritional advantages but also aid in warding off chronic diseases. The secondary metabolites’ role in combating infections caused by parasites like Ascaris lumbricoides and Ascaris suum underscored the significance of Syzygium aqueum as a potential source of anthelmintic agents and laid the groundwork for forthcoming research endeavors (Venkatachalam et al. 2018).

To address infections caused by parasitic worms, a range of traditional treatments have been recommended for patients experiencing mild to moderate symptoms, yielding unexpectedly positive outcomes in disease management (Laudisi et al. 2020). However, the precise molecular mechanisms governing the interaction between these herbal remedies and Ascaris lumbricoides as well as Ascaris suum remain unresolved. This investigation explored 76 bioactive components of Syzygium aqueum as potential contenders for managing parasitic infections using both computational and in vitro methods. Computational techniques such as molecular docking, molecular dynamics simulation, and absorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis were employed to identify promising candidates. These were followed by in vitro testing of Syzygium aqueum extract to evaluate its anthelmintic effects, focusing on mortality, paralysis, and ovicidal activity to assess its efficacy against parasitic worms. The molecular mechanisms of the top candidates identified computationally were further scrutinized, highlighting their roles in various traditional therapies aimed at inhibiting the ATP-dependent 6-PFK target. The outcomes of this study provide researchers with valuable insights into potential new treatments for combating parasitic infections caused by Ascaris lumbricoides and Ascaris suum.

Materials and methods

Preparation of ATP-dependent 6-phosphofructokinase receptor structure

The ATP-dependent 6-PFK receptor crystal structure became the choice for lead protein design. The FASTA file, which had a 3D X-ray crystallographic structure of the used target protein (Code F1KSL6), was downloaded from the Uniprot (https://www.uniprot.org/) database. The protein structure was further prepared for subsequent docking studies by removing the ligand bound to its crystallographic ensemble as well as water molecules being part of this complex, and hydrogen atoms alongside Kollmann charges were added. Protein 3D structure was generated from the amino acid sequence using Swiss-PDB Viewer software (version 4.10), and its energy minimization modeling was carried out by use of an empirical force field to obtain conformations with the lowest energy reflecting better stability (Dlamini et al. 2015). In the context of protein structure modeling, this procedure was essentially used to correct mispentors that were then introduced through modeling as one tries to improve the model’s geometry. The steepest descent algorithm with the GROMOS96 force field was implemented for geometry optimization (Schuler et al. 2001).

Validation of ATP-dependent 6-phosphofructokinase receptor structure

The model architecture of an ATP-dependent phosphofructokinase, type 6-PFK receptor, proceeded through careful evaluation with a Ramachandran plot on PROCHECK (https://www.ebi.ac.uk/thornton-srv/software/PROCHECK/) online server (Laskowski et al. 2012), used to validate the protein structure model built from the SWISS-MODEL (https://swissmodel.expasy.org/) server (Waterhouse et al. 2018). The plot displayed the amino acid of the protein and their propensity, allowed range, as well as restriction limits for these dihedral angles, which are phi (π) and psi (ψ) defined in degrees. Furthermore, the quality of this protein model was predicted using the ProSA-web (https://prosa.services.came.sbg.ac.at/prosa.php) tool. If its Z-score was in the corresponding range of native protein, it makes sure that the validity of such a high score for protein.

Acquisition of dataset

A total of 76 bioactive compounds from Syzygium aqueum were selected based on a literature review (Yassir et al. 2022). These compounds were divided into several groups: phenolic acids, hydroxycoumarins, flavonoids (such as anthocyanins and proanthocyandins), dihydrochalcones, lignans, and stilbenes, volatile chemicals of low molecular weight molecules, including fatty acid esters or steroidal type hormones.

Molecular docking assessment

Ligand molecule preparation

The ligand structures as compounds were retrieved from PubChem (https://pubchem.ncbi.nlm.nih.gov/) in structured data format (SDF), and further conversion to protein data bank (PDB) files was achieved using the software Open Babel 3.1.1 for future analysis (Smith et al. 2013). Ligands were energy minimized, hydrogen atoms added, and charges added as well, performing the number of active torsions by using AutoDock Tools version 4.2.6 (Forli et al. 2012). Files after being saved in AutoDock PDBQT format.

Receptor macromolecule preparation

The sequence of crystal structure for the ATP-dependent 6-PFK receptor was downloaded from Uniprot (https://www.uniprot.org/) in FASTA format. The water molecules, heteroatoms, as well as the nonstandard ligands, were removed using BIOVIA Discovery Studio 2024 Client for macromolecules and then converted to AutoDock PDBQT format with its additional resources (BIOVIA 2017).

Binding site determination

Prediction of binding sites on the ATP-dependent 6-PFK was demonstrated with computed atlas of surface topography of proteins (CASTp) 3.0 software accessible at http://sts.bioe.uic.edu/castp/index.html?3trg (Tian et al. 2018). It helps in the localization and analysis of critical binding sites necessary for understanding the functional mechanisms of an enzyme.

Visualization of molecular interactions through docking

Molecular docking simulation was performed by AutoDock Vina version 1.2.3 and Autodock Tools version 4.2.6 (Valdés-Tresanco et al. 2020). Ligand and ATP-dependent 6-PFK receptor data in AutoDock PDBQT format was copied into the Vina folder (Eberhardt et al. 2021). The Vina configuration was then set up and saved in Notepad++ as a text document, then run from the Microsoft Windows command prompt (cmd). The results were obtained by opening docking results in Notepad++, and the best ligand on receptor place was defined for minimum binding free energy, saved into a log file (txt). At the other end, using AutoDock Tools version 4.2.6 for the docking process, the receptor and ligand documents were opened in the Grid menu, and the GridBox was readjusted to the desired binding active site before saving the document in GPF format. The next documents that were opened are the receptor and ligand in the docking menu; however, in this case, the rigid filename is restricted by the actual name of the docking process to be saved in DPF format for the docking parameters file. After saving this file, the GPF and DPF files are saved in the same folder, and the Autogrid and AutoDock commands are executed via the command prompt. The ligand placement and orientation in the active site of the receptor were visualized along with respective amino acid interactions by BIOVIA Discovery Studio 2024 Client using both two-dimensional (2D) and three-dimensional (3D) representation modes (BIOVIA 2017).

ADMET analysis

Prediction of ADME parameters was accomplished by SwissADME (http://www.swissadme.ch/), which predicts pharmacokinetics properties and drug-like characteristics (Daina et al. 2017) and pkCSM (http://biosig.unimelb.edu.au/pkcsm/) (Yeni and Rachmania 2022). For the compounds in question, toxicity prognosis was performed using ProTox-II (https://tox-new.charite.de/protox_II/) (Pampalakis 2023). Canonical SMILES obtained from the PubChem database are necessary for input into this online tool.

Estimation of predicted IC₅₀

The IC₅₀ value was determined using the AutoDock Tools version 4.2.6 program (Valdés-Tresanco et al. 2020). Due to this, in order to describe the binding site between the ATP-dependent 6-PFK receptor and ligand, the site was fixed using GridBox. The analysis was divided into two phases: setting up molecules and performing grid-based execution. The control retains the IC₅₀ values of each docked complex DLG directory.

Simulating molecular dynamics (MD)

MD simulations were done by Gromacs version 2016.3 software of the ligand in special conformation after eventual docking (Róg et al. 2016; Kutzner et al. 2019). The pdb2gmx command in Gromacs with the CHARMM36 protein force field was applied for establishing ATP-dependent 6-PFK receptor and ligand topologies (Huang et al. 2016). The water molecules are simulated in TIP3P, and ions have been added. Ligand topologies were generated with the CHARMM General Force Field (CGenFF) II (Vanommeslaeghe et al. 2010). An empty dodecahedron box was used, allowing the protein complexes to be placed at least 1.0 nm away from the edge of the box, and a uniform distribution throughout all supports this choice box boundary. The charged systems were balanced by adding sodium ions. The simulation systems were then minimized in energy through 50,000 steps of the steepest descent minimization algorithm. Afterwards, equilibration for the solvent and ion systems was performed through two restrained phases. In this respect, the 1 ns NVT ensemble was set to a reference temperature of 300 K, with the temperature kept at this value, while the reference pressure was initially set to 1.0 bar for the 1 ns NPT ensemble. The simulation system used a time step of 2.0 fs, while an unrestrained MD simulation for the equilibrated systems was conducted. A 1.2 nm cutoff radius was used for short-range van der Waals. Temperature coupling was controlled through the Berendsen thermostat, with the pressure coupling handled by the Parrinello-Rahman barostat. Subsequently, an MD simulation of 200 ns was accomplished. The analysis consisted of RMSD, RMSF, Rg, SASA, and intermolecular hydrogen bond measurements derived from the trajectory. Short-range receptor-ligand interaction energies were determined using the Lennard-Jones potential.

In vitro testing for anthelmintic effect

Plant sample collection and preparation

The leaves of Syzygium aqueum were collected from Sarijadi, Bandung, Indonesia, and identified by a botanist. A voucher specimen was prepared and deposited at the herbarium of Universitas Islam Bandung. The extraction process was performed by maceration using 96% ethanol as the solvent. A total of 482.12 g of dried leaves were ground and soaked in 1 liter of ethanol for 24 hours at room temperature. The resulting mixture was concentrated using a rotary evaporator at 50 °C, yielding 85.12 g of crude extract, which represents 17.65% of the dried leaf mass (Mulqie et al. 2022).

Evaluation of mortality and paralysis

The effects of ferulic acid on the mortality and paralysis of male and female worms were evaluated using a controlled experimental method (Abu Hawsah et al. 2023). The method involved exposing the worms to various concentrations of ferulic acid: 0.0125%, 0.025%, 0.05%, and 0.1%. Each concentration was tested separately on groups of male and female worms, consisting of 8 male worms and 8 female worms in each group. The worms were placed in suitable containers containing the ferulic acid solutions, with a 120-min exposure time. Observations were conducted at 30-min intervals, recording the number of worms that exhibited signs of death or paralysis. Mortality was determined by checking for the absence of movement in the worms, while paralysis was measured based on their response to physical stimuli. The collected data were then analyzed by calculating the percentage of mortality and paralysis for each concentration, as well as comparing the effects between male and female worms. All experiments were conducted in triplicate to ensure the reliability and reproducibility of the results.

Assessment of ovicidal effects

A modified egg hatch test (EHT) was used to assess the effect of Syzygium aqueum extract and ferulic acid in inhibiting the growth and hatching of eggs of parasitic worms in vitro (Kļaviņa et al. 2023). Briefly, flat-bottomed 24-well microplates were employed, with each well containing 0.5 mL of distilled water and about 200 eggs. Each well was treated with 10% of Syzygium aqueum extract in 1% DMSO, 0.1% of ferulic acid in 0.1% DMSO, and 0.12% of Albendazole in 0.1% DMSO as a positive control. Meanwhile, the negative control group consisted of adding either 0.5 mL of distilled water or 1% DMSO to the egg suspension. The microplates were then kept in darkness at 25 °C with 80% humidity for 48 h. A microscope was used to count the number of eggs and first-stage larvae in each well. All tests were conducted in triplicate across three independent trials to ensure the reliability and reproducibility of the findings (Carisya et al. 2022). The results were assessed to determine if there were significant differences in the number of fertile eggs, particularly between the control group and the treated groups.

Statement of ethical approval

Ethical approval was unnecessary for the procedures performed in this research. Parasite eggs and larvae were sourced from pig fecal samples collected during routine animal examinations and forwarded to the Pharmacology Laboratory within the Department of Pharmacy of the Faculty of Mathematics and Natural Sciences at Universitas Islam Bandung. The animal examinations and sample collection were conducted under the supervision of a certified laboratory technician, following guidelines for proper animal care and welfare, ensuring no distress was caused.

Results

ATP-dependent 6-phosphofructokinase receptor sequence

The ATP-dependent 6-PFK receptor sequence was acquired in FASTA format from the UniProt database, as illustrated in Fig. 1. This retrieval of the sequence from a reputable and widely accepted source established the dependability of the receptor data employed in the subsequent molecular docking investigations, bolstering the overall trustworthiness of the study’s results.

Figure 1.

The sequence of the ATP-dependent 6-PFK receptor.

Receptor constructed by homology modeling

The three-dimensional structure of the ATP-dependent 6-PFK receptor was generated using SWISS-MODEL, achieving a robust global model quality estimation (GMQE) score of 0.89 (Fig. 2A). A GMQE score exceeding 0.70 is widely accepted as a reliable predictive indicator (Benkert et al. 2009). Furthermore, the crystal structure of ATP-dependent 6-phosphofructokinase exhibited a notable 77.59% identity resemblance to the template protein, which was the AlphaFold DB model of A0A044T2Q6. The QMEANDisCo score, representing the per-residue average derived from QMEAN measurements constrained by distances, typically should not fall below 0.6, as lower values indicate subpar protein quality. In our case, the QMEANDisCo score computed using SWISS-MODEL was 0.79 ± 0.05, signifying the high quality of the predicted protein (Studer et al. 2020). Hence, these outcomes affirmed the reliability of the ATP-dependent 6-PFK model’s quality.

Figure 2.

A. A standard SWISS-MODEL homology modeling report, and B. The Ramachandran plot depicts the phi-psi torsion angles of each residue in ATP-dependent 6-PFK.

Fig. 2B illustrates the results of a Ramachandran plot analysis conducted by PROCHECK. This analysis revealed that all residues (100 percent) were situated within the favored region (91.40 percent) and the additionally allowed region (7.60 percent), with no residues falling within the disallowed region. Consequently, this model was consistently confirmed to be of high quality and has been utilized for further computational ligand-receptor interaction studies (Gopukumar et al. 2020).

Molecular docking analysis

A total of 76 reviewed outcomes involving selected compounds sourced from Syzygium aqueum were employed in molecular docking investigations. Suppl. material 1 provides information on the binding affinities between the examined natural compounds and the ATP-dependent 6-PFK receptor using AutoDock Vina version 1.2.3. To ensure accuracy, the docking procedure was repeated using AutoDock Tools version 4.2.6, specifically focusing on the most promising interactions generated by AutoDock Vina version 1.2.3. The binding free energy as determined by AutoDock Vina version 1.2.3 was found to be a minimum of −8.00 kcal/mol. Following these criteria, a total of 18 superior compounds were identified, demonstrating binding affinities ranging from −8.10 kcal/mol to −13.50 kcal/mol. Furthermore, from the outcomes of the molecular docking analysis, three compounds were pinpointed as having the most favorable binding energies with the ATP-dependent 6-PFK receptor, namely prodelphinidin B-2,3,3”-di-O-gallate (−10.10 kcal/mol), samarangenin B (−12.90 kcal/mol), and samarangenin A (−13.50 kcal/mol).

The utilization of advanced molecular docking techniques with AutoDock Tools version 4.2.6 led to the identification of six compounds that exhibited the most prominent molecular interactions. The six compounds were myricetin-3-O-rhamnoside, chrysoeriol-7-O-glucoside, prodelphinidin B-2,3,3”-di-O-gallate, samarangenin A, samarangenin B, and butyrospermol. Suppl. material 2 illustrates the interactions between amino acid residues in the ATP-dependent 6-PFK receptor and these compounds. Notably, even though prodelphinidin B-2,3,3”-di-O-gallate and samarangenin B exhibited positive binding free energy, their interactions with the active site binding of the ATP-dependent 6-PFK receptor still surpassed those of the other compounds. Prodelphinidin B-2,3,3”-di-O-gallate formed a conventional hydrogen bond within ATP-dependent 6-PFK with A:SER585, A:PHE351, A:ASP591, A:ASP591, A:GLY765, A:SER420, A:ARG766, and A:PHE719. Additionally, this compound also established hydrophobic interactions with amino acids A:LEU586 and A:LEU763 (pi-sigma), A:PHE719 (pi-pi t-shaped), A:LEU586 (alkyl), and A:MET217, A:ILE381, A:ILE381, A:ALA350, A:LEU354, and A:LEU763 (pi-alkyl) (Fig. 3A).

Figure 3.

Diagrams in both 2D and 3D formats illustrate the interaction within A. Prodelphinidin B-2,3,3”-di-O-gallate and B. Samarangenin B with the amino acid residues of the ATP-dependent 6-PFK receptor.

Samarangenin B engaged in traditional hydrogen bonds with A:PHE421, A:ARG766, A:ASP222, A:GLY765, and A:ASP216 within the ATP-dependent 6-PFK receptor. Additionally, it formed a p-donor hydrogen bond with A:SER420, which is an important interaction that enhances the binding affinity. Furthermore, it’s important to note that hydrophobic interactions were established between the samarangenin B and the following amino acids within the receptor: A:LEU763 (pi-sigma), A:MET217, and A:LEU763 (alkyl). These hydrophobic interactions played a crucial role in stabilizing the ligand-receptor complex. Additionally, samarangenin B formed pi-alkyl interactions with A:PHE719, A:PHE719, A:MET217, A:ILE381, A:ARG766, and A:ARG766 (Fig. 3B). These diverse interactions collectively contributed to the strong binding of samarangenin B to the receptor.

ADMET analysis

The ADMET of chemicals are pivotal factors in the process of discovering and developing new drugs (Wadapurkar et al. 2018). Consequently, the pharmacokinetic and toxicity properties of the most promising compounds that exhibited the strongest interactions with the ATP-dependent 6-PFK receptor were scrutinized to ensure both their effectiveness and safety (Table 1). Notably, Notably, the average molecular weight of the chosen compounds generally falls below 500 g/mol, with the exceptions of prodelphinidin B-2,3,3”-di-O-gallate (914.73 g/mol), samarangenin A (760.61 g/mol), and samarangenin B (884.70 g/mol). A thorough evaluation covered various parameters, including assessments of carcinogenicity, hepatotoxicity, central nervous system (CNS) permeability, cytochrome P450 (CYP) inhibition, and acute oral toxicity. Toxicity levels are categorized as: class I (lethal if ingested, LD₅₀ ≤ 5 mg/kg), class II (lethal if ingested, 5 < LD₅₀ ≤ 50 mg/kg), class III (toxic if ingested, 50 < LD₅₀ ≤ 300 mg/kg), class IV (harmful if ingested, 300 < LD₅₀ ≤ 2000 mg/kg), class V (may be hazardous if ingested, 2000 < LD50 ≤ 5000 mg/kg), and class VI (non-toxic, LD₅₀ > 5000 mg/kg) (Wang et al. 2017; Kumar et al. 2021).

Table 1.

Download as

CSV

XLSX

Pharmacological characteristics of the leading prospective candidates identified using pkCSM, ProTox-II, and SwissADME.

ADMET Parameters	Myricetin-3-O-rhamnoside	Chrysoeriol 7-O-glucoside	Prodelphinidin B-2,3,3”-di-O-gallate	Samarangenin A	Samarangenin B	Butyrospermol
Molecular weight	464.38 g/mol	462.40 g/mol	914.73 g/mol	760.61 g/mol	884.70 g/mol	426.72 g/mol
H-bond acceptor	12	11	22	18	21	1
H-bond donor	8	6	16	13	15	1
CNS	−3.20	−3.86	−7.83	−6.64	−8.11	−8.02
Molar refractivity	111.02	112.60	222.17	184.52	215.44	137.04
TPSA	210.51 Å²	179.28 Å²	394.74 Å²	316.98 Å²	366.67 Å²	20.23 Å²
Substrate of CYP2D6	−	−	−	−	−	−
Substrate of CYP3A4	−	−	−	−	−	−
Inhibitor of CYP1A2	−	−	−	−	−	−
Inhibitor of CYP2C19	−	−	−	−	−	−
Inhibitor of CYP2C9	−	−	−	+	+	−
Carcinogenicity profile	+	−	−	−	−	−
Hepatotoxicity profile	−	−	−	−	−	−
p-glycoprotein substrate	+	+	+	+	+	−
Class of acute oral toxicity	V	V	IV	IV	IV	IV
Lipinski rule of five	−	−	−	−	−	+

The bioavailability radar provides an intuitive analysis of the investigated compounds (Fig. 4). This distinctive visualization in SwissADME forms a hexagonal graph where each vertex signifies a drug bioavailability determinant. The pink region shows the ideal range for six critical properties: lipophilicity (XLOGP3 from −0.7 to +5.0), size (MW from 150 to 500 g/mol), polarity (TPSA from 20 to 130 Å²), solubility (log S not exceeding 6), saturation (carbon fraction in sp3 hybridization greater than or equal to 0.25), and flexibility (less than nine rotatable bonds) (Az-Zahra et al. 2022). Drug similarity attributes are represented by the red distorted hexagons within the pink area. Notably, only three compounds (myricetin-3-O-rhamnoside, samarangenin A, and samarangenin B) slightly deviate from the pink area due to a minor flexibility mismatch. In some instances, a particular node extends towards polarit.

Figure 4.

Toxicity characteristics of the compounds under investigation were assessed through ProTox-II and SwissADME. The bioavailability radar, with the pink region denoting the ideal range for specific properties, was employed to evaluate the studied compounds. These properties included lipophilicity (XLOGP3), size (molecular weight, MW), polarity (topological polar surface area, TPSA), water insolubility (log S scale), saturation (fraction of carbons in sp³ hybridization), and flexibility (number of rotatable bonds, FLEX).

Computational inhibition constant

The anticipated halfway point of inhibitory concentration (IC₅₀) values were also assessed (Table 2) to provide a more comprehensive understanding of the potential experimental antiparasitic efficacy of the compounds under investigation. IC₅₀ serves as a crucial parameter for evaluating a compound’s capacity to impede biological processes and is widely employed to indicate the inhibitory effects of compounds (Damayanti et al. 2022). The most favorable IC₅₀ values for the ATP-dependent 6-PFK receptor were calculated to be 1.74 uM, 1.43 uM, 77.50 mM, and 332.70 nM for myricetin-3-O-rhamnoside, chrysoeriol-7-O-glucoside, samarangenin A, and butyrospermol, respectively. In contrast, neither prodelphinidin B-2,3,3”-di-O-gallate nor samarangenin B exhibited an IC₅₀ value, as both completely failed to inhibit the activity of ATP-dependent 6-PFK in the conducted assays. Nonetheless, this phenomenon necessitates further validation of its stability through molecular dynamics simulations.

Table 2.

Download as

CSV

XLSX

Anticipated half-maximal inhibitory concentration (IC₅₀) values targeting the ATP-dependent 6-PFK receptor.

Phytochemical Name	Predicted IC₅₀
5-Caffeoylquinic acid	23.26 uM (micromolar)
4-Caffeoylquinic acid	42.57 uM (micromolar)
Myricetin-3-O-rhamnoside	1.74 uM (micromolar)
Chrysoeriol 7-O-glucoside	1.43 uM (micromolar)
Quercetin 3-O-glucoside	21.28 uM (micromolar)
Quercetin 4’-O-glucoside	9.10 uM (micromolar)
Delphinidin 3-O-glucoside	11.34 uM (micromolar)
Peonidin-3-glucoside	7.90 uM (micromolar)
Delphinidin 3,5-O-diglucoside	15.68 uM (micromolar)
(−)-Epigallocatechin	24.02 uM (micromolar)
(−)-Epigallocatechin 3-gallate	22.98 uM (micromolar)
Prodelphinidin B-2,3,3”-di-O-gallate	−
Samarangenin A	77.50 mM (millimolar)
Samarangenin B	−
Phlorizin	141.92 uM (micromolar)
Pinoresinol	7.87 uM (micromolar)
Resveratrol 5-O-glucoside	4.03 uM (micromolar)
Butyrospermol	332.70 nM (nanomolar)

Molecular dynamics simulations study

The energy for all atoms between the ATP-dependent 6-PFK receptor and the ligand was estimated using molecular mechanics poisson-boltzmann surface area (MM/PBSA). The MM/PBSA values are presented as van der Waals energy, electrostatic energy, solvent-accessible surface area (SASA) energy, and total energy. In the complex form, samarangenin A and butyrospermol have lower MM/PBSA values (Table 3). Although the MM/PBSA method with an implicit solvent model has some limitations, it is widely used to estimate binding free energies. Nonetheless, binding free energy estimation is valuable for qualitatively evaluating the binding affinity between proteins and ligands.

Table 3.

Download as

CSV

XLSX

Recapitulation of MM/PBSA between the ATP-dependent 6-PFK receptor and the top-performing ligand in the complex form. Measurements were conducted at the final stage of the trajectory (200 ns).

Phytochemical Name	Van der Waals Energy	Electrostatic Energy	Polar Solvation Energy	SASA Energy	Total Energy
Native	−226.996 ± 14.651 kJ/mol	−34.963 ± 11.645 kJ/mol	167.099 ± 16.518 kJ/mol	−22.615 ± 0.989 kJ/mol	−117.475 ± 20.926 kJ/mol
Myricetin-3-O-rhamnoside	−198.427 ± 16.512 kJ/mol	−189.962 ± 26.361 kJ/mol	307.337 ± 20.324 kJ/mol	−21.933 ± 0.764 kJ/mol	−102.985 ± 15.492 kJ/mol
Chrysoeriol 7-O-glucoside	−244.162 ± 14.249 kJ/mol	−116.008 ± 19.332 kJ/mol	289.957 ± 22.464 kJ/mol	−24.054 ± 0.434 kJ/mol	−94.266 ± 18.403 kJ/mol
Prodelphinidin B-2,3,3”-di-O-gallate	−364.770 ± 24.456 kJ/mol	−238.461 ± 17.433 kJ/mol	528.324 ± 23.041 kJ/mol	−38.291 ± 1.158 kJ/mol	−113.197 ± 28.904 kJ/mol
Samarangenin A	−295.114 ± 20.092 kJ/mol	−251.603 ± 22.658 kJ/mol	424.562 ± 19.999 kJ/mol	−30.310 ± 1.171 kJ/mol	−152.465 ± 20.846 kJ/mol
Samarangenin B	−329.815 ± 14.202 kJ/mol	−181.740 ± 37.492 kJ/mol	437.652 ± 45.254 kJ/mol	−35.335 ± 1.506 kJ/mol	−109.238 ± 40.640 kJ/mol
Butyrospermol	−251.045 ± 21.788 kJ/mol	−18.174 ± 8.139 kJ/mol	127.341 ± 22.547 kJ/mol	−25.073 ± 0.646 kJ/mol	−166.951 ± 32.689 kJ/mol

The molecular dynamics simulation study spanned 200 ns to evaluate how the interaction between the ligand and receptor behaves in a water-based environment, focusing on stability and dynamics. As depicted in Fig. 5, the root-mean-square deviation (RMSD) profile for the apo-protein complex and native protein indicates improved stability after 60 to 200 ns. The root-mean-square fluctuation (RMSF) values remained below 1.5 nm throughout the simulation process. Meanwhile, the radius of gyration (Rg) values of the third complex became more flexible after 100 ns, whereas the apo-protein complex experienced fluctuations from 150 ns until the end of the simulation. The solvent-accessible surface area (SASA) profiles for both complexes exhibited similar patterns during the simulation process.

Figure 5.

A. RMSD; B. RMSF; C. Rg; D. SASA; E. Number of intermolecular hydrogen bonds; F. Analysis of short-range Lennard-Jones receptor-ligand interaction energy between the apo-protein, native-protein, and Samarangenin A-bound form of the ATP-dependent 6-PFK receptor over a period of 200 ns.

As observed in Fig. 6, the RMSD of the butyrospermol complex exhibited a higher deviation compared to the apo-protein and ligand during the period of 50 to 200 ns, but then the complex stabilized. RMSF shows low fluctuations. The Rg profile for the butyrospermol complex, apo-protein, and ligand stabilized after 100 ns. The SASA profile for all three systems showed fluctuations during the simulation but eventually reached stability by the conclusion of the experiment. Thus, the simulation results demonstrated the stable behavior of the butyrospermol complex in interacting with the receptor. These findings underscore the importance of understanding molecular dynamics in studying ligand-receptor interactions.

Figure 6.

A. RMSD; B. Residue; C. Rg; D. SASA; E. Number of intermolecular hydrogen bonds, and F. Analysis of short-range Lennard-Jones receptor-ligand interaction energy were conducted for both the apo-protein, native-protein, and Butyrospermol holo form of the ATP-dependent 6-PFK receptor over a period of 200 ns.

In vitro testing for anthelmintic effect

Evaluation of mortality and paralysis

The choice of ferulic acid as a sample for analysis was based on previous studies that reported that this compound was present in the extract of Syzygium aqueum and exhibited potential antiparasitic properties (Kiran et al. 2021; Amir Rawa et al. 2022; Qiu et al. 2022). At concentrations (0.0125%, 0.025%, and 0.050%) of ferulic acid, there were no observed fatalities among the male and female worms after a 120-min observation period. This indicated that ferulic acid at low concentrations did not exert a lethal effect on the worm population, suggesting a potential safety margin at these concentrations. Conversely, at the highest concentration of 0.1%, a single male worm (12.5% of the group) succumbed, highlighting that while at a higher concentration it might pose a risk, it did not uniformly affect all individuals within the test group. Additionally, paralysis was noted in male worms at both 0.050% and 0.1% concentrations, with 12.5% of worms experiencing paralysis at 0.050% and 37.5% at 0.1% concentration (Table 4). There was a dose-dependent relationship between ferulic acid concentration and the incidence of paralysis, suggesting that higher concentrations not only increased the likelihood of mortality but also exacerbated the severity of paralysis. The LC₅₀ of ferulic acid in male worms was 0.364% w/v. The results of this study provided valuable insights into the effects of ferulic acid on male and female worms at various concentrations.

Table 4.

Download as

CSV

XLSX

Effects of different concentrations of ferulic acid on mortality and paralysis in male and female Ascaris suum.

Groups of Worms	Consentration (% b/v)	Death	Paralysis
Observations on male Ascaris suum	0.0125	0	0
	0.025	0	0
	0.050	0	1 (12.5%)
	0.1	1 (12.5%)	3 (37.5%)
Observations on female Ascaris suum	0.0125	0	0
	0.025	0	0
	0.050	0	0
	0.1	0	1 (25%)

Assessment of ovicidal effects

Table 5 illustrates varying levels of ovicidal activity among different treatments on worm eggs. The control group, with no treatment, had a high average of fertile eggs, showing a baseline fertility rate. Ferulic acid at a concentration of 0.1% showed a minimal decrease in egg fertility, suggesting it has negligible ovicidal effects. In contrast, the 10% extract of Syzygium aqueum significantly reduced the number of fertile eggs, indicating potent ovicidal properties, though less effective than 0.12% albendazole, which exhibited the strongest ovicidal activity among the treatments. These findings highlighted the potential of Syzygium aqueum extract as a natural alternative to synthetic anthelmintics like albendazole, although further research is needed to explore its safety and mechanism of action.

Table 5.

Download as

CSV

XLSX

Ovicidal activity of Syzygium aqueum extract and ferulic acid on worm eggs of Ascaris suum.

Group	Average Number of Fertile Eggs
Negative control	1,264.21 ± 352.31
Ferulic acid 0.1%	1,248.27 ± 456.24
Ethanol extract of Syzygium aqueum 10%	487.50 ± 350.00
Albendazole 0.12%	350.00 ± 132.29

Discussion

The present in silico investigation encompassed an examination of 76 bioactive compounds originating from Syzygium aqueum. The docking outcomes yielded by AutoDock Vina version 1.2.3 and AutoDock Tools version 4.2.6 are delineated in Suppl. materials 1, 2, respectively. The consistency in binding free energy values between the two tools indicated the reliability of the docking results. Both platforms are widely used for studying interactions between ATP-dependent 6-PFK receptors and ligands. The outcomes indicated comparable performance between Vina and AutoDock in distinguishing active compounds from decoys. Discrepancies in binding free energy among different receptor-ligand pairs suggested variability in their interaction strengths. From a pharmacological and biological standpoint, these molecules posited as ATP-dependent 6-PFK receptor inhibitors exhibited promising antiparasitic properties based on prior literature and experimental findings. Numerous investigations have spotlighted ATP-dependent 6-PFK as prospective targets for small-molecule therapeutics against infectious diseases, encompassing those caused by parasites such as Ascaris lumbricoides and Ascaris suum.

The binding scores for compounds binding to the ATP-dependent 6-PFK receptor are presented in Suppl. material 1. Samarangenin A and samarangenin B exhibited significant binding affinity to ATP-dependent 6-PFK with binding free energies of −13.50 kcal/mol each. Both compounds have shown potential as suppressors of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) (Kuo et al. 2002; Cheng et al. 2004). Peonidin-3-glucoside and epigallocatechin, and other compounds belonging to the anthocyanins group, demonstrated activity against ATP-dependent 6-PTK. They are promising inhibitors targeting proteins of Ascaris lumbricoides and Ascaris suum. These flavonoids are renowned for their antioxidant properties and have been found to possess antiparasitic, antiprotozoal, antibacterial, and antiviral properties. Quercetin 3-O-glucoside and quercetin 4-O-glucoside have also been proven to inhibit the ATP-dependent 6-PTK receptor. This discovery highlights the potential of natural compounds as candidates for treating various infectious diseases.

The interaction patterns for both examined receptors are presented in Suppl. material 2. These compounds were observed binding to the substrate active site of ATP-dependent 6-PFK. In models of molecular docking, interactions such as hydrogen bonding, pi-pi stacking, and cation-pi stacking are widely acknowledged. Ligands that engage in hydrophobic interactions make a significant contribution to binding affinity (Yadav et al. 2022; Tsai et al. 2023). The lower occurrence of hydrogen bonds indicates that the binding active pocket is more hydrophobic. The total ligand-receptor binding free energy can be explained by the combined effects of electrostatic, preorganized electrostatic, and non-electrostatic interactions. Electrostatic contributions, which encompass hydrogen bonding interactions, can be assessed using Coulomb’s potential, while non-electrostatic contributions can be evaluated using Lennard-Jones’ potential (Arkundato et al. 2023). In determining prearranged electrostatic impacts, Coulomb’s potential is utilized to analyze geometries sampled from molecular dynamics simulations where the ligand’s partial atomic charges are set to zero. Entropic factors are implicitly considered across these geometries. The relative importance of electrostatic versus non-electrostatic contributions hinges on the compatibility of charges and shapes between interacting molecules. Notably, prearranged electrostatic effects assess the stability of correctly aligned ligand binding groups within both the protein and the surrounding solvent. Conformations exhibiting stronger electrostatic interactions often exhibit weaker non-electrostatic interactions, and vice versa.

The process of binding entails exploring a cavity, where hydrophobic interactions are pivotal in determining the binding strength of the docked complex within a specific solvent environment, influenced by entropy-driven interactions (Yu et al. 2021). Hydrophobic connections include pi-cation, pi-pi, and various other nonspecific interactions, crucial for maintaining the stability and biological functions of proteins while minimizing unfavorable interactions with water. Pi-pi T-shaped interactions involve the pi-electron cloud interaction between two aromatic groups in a T-shaped configuration, encompassing the sideways electron cloud of one ring and the front-facing electron cloud of another ring. This interaction was observed between 4-caffeoylquinic acid, myricetin-3-O-rhamnoside, quercetin 4’-O-glucoside, peonidin-3-glucoside, prodelphinidin B-2,3,3”-di-O-gallate, samarangenin A, and resveratrol 5-O-glucoside with the ATP-dependent 6-PFK receptor. Conversely, hydrogen bonding significantly influences the interaction. The formation of more hydrogen bonds with amino acid residues results in stronger bonds, leading to lower energy scores and enhanced stability. In hydrogen bonding, interactions occur between hydrogen atoms (H) covalently bonded with atoms such as fluorine (F), nitrogen (N), and oxygen (O) (Yuniarta et al. 2023).

The estimated inhibition constant was assessed for the chosen compounds. A higher IC₅₀ value implies a greater inhibitory dosage needed to achieve the intended effect, thereby raising the likelihood of off-target effects and potential toxicity of the drug candidate. Furthermore, in this research, a higher IC value corresponded to a lower binding free energy value. On the other hand, a high IC value indicates that a compound has limited affinity for its target. The compounds recognized for their strong binding to the studied receptors show an average toxicity classification of class IV or V (potentially harmful if ingested), except withanone, which falls into class II (lethal if ingested) (Elseginy et al. 2021; Silva et al. 2022). Several methods can be employed in drug development during the decision-making phase to minimize the toxicity and metabolic variability of potential drugs.

The MD simulation is frequently employed to forecast the stability of ATP-dependent 6-PFK receptors and ligands. Further investigation into the interaction of the best protein-ligand complexes obtained from AutoDock Vina version 1.2.3 and AutoDock Tools version 4.2.6 was conducted in the MD simulation. Apo-protein and native-receptor complexes were utilized as benchmarks to assess alterations in protein stability following ligand binding. The RMSD of the backbone atoms from the ATP-dependent 6-PFK receptor system was scrutinized to understand the structural disparities within the complexes. As illustrated in Figs 5A, 6A, although the initial RMSD profile of the samarangenin A and butyrospermol complexes with the protein remained stable (0 to 50 ns), they expanded their RMSD profiles, exhibiting substantial deviation from 50 to 100 ns, and then reverted to lower RMSD profiles. These complexes began to stabilize after 100 ns and sustained their stability until 200 ns. Similarly, the apo-protein and native-receptor structures exhibited stable profiles but lacked the pronounced fluctuations observed with samarangenin A and butyrospermol in the initial phase. This could be because of unstable changes in the main receptor structure shortly after the ligand binds.

Additionally, the RMSF values of these complexes were scrutinized to comprehend the flexible amino acid residues along the ATP-dependent 6-PFK receptor. As depicted in Figs 5B, 6B, these residues exhibited RMSF values below 1.5 nm consistently throughout the simulation period. Consequently, the Rg values of the simulated complexes were assessed to probe the dynamic nature of the receptor systems, where higher Rg values are associated with increased receptor mobility, while lower Rg values indicate system stability. Figs 5C, 6C illustrate that both the apo-protein, native-receptor, and ligand complexes manifested similar profiles from 0 to 50 ns. However, beyond this period, the apo-protein complexes witnessed a rise in their Rg values, indicative of heightened flexibility. Conversely, the Rg values of the ligand complexes diminished, emphasizing the contracted state of these complexes. SASA, which reflects alterations in protein surface area, shows a positive correlation with high SASA values denoting surface area expansion and lower SASA values indicating reduced protein volume (Aris et al. 2024). Figs 5D, 6D reveal that the SASA profiles of both systems remained consistent from the initial to final simulation phases, with no significant deviations observed.

The hydrogen bond between the ATP-dependent 6-PFK receptor and the ligand was also analyzed because they play a crucial role in determining the stability of the complex. As depicted in Figs 5E, 6E, the hydrogen bond patterns of the samarangenin A complex remained stable and did not undergo significant changes in the simulation environment. Furthermore, the intensity of interaction between the compound and the protein under investigation was computed as nonbonded interaction energy. The mean short-range Lennard-Jones interaction energy measured −139.933 kJ/mol. These interaction energies were consistently stable without any variation observed. The lower Lennard-Jones interaction energies were associated with enhanced stability and increased efficacy of these systems.

Bioactive compounds derived from plants play a pivotal role in phytotherapy due to their potent antiparasitic properties. Syzygium aqueum extract contains tannins, flavonoids, and gallic acid. Tannins have the ability to bind with proteins, impacting the feeding and movement of parasites, and also stimulate the immune response in the host animal. However, the effects of tannins vary across different animal species; ruminants such as sheep and cattle are more susceptible to tannins compared to goats and deer, owing to differences in their digestive systems and microbial populations. In alignment with computational research, proanthocyanidins, a type of tannin, have been investigated for their potential antiparasitic activity, including against worms. Proanthocyanidins have demonstrated effectiveness against various parasite types, including worms. The primary proposed mechanisms involve anthelmintic effects, which could disrupt the nervous system, alter energy metabolism, or affect the structure of parasite membranes (González-Quilen et al. 2020).

Finally, the results of the in vitro study also showed that ferulic acid, a compound predicted to be present in the ethanol extract of Syzygium aqueum, at lower concentrations (0.0125%, 0.025%, and 0.050%) did not have lethal effects on male worms, indicating a potential safety margin for these doses in therapeutic applications. However, at a concentration of 0.1%, ferulic acid exhibited a dose-dependent increase in paralysis and mortality, suggesting that higher concentrations could pose risks (Wang et al. 2020). Furthermore, although ferulic acid exhibited minimal ovicidal effects at a concentration of 0.1%, the 10% concentration of Syzygium aqueum extract significantly reduced the number of fertile eggs, although less effective than 0.12% albendazole (Klimek-Szczykutowicz et al. 2018). These findings highlighted the potential of Syzygium aqueum extract as a natural alternative to synthetic anthelmintics, though further research is needed to explore its mechanisms and safety.

Conclusions

At present, there is a significant focus on exploring novel small-molecule therapeutics to combat infectious diseases, particularly those caused by parasites like Ascaris lumbricoides and Ascaris suum. The potential of natural products as potential sources of new antihelmintic agents has propelled scientists to search for compounds capable of preventing infections induced by parasites such as Ascaris lumbricoides and Ascaris suum. The outcomes of molecular docking on phytochemicals of Syzygium aqueum have yielded highly promising results, unveiling 18 molecules of great interest from both chemical and biological standpoints. Consequently, these molecules are proposed as potential inhibitors of the ATP-dependent 6-PFK receptor. The in vitro studies demonstrated that the 10% concentration of Syzygium aqueum extract significantly reduced the development of viable eggs, exhibiting potent ovicidal activity, while ferulic acid showed increased mortality and incidence of paralysis at higher doses against male worms. Therefore, to fully understand the potential of their mechanisms of action, further studies are warranted to evaluate their efficacy against these parasites in both cell and animal models.

Author contributions

Conceptualization, S.S., I.J., and T.M.F.; methodology, S.S., I.J., and T.M.F.; software, T.M.F.; validation, T.M.F.; formal analysis, T.M.F., D.M., and S.E.P.; investigation, T.M.F., D.M., and S.E.P.; resources, T.M.F., D.M., and S.E.P.; data curation, S.S., I.J., and T.M.F.; writing—original-draft preparation, S.S., I.J., T.M.F., D.M., and S.E.P.; writing—review and editing, S.S., I.J., and T.M.F.; visualization, T.M.F.; supervision, S.S., I.J., and T.M.F.;. All authors have read and agreed to the published version of the manuscript.

Funding

This research received funding from the Institute for Research and Community Service (LPPM) at Universitas Islam Bandung through the Penelitian Kolaborasi Luar Negeri (PKLN) 2023, under the reference number 011/B.04/LPPM/I/2023.

Conflicts of interest

The authors declare no conflict of interest.

Acknowledgments

The authors express their gratitude to the Institute for Research and Community Service (LPPM) at Universitas Islam Bandung for the financial support provided through the Penelitian Kolaborasi Luar Negeri (PKLN) 2023, under the reference number 011/B.04/LPPM/I/2023.

References

Abu Hawsah M, Al-Otaibi T, Alojayri G, Al-Shaebi EM, Dkhil MA, Elkhadragy MF, Al-Quraishy S, Abdel-Gaber R (2023) In vitro studies for the antiparasitic activities of Azadirachta indica extract. Food Science and Technology (Brazil) 43: e117122. https://doi.org/10.1590/fst.117122

Amir Rawa MS, Mazlan MKN, Ahmad R, Nogawa T, Wahab HA (2022) Roles of syzygium in anti-cholinesterase, anti-diabetic, anti-inflammatory, and antioxidant: from alzheimer’s perspective. Plants 11: 1476. https://doi.org/10.3390/plants11111476

Aris P, Mohamadzadeh M, Zarei M, Xia X (2024) Computational design of novel griseofulvin derivatives demonstrating potential antibacterial activity: Insights from molecular docking and molecular dynamics simulation. International Journal of Molecular Sciences 25: 1039. https://doi.org/10.3390/ijms25021039

Arkundato A, Monado F, Sugihartono I, Rivai AK, Suud Z (2023) Diffusion coefficient calculation of iron in liquid lead using molecular dynamics method with new mixing rule for Lennard-Jones potential parameters. Kuwait Journal of Science 50. https://doi.org/10.48129/kjs.17205

Az-Zahra F, Afidika J, Diamantha SDA, Rahmani AE, Fatimah S, Aulifa DL, Elaine AA, Sitinjak BDP (2022) Indonesian journal of biological pharmacy in silico study of betel leaves compound (Piper betle L.) as Acetylcholinesterase (AChE) enzyme inhibitor in alzheimer disease. Indonesian Journal of Biological Pharmacy 2. https://doi.org/10.24198/ijbp.v2i2.40462

Benkert P, Tosatto SCE, Schwede T (2009) Global and local model quality estimation at CASP8 using the scoring functions QMEAN and QMEANclust. Proteins: Structure, Function and Bioinformatics 77: 173–180. https://doi.org/10.1002/prot.22532

BIOVIA (2017) Dassault Systèmes BIOVIA, Discovery Studio Modeling Environment, Release 2017. Dassault Systèmes San Diego.

Carisya IV, Koesdarto S, Restiadi TI, Hidajati N (2022) Ovicidal effects of pineapple peel juice (Ananas comosus L.) as a control of ascaridians in chicken (Gallus domesticus) in vitro. Journal of Parasite Science 6. https://doi.org/10.20473/jops.v6i2.35321

Charitha VG, Adeppa J, Malakondaiah P (2017) In vitro anthelmintic activity of Syzygium aromaticum and Melia dubia against Haemonchus contortus of sheep. Indian Journal of Animal Sciences 87: 968–970. https://doi.org/10.56093/ijans.v87i8.73453

Cheng HY, Lin TC, Yang CM, Wang KC, Lin CC (2004) Mechanism of action of the suppression of herpes simplex virus type 2 replication by pterocarnin A. Microbes and Infection 6: 738–744. https://doi.org/10.1016/j.micinf.2004.03.009

Daina A, Michielin O, Zoete V (2017) SwissADME: A free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Scientific Reports 7: 42717. https://doi.org/10.1038/srep42717

Damayanti MM, Rachmawati M, Widiyastuti E, Kharisma Y, Fakih TM, Arfan A, Ramadhan DSF (2022) Structure-based design through molecular dynamics approaches of the small-molecule bioactive compounds in cinnamon as interleukin-6 (il-6) inhibitors. Rasayan Journal of Chemistry 2022: 158–166. https://doi.org/10.31788/RJC.2022.1558192

Dixit A, Tanaka A, Greer JM, Donnelly S (2017) Novel therapeutics for multiple sclerosis designed by parasitic worms. International Journal of Molecular Sciences 18: 2141. https://doi.org/10.3390/ijms18102141

Djune-Yemeli L, Nana-Djeunga HC, Lenou-Nanga CG, Donfo-Azafack C, Domche A, Fossuo-Thotchum F, Niamsi-Emalio Y, Ntoumi F, Kamgno J (2020) Serious limitations of the current strategy to control Soil-Transmitted Helminths and added value of Ivermectin/Albendazole mass administration: A population-based observational study in Cameroon. PLoS neglected tropical diseases 14(11): e0008794. https://doi.org/10.1371/journal.pntd.0008794

Dlamini Z, Ntlabati P, Mbita Z, Shoba-Zikhali L (2015) Pyruvate dehydrogenase kinase 4 (PDK4) could be involved in a regulatory role in apoptosis and a link between apoptosis and insulin resistance. Experimental and Molecular Pathology 98: 574–584. https://doi.org/10.1016/j.yexmp.2015.03.022

Eberhardt J, Santos-Martins D, Tillack AF, Forli S (2021) AutoDock Vina 1.2.0: New Docking Methods, Expanded Force Field, and Python Bindings. Journal of Chemical Information and Modeling 61. https://doi.org/10.1021/acs.jcim.1c00203

Elseginy SA, Fayed B, Hamdy R, Mahrous N, Mostafa A, Almehdi AM, S. M. Soliman S (2021) Promising anti-SARS-CoV-2 drugs by effective dual targeting against the viral and host proteases. Bioorganic and Medicinal Chemistry Letters 43: 128099. https://doi.org/10.1016/j.bmcl.2021.128099

Fitzsimmons CM, Falcone FH, Dunne DW (2014) Helminth allergens, parasite-specific IgE, and its protective role in human immunity. Frontiers in Immunology 5: 61. https://doi.org/10.3389/fimmu.2014.00061

Forli W, Halliday S, Belew R, Olson A (2012) AutoDock Version 4.2. Citeseer.

Gayen PR, Monjur-Al-Hossain ASM, Saifuzzaman M, Faroque ABM (2016) Anthelmintic activity of ethanolic extract of Syzygium samarangense (Blume) Merril & Perry. Dhaka University Journal of Pharmaceutical Sciences 15(1): 109–111. https://doi.org/10.3329/dujps.v15i1.29204

González-Quilen C, Rodríguez-Gallego E, Beltrán-Debón R, Pinent M, Ardévol A, Blay MT, Terra X (2020) Health-promoting properties of proanthocyanidins for intestinal dysfunction. Nutrients 12(1): 130. https://doi.org/10.3390/nu12010130

Gopukumar ST, Nair SG, Radha R, Sugathan NV, Anooj ES, Gangadhar L (2020) Three dimensional structure modeling and ramachandran plot analysis of autographa californica nucleopolyhdro viral protein. Annals of Tropical Medicine and Public Health 23. https://doi.org/10.36295/ASRO.2020.23626

Hansen T, Schönheit P (2001) Sequence, expression, and characterization of the first archaeal ATP-dependent 6-phosphofructokinase, a non-allosteric enzyme related to the phosphofructokinase-B sugar kinase family, from the hyperthermophilic crenarchaeote Aeropyrum pernix. Archives of Microbiology 177: 62–69. https://doi.org/10.1007/s00203-001-0359-1

Hansen T, Musfeldt M, Schönheit P (2002) Atp-dependent 6-phosphofructokinase from the hyperthermophilic bacterium Thermotoga maritima: Characterization of an extremely thermophilic, allosterically regulated enzyme. Archives of Microbiology 177: 401–409. https://doi.org/10.1007/s00203-002-0405-7

Huang J, Rauscher S, Nawrocki G, Ran T, Feig M, De Groot BL, Grubmüller H, MacKerell AD (2016) CHARMM36m: An improved force field for folded and intrinsically disordered proteins. Nature Methods 14: 71–73. https://doi.org/10.1038/nmeth.4067

Jin L, Zhang BW, Lu JW, Liao JA, Zhu QJ, Lin Y, Yu XQ (2023) The mechanism of Cry41-related toxin against Myzus persicae based on its interaction with Buchnera-derived ATP-dependent 6-phosphofructokinase. Pest Management Science 79: 1684–1691. https://doi.org/10.1002/ps.7340

Kiran Z, Begum S, Sara S, Bano Z, Siddiqui BS (2021) Novel ferulic acid and benzophenone derivatives from the flower buds of Syzygium aromaticum. Natural Product Research 35: 3301–3306. https://doi.org/10.1080/14786419.2019.1698570

Kļaviņa A, Keidāne D, Ganola K, Lūsis I, Šukele R, Bandere D, Kovalcuka L (2023) Anthelmintic activity of Tanacetum vulgare L. (leaf and flower) extracts against Trichostrongylidae Nematodes in sheep in vitro. Animals 13: 2176. https://doi.org/10.3390/ani13132176

Klimek-Szczykutowicz M, Szopa A, Ekiert H (2018) Chemical composition, traditional and professional use in medicine, application in environmental protection, position in food and cosmetics industries, and biotechnological studies of Nasturtium officinale (watercress) – a review. Fitoterapia 129: 283–292. https://doi.org/10.1016/j.fitote.2018.05.031

Kumar N, Singh A, Gulati HK, Bhagat K, Kaur K, Kaur J, Dudhal S, Duggal A, Gulati P, Singh H, Singh JV, Bedi PMS (2021) Phytoconstituents from ten natural herbs as potent inhibitors of main protease enzyme of SARS-COV-2: In silico study. Phytomedicine Plus 1: 100083. https://doi.org/10.1016/j.phyplu.2021.100083

Kuo YC, Lin LC, Tsai WJ, Chou CJ, Kung SH, Ho YH (2002) Samarangenin B from Limonium sinense suppresses herpes simplex virus type 1 replication in vero cells by regulation of viral macromolecular synthesis. Antimicrobial Agents and Chemotherapy 46. https://doi.org/10.1128/AAC.46.9.2854-2864.2002

Kutzner C, Páll S, Fechner M, Esztermann A, de Groot BL, Grubmüller H (2019) More bang for your buck: Improved use of GPU nodes for GROMACS 2018. Journal of Computational Chemistry 40: 2418–2431. https://doi.org/10.1002/jcc.26011

Laskowski RA, MacArthur MW, Thornton JM (2012) PROCHECK: validation of protein-structure coordinates. International Tables for Crystallography, Vol. F, ch. 21.4, 684–687. https://doi.org/10.1107/97809553602060000882

Laudisi F, Marônek M, Di Grazia A, Monteleone G, Stolfi C (2020) Repositioning of anthelmintic drugs for the treatment of cancers of the digestive system. International Journal of Molecular Sciences 21: 4957. https://doi.org/10.3390/ijms21144957

Liu GH, Wu CY, Song HQ, Wei SJ, Xu MJ, Lin RQ, Zhao GH, Huang SY, Zhu XQ (2012) Comparative analyses of the complete mitochondrial genomes of Ascaris lumbricoides and Ascaris suum from humans and pigs. Gene 492: 110–116. https://doi.org/10.1016/j.gene.2011.10.043

Longevity OMAC (2024) Retracted: Watery rose apple: A comprehensive review of its traditional uses, nutritional value, phytochemistry, and therapeutic merits against inflammation-related disorders. Oxidative medicine and cellular longevity 2024: 1. https://doi.org/10.1155/2024/9834349

Loukas A, Maizels RM, Hotez PJ (2021) The yin and yang of human soil-transmitted helminth infections. International Journal for Parasitology 51: 1243–1253. https://doi.org/10.1016/j.ijpara.2021.11.001

Mulqie L, Suwendar S, Rajih MF, Mardliyani D, Yumniati I, Widiasari W, Nurrosyidah Z (2022) Aktivitas antibakteri ekstrak etanol daun jambu air [Eugenia aqueum (Burm. F) Alston] dengan mikrodilusi agar. Jurnal Ilmiah Farmasi Farmasyifa 5: 1–8. https://doi.org/10.29313/jiff.v5i1.7849

Pampalakis G (2023) Underestimations in the in silico-predicted toxicities of V-agents. Journal of Xenobiotics 13: 615–624. https://doi.org/10.3390/jox13040039

Qiu J, Chen X, Liang P, Zhang L, Xu Y, Gong M, Qiu X, Zhang J, Xu W (2022) Integrating approach to discover novel bergenin derivatives and phenolics with antioxidant and anti-inflammatory activities from bio-active fraction of Syzygium brachythyrsum. Arabian Journal of Chemistry 15: 103507. https://doi.org/10.1016/j.arabjc.2021.103507

Rennie C, Fernandez R, Donnelly S, McGrath KCY (2021) The Impact of Helminth Infection on the Incidence of Metabolic Syndrome: A Systematic Review and Meta-Analysis. Frontiers in Endocrinology 12. https://doi.org/10.3389/fendo.2021.728396

Róg T, Orłowski A, Llorente A, Skotland T, Sylvänne T, Kauhanen D, Ekroos K, Sandvig K, Vattulainen I (2016) Data including GROMACS input files for atomistic molecular dynamics simulations of mixed, asymmetric bilayers including molecular topologies, equilibrated structures, and force field for lipids compatible with OPLS-AA parameters. Data in Brief 7: 1171–1174. https://doi.org/10.1016/j.dib.2016.03.067

Schuler LD, Daura X, Van Gunsteren WF (2001) An improved GROMOS96 force field for aliphatic hydrocarbons in the condensed phase. Journal of Computational Chemistry 22: 1205–1218. https://doi.org/10.1002/jcc.1078

Silva AM, Martins-Gomes C, Silva TL, Coutinho TE, Souto EB, Andreani T (2022) In vitro assessment of pesticides toxicity and data correlation with pesticides physicochemical properties for prediction of toxicity in gastrointestinal and skin contact exposure. Toxics 10: 378. https://doi.org/10.3390/toxics10070378

Sirisha KS, Shreeja K (2019) Rose Apple: A Systematic Review. The Pharma Innovation Journal 8.

Smith R, Williamson R, Ventura D, Prince JT (2013) Rubabel: Wrapping open Babel with Ruby. Journal of Cheminformatics 5: 35. https://doi.org/10.1186/1758-2946-5-35

Sonawane MS (2018) Dietary Benefits of Watery Rose Apple (Syzygium aqueum (Burm.f.) Alston). International Archive of Applied Sciences and Technology 9.

Studer G, Rempfer C, Waterhouse AM, Gumienny R, Haas J, Schwede T (2020) QMEANDisCo—distance constraints applied on model quality estimation. Bioinformatics 36: 1765–1771. https://doi.org/10.1093/bioinformatics/btz828

Tian W, Chen C, Lei X, Zhao J, Liang J (2018) CASTp 3.0: Computed atlas of surface topography of proteins. Nucleic Acids Research 46: W363–W367. https://doi.org/10.1093/nar/gky473

Tsai YC, Chen WY, Chiu C cheng (2023) Molecular effects of site-specific phosphate-methylated primer on the structure and motions of Taq DNA polymerase. Computational and Structural Biotechnology Journal 21: 1820–1827. https://doi.org/10.1016/j.csbj.2023.02.043

Valdés-Tresanco MS, Valdés-Tresanco ME, Valiente PA, Moreno E (2020) AMDock: a versatile graphical tool for assisting molecular docking with Autodock Vina and Autodock4. Biology Direct 15: 12. https://doi.org/10.1186/s13062-020-00267-2

Vanommeslaeghe K, Hatcher E, Acharya C, Kundu S, Zhong S, Shim J, Darian E, Guvench O, Lopes P, Vorobyov I, Mackerell AD (2010) CHARMM general force field: A force field for drug-like molecules compatible with the CHARMM all-atom additive biological force fields. Journal of Computational Chemistry 31: 671–690. https://doi.org/10.1002/jcc.21367

Venkatachalam K, Techakanon C, Thitithanakul S (2018) Impact of the Ripening Stage of Wax Apples on Chemical Profiles of Juice and Cider. ACS Omega 3. https://doi.org/10.1021/acsomega.8b00680

Wadapurkar RM, Shilpa MD, Katti AKS, Sulochana MB (2018) In silico drug design for Staphylococcus aureus and development of host-pathogen interaction network. Informatics in Medicine Unlocked 10: 58–70. https://doi.org/10.1016/j.imu.2017.11.002

Wang JY, Chen H, Wang YY, Wang XQ, Chen HY, Zhang M, Tang Y, Zhang B (2017) Network pharmacological mechanisms of Vernonia anthelmintica (L.) in the treatment of vitiligo: Isorhamnetin induction of melanogenesis via up-regulation of melanin-biosynthetic genes. BMC Systems Biology 11: 103. https://doi.org/10.1186/s12918-017-0486-1

Wang N, Zhou Y, Zhao L, Wang C, Ma W, Ge G, Wang Y, Ullah I, Muhammad F, Alwayli D, Zhi D, Li H (2020) Ferulic acid delayed amyloid β-induced pathological symptoms by autophagy pathway via a fasting-like effect in Caenorhabditis elegans. Food and Chemical Toxicology 146: 111808. https://doi.org/10.1016/j.fct.2020.111808

Waterhouse A, Bertoni M, Bienert S, Studer G, Tauriello G, Gumienny R, Heer FT, De Beer TAP, Rempfer C, Bordoli L, Lepore R, Schwede T (2018) SWISS-MODEL: Homology modelling of protein structures and complexes. Nucleic Acids Research 46: W296–W303. https://doi.org/10.1093/nar/gky427

Wiedemann M, Voehringer D (2020) Immunomodulation and immune escape strategies of gastrointestinal helminths and schistosomes. Frontiers in Immunology 11. https://doi.org/10.3389/fimmu.2020.572865

Williams AR, Fryganas C, Ramsay A, Mueller-Harvey I, Thamsborg SM (2014) Direct anthelmintic effects of condensed tannins from diverse plant sources against Ascaris suum. PLoS ONE 9(5): e97053. https://doi.org/10.1371/journal.pone.0097053

Yadav A, Ojha MD, Hariprasad P (2022) Computational studies evidenced the potential of steroidal lactone to disrupt surface interaction of SARS-CoV-2 spike protein and hACE2. Computers in Biology and Medicine 146: 105598. https://doi.org/10.1016/j.compbiomed.2022.105598

Yassir M, Bakrim W Ben, Mahmoud MF, Drissi B, Kouisni L, Sobeh M (2022) Watery rose apple: A comprehensive review of its traditional uses, nutritional value, phytochemistry, and therapeutic merits against inflammation-related disorders. Oxidative Medicine and Cellular Longevity 2022: 1–18. https://doi.org/10.1155/2022/7502185

Yeni Y, Rachmania RA (2022) The prediction of pharmacokinetic properties of compounds in Hemigraphis alternata (Burm.f.) t. ander leaves using pkCSM. Indonesian Journal of Chemistry 22: 1081–1089. https://doi.org/10.22146/ijc.73117

Yu AC, Lian H, Kong X, Lopez Hernandez H, Qin J, Appel EA (2021) Physical networks from entropy-driven non-covalent interactions. Nature Communications 12: 746. https://doi.org/10.1038/s41467-021-21024-7

Yumita A, Hikmawanti NPE, Hanani E, Saputri CW, Hanana PH, Ero JND, Marcelena M, Baytisani T, Sofiana FA, Shania AF, Saputri ESA, Islami FPN (2023) Exploring the polyphenol contents and antioxidant capacity of the leaf extracts of selected indonesian Syzygium species. Tropical Journal of Natural Product Research 7: 6. https://doi.org/10.26538/tjnpr/v7i6.8

Yuniarta TA, Sumartha IGA, Fakih TM, Handayani R, Ramadhan DSF (2023) Discovery of potential prolyl-trna synthetase allosteric inhibitor through virtual screening and in vitro assay against plasmodium falciparum. Jordan Journal of Pharmaceutical Sciences 16: 4. https://doi.org/10.35516/jjps.v16i4.1027

Zhang X, Bai Y, Zhu W, Xinyue LV, Pei W (2022) ApoM regulates PFKL through the transcription factor SREBF1 to inhibit the proliferation, migration and metastasis of liver cancer cells. Oncology Letters 24: 1. https://doi.org/10.3892/OL.2022.13331

Supplementary materials

Supplementary material 1

Examination of the ligand binding to ATP-dependent 6-PFK receptors utilizing AutoDock Vina version 1.2.3

Suwendar Suwendar, Sani Ega Priani, Dina Mulyanti, Taufik Muhammad Fakih, Ibrahim Jantan

Data type: docx

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

Download file (22.08 kb)

Supplementary material 2

The interactions at the molecular level between the most effective ligands and the ATP-dependent 6-PFK receptor were investigated using AutoDock Tools version 4.2.6

Suwendar Suwendar, Sani Ega Priani, Dina Mulyanti, Taufik Muhammad Fakih, Ibrahim Jantan

Data type: docx

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

Download file (22.27 kb)

﻿Abstract

Keywords

﻿Introduction

﻿Materials and methods

﻿Preparation of ATP-dependent 6-phosphofructokinase receptor structure

﻿Validation of ATP-dependent 6-phosphofructokinase receptor structure

﻿Acquisition of dataset

﻿Molecular docking assessment

﻿Ligand molecule preparation

﻿Receptor macromolecule preparation

﻿Binding site determination

﻿Visualization of molecular interactions through docking

﻿ADMET analysis

﻿Estimation of predicted IC50

﻿Simulating molecular dynamics (MD)

﻿In vitro testing for anthelmintic effect

﻿Plant sample collection and preparation

﻿Evaluation of mortality and paralysis

﻿Assessment of ovicidal effects

﻿Statement of ethical approval

﻿Results

﻿ATP-dependent 6-phosphofructokinase receptor sequence

﻿Receptor constructed by homology modeling

﻿Molecular docking analysis

﻿ADMET analysis

﻿Computational inhibition constant

﻿Molecular dynamics simulations study

﻿In vitro testing for anthelmintic effect

﻿Evaluation of mortality and paralysis

﻿Assessment of ovicidal effects

﻿Discussion

﻿Conclusions

﻿Author contributions

﻿Funding

﻿Conflicts of interest

﻿Acknowledgments

﻿References

Supplementary materials

Abstract

Introduction

Materials and methods

Preparation of ATP-dependent 6-phosphofructokinase receptor structure

Validation of ATP-dependent 6-phosphofructokinase receptor structure

Acquisition of dataset

Molecular docking assessment

Ligand molecule preparation

Receptor macromolecule preparation

Binding site determination

Visualization of molecular interactions through docking

ADMET analysis

Estimation of predicted IC₅₀

Simulating molecular dynamics (MD)

In vitro testing for anthelmintic effect

Plant sample collection and preparation

Evaluation of mortality and paralysis

Assessment of ovicidal effects

Statement of ethical approval

Results

ATP-dependent 6-phosphofructokinase receptor sequence

Receptor constructed by homology modeling

Molecular docking analysis

ADMET analysis

Computational inhibition constant

Molecular dynamics simulations study

In vitro testing for anthelmintic effect

Evaluation of mortality and paralysis

Assessment of ovicidal effects

Discussion

Conclusions

Author contributions

Funding

Conflicts of interest

Acknowledgments

References