Molecular dynamics simulations, molecular docking, and kinetics study of kaempferol interaction on Jack bean urease: Comparison of extended solvation model

Abstract Since the urease enzyme creates gastric cancer, peptic ulcer, hepatic coma, and urinary stones in millions of people worldwide, it is essential to find strong inhibitors to help patients. Natural products are well known for their beneficial effects on health and efforts are being made to isolate the ingredients, the so‐called flavonoids. Flavonoids are now considered as an indispensable component in a variety of nutraceutical, pharmaceutical, and cosmetic applications. Kaempferol (KPF) is an antioxidant found in many fruits and vegetables. Many reports have explained the significant effects of dietary KPF in reducing the risk of chronic diseases such as cancer, ischemia, stroke, and Parkinson’s. The current study aimed at investigating the inhibitory impact of KPF on Jack bean urease (JBU) using molecular dynamics (MD) simulations and molecular mechanics Poisson–Boltzmann surface area (MM‐PBSA) calculations to confirm the results obtained from isothermal titration calorimetry (ITC), extended solvation model, and docking software. In addition, UV–VIS spectrophotometry was used to study the kinetics of urease inhibition. Calorimetric and spectrophotometric determinations of the kinetic parameters of this inhibition indicate the occurrence of a reversible and noncompetitive mode. Also, the docking and MD results indicated that the urease had well adapted to the kaempferol in the binding pocket, thereby forming a stable complex. Kaempferol displayed low binding energy during MMPBSA calculations. The inhibitory potential of kaempferol was confirmed by experimental and simulation data, but in vivo investigations are also recommended to validate our results.


| INTRODUC TI ON
Flavonols are flavonoids with a ketone group. They are building blocks of proanthocyanins. The most studied flavonols are kaempferol, quercetin, myricetin, and fisetin with specific clinical characteristics such as antiallergic, anti-inflammatory, antifungal, antiviral, antitumor, anti-H. pylori, and antibacterial properties(5-7) have recently attracted greater attention for treating cancer (Harris et al., 2016;Qiu et al., 2018). Kaempferol derivatives have also depicted antibacterial activity (10) and considerable inhibitory effects against the growth of H. pylori (11). Kaempferol (3,4′,5,7-tetrahydroxy flavone), a yellow crystalline solid with a melting point of 276-278 °C (529-532 °F), is a natural flavonol, a kind of flavonoid, that is found in a specific variety of plants and plant-derived foods such as kale, beans, tea, spinach, and broccoli (Holland et al., 2020;Sen et al., 2016). One of the most well-known properties of Kaempferol is its anti-inflammatory and anticancer properties. Urease (urea amidohydrolase; E.C. 3.5.1.5) is a nickelcontaining enzyme that catalyzes the hydrolysis of urea to the formation of ammonia and carbon dioxide (Mobley et al., 1995). The ammonia that is produced there does serious damage to gastric epithelium through its interactions with the immune system in humans while also creating severe metabolic disorders (Preininger & Wolfbeis, 1996;Suzuki et al., 1992). Also, urease creates several pathogenic states in both humans and animals, such as gastric cancer, stone formation in kidneys, urinary and GIT infections, pyelonephritis, ammonia encephalopathy, catheter encrustation, hepatic coma (Aidoo et al., 2013;Ciurli et al., 1999;Jang et al., 2008;Mobley et al., 1995). The high activity of urease in agriculture creates major environmental and economic problems by releasing abnormally large amounts of ammonia into the atmosphere during urea fertilization.
Furthermore, the high activity damages plant essentially by robbing plants of their essential nutrients and creating ammonia toxicity thereby increasing the pH of the soil (Bremner, 1995). Urease inhibitors making up several compounds, e.g., hydroxamic acid and its derivatives, were used in agriculture and medicine (Muri et al., 2004).
Bioinformatics plays an essential role in drug development. The discovery of herbal drug candidates requires a strong assessment of pharmacological quality, including absorption, distribution, metabolism, excretion, and toxicity (Ferreira et al., 2015). The inhibition mechanism has not been fully understood despite urease being the first crystallized enzyme. The logical design of urease inhibitors is strongly enforced by the knowledge of crystal structures of this enzyme. From the published crystal structures, it can be understood that the presence of nickel-complexing moiety alongside properly placed, network of hydrogen-bond donors and acceptors attached to flexible scaffold is effective in inhibiting this enzyme. Several an in silico investigations were performed with the aim of finding potent inhibitor molecules that could strongly bind to the active site of urease; such as (PDB4H9M, Kaempferol 3 -O-(6"-O-trans-coumaryl) glucoside 7-O-(6″'-O-trans-coumaryl) glucoside, Kaempferol-3-O-(6"-O-trans-coumaryl) glucoside (Tiliroside), (PDB 4GY7), carbonic anhydrase-II from Iris species), (PDB 4H9M, Luteolin) or (PDB 4CEX, fluoride) (Benini et al., 2014;Eftekhari et al., 2021;Mazinani et al., 2020;Saleem et al., 2019). Also, an in silico evaluation of bioactive molecules of tea such as kaempferol indicated that it can be a strong inhibitor of protein-15 SARS-COV-2 (Sharma et al., 2021).
In this study, we will challenge our previous thermodynamic results of kaempferol binding to urease, performed with isothermal titration calorimetry (ITC) and an extended solvation model (Zolghadr & Behbehani, 2019). A previous in vitro study reported that kaempferol was a good inhibitor; therefore, the power of molecular dynamics and molecular docking was used for confirmation. We were using the GROMACS 4.5.4 package and Auto Dock 4 (version 1.5.6) software and inhibition kinetics investigation through molecular dynamics evaluation. After MD simulation studies, the MM-PBSA approach was applied to interpret the free binding energy.

| MATERIAL S AND ME THODS
Kaempferol and purified Jack bean urease were purchased from Sigma.

| Kinetic studies
Calorimetric experiments were carried out with a high-sensitivity ITC (ITC: isothermal titration calorimetry) micro-calorimeter (Thermal Activity Monitor 2277, Thermometric, Sweden). The Hamilton syringe was used to inject kaempferol solution (8000 μmol/L) into the vessel containing 5 μmol/L enzymes, and 30 injections were performed consecutively. In the next step, the heat of each injection was calculated by the "Thermometric Digitam3" software program.
In addition, the heat of the biomolecules ligands' interactions (q) in the aqueous solvent system could be precisely calculated through the following Equation: The inhibitory effect of kaempferol on Jack bean urease was investigated in a study to examine the kinetic studies, inhibitory potential, and mechanism of inhibition in phosphate buffer and 1 mM EDTA at pH 6.8. Lineweaver-Burk plots were constructed from kinetic data to determine the mechanism of enzyme inhibition by varying the concentrations of substrate urea in the presence of different concentrations of kaempferol compound (0, 0.5, 1, 2.5, and 5 μM).
Inhibition constant (K i ) was determined, different concentrations of inhibitor were obtained from the Lineweaver-Burke plot, and all experiments were conducted in triplicate. The IC50 values of inhibitors were calculated. Inhibition was found noncompetitive.

| Protein-ligand docking process
To get insight into possible interaction modes between active sites of Jack bean urease, the molecular docking study was performed using AutoDock4 software. We obtained geometries of kaempferol from PubChem (CID: 5280863), while the 3D structure of receptor urease (jack bean) was obtained from protein data bank (PDB) with PDB4H9M (http://www.rcsb.org). The molecules of water, acetohydroxamic acid, and 1,2-ethanediol were cleaned from the enzyme structure, and hydrogens were added while calculating

PBSA analysis
The molecular dynamics simulation process of the studied complex was carried out using GROMACS 2020.1 software suite (Hess et al., 2008). For the water solvent model, TIP3P and force field are also CHARMM (Jorgensen et al., 1983). To perform the simulation in the neutralization and minimization stages of the system, the (Steep = Steepest descent minimization) algorithm was used for integration into 50,000 steps. Then, the equilibration steps were performed with the help of canonical (NVT) and isothermal-isobaric (NPT) ensembles with the help of the Leap-frog algorithm for integration into 500,000 steps (Hockney, 1970). The system configuration was saved every 0.2 ps. The restriction was also performed on all links. The primary sampling step was performed with the help of an NPT set for 30 ns. To fix the temperature of the system at 300 K, the Nose-Hoover thermostat constant was applied. The Parrinello-Rahman pressure coupling method was applied to preserve the system pressure at a fixed 1 bar. The structure of the rigid water model was constrained using SETTLE (Vermaas et al., 2016). The biding of bond length containing hydrogen atoms was performed using the LINCS algorithm (Hess et al., 2008). Also, to measure the electrostatic interactions, the particle mesh Ewald (SPME) method was applied with 1.0 nm short-range electrostatic and van der Waals cut-offs (Essmann et al., 1995). Finally, while taking time steps of 2 fs on equilibrated systems, 100 ns MD simulation for the complex of urease-kaempferol was done. The stored paths in the simulation are used to analyze the structural parameters of the studied complex. In the next step, six parameters including analysis of hydrogen bonds (H-bonds) and surface accessible solvent area (SASA), the root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (R g ), and dictionary of secondary structure of protein (DSSP) were studied.

| MMPBSA calculations
The molecular mechanics Poisson-Boltzmann surface area (MMPBSA) approach has been widely applied as an efficient and reliable free energy simulation method to model molecular recognition, such as for protein-ligand binding interactions (Arba et al., 2017;Duan et al., 2016;Sharma et al., 2021). The gmmpbsa module of GROMACS was used for MMPBSA calculations on the simulated urease-kaempferol complex to obtain the final binding free energies and residue contribution energies of the complex (Kumari et al., 2014). Equations were used in this module for calculation of binding free energy (BFE) as follows: In these equations, ∆G binding, ∆E electrostatic, ∆E vd W, ∆G polar , and ∆G polar are the BFE, the electrostatic contribution, the Vander Waalse contribution, and polar and nonpolar solvation terms, respectively. The nonpolar solvation term is more commonly referred to as the SASA contribution .

| RESULTS AND DISCUSSION
Previously, it was demonstrated by Behbehani et al. that the heats of the biomolecules ligands interactions (q) in the aqueous solvent system could be precisely calculated through the following Equation (Behbehani et al., 2008;Rezaei Behbehani et al., 2011;Rezaei Behbehani, Saboury, Barzegar, et al., 2010): In this research, we used this equation and isothermal titration calorimetry (ITC) to study the thermodynamics of kaempferol binding to urease. To further confirm our results, we investigate and describe inhibition using kinetics studies and mechanism of inhibition and molecular dynamics simulations, and MM-PBSA calculations in the present study.

| Investigation of positive cooperativity of kaempferol with urease
The inhibition of urease by kaempferol was investigated using isothermal titration calorimetry (ITC), a method that is becoming a major tool to aid enzyme inhibitor screening and design. As mentioned, we used Equation 1 to calculate the heats of the JBU + KPF interactions. To investigate the type of ligand-enzyme cooperation, we needed to obtain the value of the parameter P. We used the following two equations: The results of fitting the heat of urease and kaempferol interactions are shown in Figure (1a) and Table 1 Table 2 and Figure (1.b, c).
As you can see in Table 2, the value of n in two regions is more significant than 1 (n > 1) and confirms this positive cooperation, which is the same result we obtained from Equation 1 .

| Kaempferol noncompetitive inhibition of urease
The results of our kinetic studies showed that urease activity is dose dependent. The IC 50 value was calculated as 6.96 μM; the results showed reversible inhibition since all straight lines passed through the origin. Lineweaver-Burk analysis was performed, and inhibition was evaluated. The change in Vmax showed that the inhibition was noncompetitive while Km was constant. The K i value was 6.06 μM (Figure 2). The study of the kinetics of Morin (3, 5, 7, 2′, 4′-pentahydroxyflavone), that is "a bioflavonoid" by Ritu Kataria

| Molecular Docking (Interaction analysis)
No published reports have described the urease-kaempferol.
Thus, this work aimed to gain further insight into the working molecular mechanism of the inhibitory effect of kaempferol on Jack bean urease. Initially, in the current study, the interaction between kaempferol and specific binding sites on urease through hydrogen bonding, metal/ion contact with Ni ions, and hydrophobic interac- modifications that occur within the receptor may impart major concepts and demonstrate that receptor flexibility is an important part of computational drug design that has been mentioned above (Mohan et al., 2005). F I G U R E 2 Lineweaver-Burk plot for kaempferol (0, 0.5, 1, 2.5, 5 μM) F I G U R E 3 Binding interactions of kaempferol with the active binding site of urease PDBID 4H9M generated using Ligplot and discovery. (a, c) Show the 3D docking of kaempferol in a binding pocket (b). (d) Shows the two-dimensional ligand-protein interactions. Legend inset represents the type of interaction between the ligand atom and the amino acid residues of the protein

O-trans-coumaroyl)glucopyranoside7-O-6′coumaroylglucopyranoside
inhibits urase with a free binding energy equal to −6.11 kcal/mol and K i equal to 33.28 μM, the hydroxyl groups of rings no interacted with crucial residues and Ni ions (Eftekhari et al., 2021). In the present study, kaempferol with the lowest free binding energy −6.48 kcal/ mol showed an appropriate docking score and suitable interactions in agreement with the biological activity, suggesting that kaempferol could be an efficient inhibitor in comparison to kaempferol-3-O-(6"-

O-trans-coumaroyl)glucopyranoside7-O-6′coumaroylglucopyranoside and Kaempferol 3-Oβ -Oneohesperidoside-7-O-[2-O-(cis-feruloyl)] β
-D-glucopyranoside (free binding energy = −1.60 kcal/mol). To sum up, these synthetic compounds bearing bulky substitutions were understood to be weaker inhibitors than kaempferol. The OH group of the neohesperidoside moiety of these compounds mediated bidentate interactions with Ni ions, while the kaempferol part of these compounds showed an inert behavior which may be attributed to the spatial hindrance of some groups, resulting in weak inhibition. In another report, evaluation of different bioactive molecules of tea for inhibition potency SARS-COV-2, results molecular docking results, indicated that kaempferol could be considered as potential inhibitor of NSp15 (total binding energy: −12.9 kcal/mol) (Sharma et al., 2021).
Weak intermolecular interactions such as hydrogen bonding and hydrophobic interactions are key players in stabilizing energetically favored ligands, in an open conformational environment of protein structures (Patil et al., 2010). Our results indicate that both interactions stabilize the kaempferol at the active site, and help alter binding affinity.

| MD simulations and MM-PBSA analysis
MD simulations are important to closely examine the stability, conformational changes, internal motions, etc. of protein-ligand complexes, which have been shown to be effective in mutational analysis and inhibitor designing (Gupta et al., 2021;Shin et al., 2020). The MD simulations of free urease and urease-kaempferol complex were performed, and the results were compared to measure the structural changes caused by ligand binding. RMSD is deemed a crucial structural and dynamical parameter to evaluate the conformational stability despite investigating the quality, equilibration, and convergence of an MD run (Gupta et al., 2021). A more considerable RMSD value demonstrates the lower strength of a protein complex and contrariwise. On the other hand, higher fluctuations indicate low stability.
Highly deviated RMSD graphs can also imply major conformational transitions occurring in the protein to obtain stable conformation with the ligand. In this study, the RMSD of the kaempferol-urease complex and urease free concerning the Cα atom was calculated versus time, Figure 4a.  (Saeed, Mahesar, et al., 2017).
As you can see in Figure 4b, the highest average fluctuations are observed in the active site areas (His409, His 519, Glys550, Asp633, Arg439, Cme 592, and Met 737). This result was acceptable given that we focused on the dynamic behavior of the active site of urease. Moreover, two significant peak fluctuations were also observed by the residues other than the active site residues, indicating their increased interaction potential implying that the kaempferol able to adapt well in the binding pocket of the urease.
The radius of gyration (R g ) determines the protein compaction level. According to its definition, it is considered as the mass-weighted root-mean-square distance of a set of atoms from their common center of the mass. Hence, the evolution of the overall protein dimension during dynamics is represented by the trajectory analysis of the radius of gyration. Since it is not easy to determine the relative distance of each atom to the center of mass of the protein, we use the radius of gyration (R g ) factor to investigate the folding or unfolding of the protein. As shown in Figure 5a, the R g of urease is greater than R g of the urease-kaempferol complex at the end of the simulation time when the system reaches equilibrium. The urease-ligand structure saw four significant ascents of 53 ns. In less than 19 ns, the R g of complexation increases, related to hydrophobic interactions.
The R g increases from 1.58 to 1.65, making the system more open.
The change in system state from the fold to open indicates the system's instability. After 50 ns, little change is seen, which means the system is in equilibrium. Totally, concerning urease-ligand, the average value of R g is 1.6502 nm, and the ascent is significant with a size of about 3.2 ns (Figure 5a). However, concerning the urease, the average value of R g is about 1.58 nm, and different decays occur in the R g plot, as presented in Figure 5a. In general, MD results showed that urease in the complex is more unstable than urease in the free state,  (Pretzler et al., 2017).
The binding free energy consists of polar solvation, Van der Waals interactions, electrostatic, and SASA energy. Contribution of electrostatic, Van der Waals, and SASA energy were negative, whereas positive contributions were shown by polar solvation energy to the overall free binding energy. As shown in Table 3, Van der Waals energy was observed to be the primary benefactor for the interaction of kaempferol with urease. These results are also in accordance with the effect of kaempferol on protein-15 of SARS-CoV-2 (Sharma et al., 2021). The contribution of each residue was also explored in Figure 2s, where the higher the contribution of a residue toward favorable interaction, more negative is energy value, whereas unfavorable contribution attains a positive energy value.

| Comparison of molecular dynamics with extended solvation model
In general, the results of molecular dynamics and kinetics confirm the inhibition of the urease enzyme by kaempferol. It is fascinating that these results have been developed following the thermodynamic study of inhibition by the extended solvation model. As shown in Tables 1 and 2, the interaction is entropy driven, indicating that the vdW energy is dominant, and docking and MMPBSA confirm this.
Negative δ A and δ B values indicate the formation of an unstable complex of urease with kaempferol. In parallel, the results of molecular dynamics and increasing R g value and hydrophobic amino acids accessible in the solvent confirmed the system's unfolding in the presence of kaempferol. On the other hand, close changes in the amount of δ A and δ B characterize particular space interactions in the system. P > 1 and negative δA and δB show that kaempferol causes a few reversible changes in the urease structure. Affinity binding kaempferol to urease, reduction in activity urease, and binding confirmations from molecular simulation studies also confirm the noncompetitive inhibition type, as shown in Figure 2. Meanwhile, the total binding free energy obtained from ITC and extended solvation model was almost close to the MMPBSA result, as shown in Table 2

| CON CLUS ION
This study reports the molecular dynamics of urease-kaempferol complexes, molecular docking, MM-PBSA, and urease inhibitory activities. The molecular docking and molecular dynamics studies on the kaempferol versus Jack bean urease valuably led to F I G U R E 7 The secondary structure (DSSP algorithm) for urease-kaempferol and urease TA B L E 3 MMPBSA-based total binding free energies along with its constituent energies for urease-kaempferol der Waal interactions with other residues, which shows that the selected molecule has significant binding interactions within the active site residues. The inhibitory activity tested in vitro against Jack bean urease shows that kaempferol exhibits a partially good inhibitory activity of IC50, 6.96 μM. Importantly, we only focused on comparing and confirming the results; detailed research continuously examined the toxicity of these complexes of urease inhibitory activity posed to the environment and humans.

ACK N OWLED G EM ENTS
We thank the Cellular and Molecular Research Center of the University of Medical Sciences and the Department of Chemistry and Simulation Center of Qazvin Imam Khomeini International University.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.