Dynamics based screening of Therapeutic compounds against Helicobacter pylori targeting TNF-α inducing protein

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Chidhambara Priyadarshini, Shalini Yadav, Sukriti Singh, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5983215/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract In about half of the world's population, Helicobacter pylori (H. pylori) is the cause of stomach ulcers. Due to the extensive use of different drugs to treat H. pylori infections, multidrug resistance has developed, making treatment more difficult. Finding a viable therapeutic target becomes increasingly important as antibiotic resistance keeps increasing. A distinct protein of H. pylori called Tumour Necrosis Factor-alpha Inducing Protein (Tip-α) is important in the development of gastric cancer, especially in patients with chronic ulcers. Because of its vital role, Tip-α may be a good target for medication. Using molecular docking experiments, two different binding sites on Tip-α were found, and possible inhibitors were assessed. ZINC11981988 and ZINC04243820, two ZINC database hits among the screened compounds, showed a significant binding affinity to Tip-α, with glide scores of -9.116 kcal/mol and -10.455 kcal/mol, respectively. A MM-GBSA analysis was conducted to have a better understanding of their binding mechanisms. The findings showed that the selectivity of Tip-α inhibitors was more influenced by electrostatic energy contributions than by van der Waals interactions. The stability of these ligands in their respective binding sites was evaluated using molecular dynamics (MD) simulations upto 100 ns. In contrast to ZINC11981988, ZINC04243820 showed a more stable binding association with Tip-α, according to the MD analysis. According to this, ZINC04243820 exhibits excellent binding stability and is therefore a promising candidate for additional research. Considering these results, ZINC04243820 could be proposed as potential lead to construct Tip-α inhibitors against H. pylori which shows favourable interaction properties and persistent binding. Further, in vivo and in vitro investigations are required to examine and validate its therapeutic potential. Helicobacter pylori Gastric ulcer Tumour Necrosis Factor Alpha Inhibitors Docking studies Molecular dynamics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. INTRODUCTION Helicobacter pylori ( H.pylori ) is a Gram-negative microaerophilic bacterium that dwells in the human stomach and is responsible for 85 percent of gastric ulcers worldwide. Antibiotics have been used indefinitely to eradicate H.pylori , which has raised the rate of multidrug resistance in them. This needs the development of a prospective pathogen-eradicating medication. Identification of a potential drug target is thus critical in the chemotherapeutics process. Tip- α is a virulence protein identified from H. pylori which is immunogenic, and similar to other virulence factors (CagA, UreaseA, Vac A) of the bacterium [ 1 ]. It is produced by the HP0596 gene of the bacterium and is found to be unique to this bacterium [ 2 ]. Among the various virulence factors characterised in H. pylori, Tip-α has been characterised to be carcinogenic and found to induce potentially TNF-α and chemokine genes through the activation NF-Kapp B cells, where NF- kappa B (nuclear factor kappa B), the Rel family of proteins is made up of a collection of dimeric transcription factors that play an important role in inflammation and immune control [ 3 – 7 ]. Several studies have proved the apoptosis inducement of Tip- α on human gastric epithelial cell lines, where the protein modulates the sensitivity of cell lines’ apoptosis [ 8 , 9 ]. Tip- α is a newly identified gene in H. pylori. It is also known as H. pylori 0596 protein as it is found in the 0596 open reading frame of the H. pylori 26695 strain. It has a 519-bp open reading frame that contains 173 amino acids [ 2 ]. Tip-α (Tip-alpha) is a protein that resembles tumour necrosis factor (TNF) and is mostly linked to Helicobacter pylori, a bacteria that is linked to gastritis, peptic ulcers, and stomach cancer. This protein helps H. pylori survive and remain in the stomach mucosa by influencing the host's immune system and encouraging inflammation. The cytokine-like action of tip-α, which is released by H. pylori, can trigger the production of chemokines and activate host cell nuclear factor-kappa B (NF-κB) pathways. These actions add to the inflammatory milieu that is a feature of an H. pylori infection. Tip-α increases the expression of pro-inflammatory cytokines including interleukin-8 (IL-8) by binding to nucleolin, a protein present on the surface of host cells, and promoting cellular signalling pathways (Mizuno et al., 2005). Increased IL-8 levels cause neutrophil recruitment, which exacerbates tissue damage and starts an inflammatory cycle. The advancement of gastric carcinogenesis is linked to Tip-α, which is interesting because it is not just an inflammatory mediator. Its capacity to trigger anti-apoptotic reactions, genetic instability, and cell proliferation highlights its carcinogenic potential (Chaturvedi et al., 2014). Furthermore, research indicates that Tip-α may decrease host T-cell responses to control immune evasion, which would enable H. pylori to colonise for a longer period of time (Watanabe et al., 2010). Tip-α has been shown to have a structure different from penicillin-binding proteins. It is composed of three closely linked domains that interact with other proteins and RNAs [ 10 ]. Tip- α protein exists in a sand-witched form of α-β, combined with helical bundles [ 11 ]. The topology includes β1, α1, α2, β2, β3 α3, α4 and a 310 helix at the N-terminal portion. α2, α3, and α4 form a helix bundle at the C-terminal and are connected by a loop of α1, α2 at the top of the helix bundle. Further, the N-terminal half of α1 was wrapped and covered by antiparallel β sheets 1, 2 and 3. A literature survey revealed Cys25-cys26, Arg81, and Leu50 as important residues for the binding of protein to DNA and dimeric formation [ 11 , 12 ]. There are limited studies on the H. pylori Tip- α pockets and their inhibitor having efficient binding. In the present work, we have conducted structural and functional studies using bio-computational approaches including docking and molecular dynamics simulations to investigate the target protein and potential inhibitors of natural origin. Thereby, this analysis proposes that natural molecules as the probable drug lead having the affinity for Tip-α of H. pylori . Previously, our group has conducted some similar studies (27, 28, 29). 2. MATERIALS AND METHODS 2.1 Retrieval of Target Protein and Preparation of Protein A repository of four crystal structures of Tip- α protein (HP0596) was deposited in PDB. 2WCQ crystal structure with a resolution of 1.9Aº was taken as reference molecule (Berman et al., 2000). As detailed study on the active binding site of this protein was absent, Glide was employed to identify the binding site information of Tip- α. To achieve this, the protein was prepared using protein preparation wizard of Glide During preparation, the Schrodinger prepares the protein by fixing major errors like incorporating missing residues near the active site, adjusting the ionization and tautomerization state of protein and further by refining the structure by relieving any strain from the adjustments [ 13 ]. 2.2 Active Site Prediction When considering the drug discovery line, the identification of substrate active sites and inhibitor binding site residues are crucial as functional change is imposed by perturbing these sites on a molecule by the inhibitors/activators [ 14 ]. As literature surveys were absent on active residue with binding potential, the Qsite tool of Glide was employed (Sankar et al., 2022). With the information on a series of physical parameters such as size, tightness, hydrophobic/hydrophilic character, degree of enclosure/exposure, and hydrogen bonding possibilities of Schrodinger, the sitemap assigns numerical descriptors to evaluate predicted binding site pockets. To prioritize possible binding sites, a weighted average of these measurements was then assigned. 2.3. Ligand preparation : For the ligand selection and screening, we have utilized the Zinc Database was used. The ZINC database is a free, open-access collection of chemical compounds that are sold commercially and are selected for online screening investigations (Irwin et al., 2012). With millions of tiny compounds pre-filtered for drug-likeness, this database is a perfect tool for finding possible inhibitors of bacterial virulence factors like Helicobacter pylori's Tumour Necrosis Factor Alpha Inducing Protein (Tip-α). Targeting Tip-α with natural inhibitors may offer an alternate therapeutic approach to treat H. pylori infections, given its critical role in gastric inflammation and carcinogenesis. Utilising the ZINC Natural Products Subset, phytochemical compounds with structural characteristics that support Tip-α inhibition were recovered. These compounds' binding potential, bioavailability, and safety were evaluated by computational screening, which included molecular docking and ADMET analysis. Using the ZINC database to find natural inhibitors of Tip-α, presents a possible non-antibiotic strategy to treat H. pylori -associated illnesses while addressing resistance issues. 2.3 Docking Analysis 2WCQ was used to specify the grid. For Grid generation, active residues predicted from the q-site were given. Two grid files were generated for the sites one and two. All the docking calculations were performed using the HTVS, Standard Precision (SP) and ‘Extra Precision’ (XP) mode of GLIDE program [ 15 , 16 ]. After XP mode, IFD and QPLD were conducted to identify various interactions and bindings of hit molecules in the active site of Tip- alpha. G-scores and Glide energy were used to rank the screened compounds in ascending order. The virtual screening technique penalises steric conflicts while recognising favourable hydrophobic, hydrogen-bonding, and metal-ligation interactions. Finally, Schrödinger's proprietary Glide Chem Scoring programme re-scored the minimised poses. $$\:\mathbf{G}\mathbf{S}\mathbf{c}\mathbf{o}\mathbf{r}\mathbf{e}\:=\:0.065\text{*}\mathbf{v}\mathbf{d}\mathbf{W}\:+\:0.130\text{*}\mathbf{C}\mathbf{o}\mathbf{u}\mathbf{l}\:+\:\mathbf{L}\mathbf{i}\mathbf{p}\mathbf{o}\:+\:\mathbf{H}\mathbf{b}\mathbf{o}\mathbf{n}\mathbf{d}\:+\:\mathbf{M}\mathbf{e}\mathbf{t}\mathbf{a}\mathbf{l}\:+\:\mathbf{B}\mathbf{u}\mathbf{r}\mathbf{y}\mathbf{P}\:+\:\mathbf{R}\mathbf{o}\mathbf{t}\mathbf{B}\:+\:\mathbf{e}$$ Where vdW denotes the energy of Vander Walls, Columbic energy is denoted by the term coul. Lipophilic is denoted as Lipo. The phrase "hydrogen bonding" is defined by Hbond. Metal is a term that refers to a metal-binding substance. BuryP is a programme that calculates a penalty for buried polar groups. Site represents active site polar interactions, while RotB is a penalty for freezing rotatable bonds. In this study, natural compounds from phase screens were virtually screened using HTVS and XP docking, respectively. 2.4 Drug-likeness Properties Analysis and ADMET Screening The ADMET properties for the lead-zinc ligands against Tip- α was predicted in-silico by utilizing qikprop module of Schrödinger. Meanwhile their drug-likeness properties were analysed through SwissADME online server (Daina et al., 2017). 2.5 Molecular Dynamics 2.5.1 System Preparation Molecular dynamics simulation had been performed on the receptor-ligand complex obtained after docking. The lack of hydrogen atoms in the protein was fulfilled by the LEAP module of AMBER 20. Ligand molecules have been parametrized by the antechamber module employed with GAFF (generalized amber force field). Partial atomic charges on the ligand atom had been assigned by the semi empirical AM1-BCC charge method. An appropriate number of positively charged sodium (Na+) was added to the system to make the complex electrically neutral. The resulting complex system was solvated with a TIP3P water model, into a box having an octahedral shape stretched up to 10 Å from the protein boundary. The FF14SB force field was employed on the protein during the MD simulation. Following system setup and parametrization of the two complexes, system geometries were minimized into two stages. i) First, all the solvent water molecules were minimized by keeping the protein fixed. ii) Second, the entire complex was minimized (5000 steps of steepest descent and 5000 steps of conjugate gradient) to get rid of bad contacts and to relax the system geometry. Under the NVT ensemble, system was gently annealed from 10 K to 300 K for 50 ps. To attain a uniform density, 1 ns of density equilibration was performed. During this, to maintain a constant temperature of 300K and the constant pressure of 1.0 atm, Langevin thermostat with a collision frequency of 2ps and Berendson barostat with a pressure relaxation time of 1ps were applied respectively. Afterwards, all the restraints had been removed and systems were further equilibrated for 3ns followed by 100ns of production MD. During MD simulation all the covalent bonds containing hydrogens were kept constrained using the SHAKE algorithm and a Particle Mesh Ewald (PME) was used to treat the long-range electrostatic interactions. We used an integration step of 2fs during the entire simulation. To get more accurate and reliable results we have performed two replicas of 100ns of both systems started from different initial velocities. All simulations had been performed by GPU version of AMBER 20 package. For the analyses purpose trajectory obtained by production run was used. CPPTRAJ module of AMBER 20 and the visualizing software VMD, Pymol were used for the analysis of the MD trajectories. 2.5.2 MMGBSA To quantify the observed binding stability of ligand in both the complexes, further, performed a thermodynamical free energy calculation i.e., MMGBSA (Molecular Mechanics Generalized Boltzmann Surface Area). This free energy calculation was carried over the 2000 frames selected from the trajectory in which RMSD of the complex was found well converged. Water molecules and all ions present in the system had been removed before preceding the MMPBSA analysis. For solute, the dielectric constant was 1 and for the surrounding solvent molecules, it had been kept at 80. MMGBSA calculation was carried out using the MMPBSA.py module of AMBER 20 3. RESULTS 3.1 Active Site Identification Tip- α appears to play a significant role in H.pylori -induced inflammation and carcinogenesis, according to previous researches conducted so far [ 17 , 18 ]. This is due to the increased expression of extra pro-inflammatory chemokines and the stimulation of TNF via Nuclear Factor kB (NFkB) [ 19 ]. The unique presence and carcinogenic activity of this protein and the absence of homologous parts in any other gram negative pathogen other than H.pylori , raises its important to be considered as a potential drug target The site map tool predicted three groups of sites with active binding residues in Tip- α. From these, two sites were selected based on the fitness score, as the third one was short, and the residues were the same as of the above two. First site included residues 47, 48, 49, 51, 52, 54, 55, 56, 81, 84, 85, 87, 88, 89, 91, 92, 111 of Chain a with a site score of 0.884 and second site included residues 90, 93, 94, 98, 99, 100, 101, 168, 169, 171, 172, 175, 133,134,135,136 with a site score of 0.823. Both the group was examined and employed further to analyse the binding scores (Table: 1). the residues at the pocket regions were visualised and characterised on Figure: 1 Table 1 Binding site residues with fitness score generated by sitemap. S.No. Chain Site Residues Fitness score 1 Site 1 47,48,49,51,52,54,55,56,81,84,85,87,88,89,91,92,111 0.884 2 Site 2 90,93,94,98,99,100,101,168,169,171,172,175, 133,134,135,136 0.823 3.2 Docking Analysis Docking analysis was performed using the Natural Compounds from the Zinc database using GLIDE. Initially HTVS (High Through put Virtual screening) was performed for 10,000,00 of naturals compounds towards both the selected sites. HTVS offers an accurate and selective filtration of ligands based on their efficiency to interact with the binding cavity residues [ 20 ]. HTVS successfully retrieved compounds binding to the pockets. Best scoring hits among the database compounds were retained. Leads of site 1 produced maximum glide score of -8.05kcal/mol and site 2 of -8.25kcal/mol. Leads having glide score more than − 5.00 was taken for XP and Induced fit dockings (Table: 2 and Table: 3). Table 2 Energy values of lead ZINC compounds obtained from docking on site 1 Compound Glide Score (kcal/mol) Glide Energy (kcal/mol) Zinc Natural subset XP SP XP SP ZINC11981988 -10.232 -9.065 -50.236 -52.134 ZINC 14455323 -8.045 -8.023 .-39.336 -35.987 ZINC 04276931 -6.496 -6.215 -24.456 -23.655 ZINC 01985795 -6.356 6.277 -20.123 -19.089 ZINC 14013067 -6.247 -6.138 -19.234 -19.232 Table 3 Energy values of lead ZINC compounds obtained from docking on site 2 Compound Glide Score (kcal/mol) Glide Energy (kcal/mol) Zinc Natural subset XP SP XP SP ZINC 04243820 -7.120 -8.027 -43.238 -44.225 ZINC 19735228 -6.445 -9.287 -55.347 -40.899 ZINC 19735640 -6.389 -9.116 -55.212 -37.899 ZINC 18067210 -5.114 -7.876 -30.149 -27.090 ZINC 39925369 -5.649 -7.765 -32.139 -31.098 3.2.1 Induced Fit Docking of Screened Compounds The best fitness scored compounds from XP dock were subjected for IFD and QPLD on Schrodinger Glide tool. Hits from visual screening output were docked into the 2WCQ active site. Top twenty ligands with glide score > -6 from the XP dock output were selected. Among the top five ligands glide scores are tabulated on Table: 3. After IFD, top ten hits >-7 were selected. Among them from site 1, lead was ZINC11981988 which produced a high glide score of -10.347. From Site 2 docking, lead was ZINC 04243820 with a glide score of -8.975kcal/mol. The values are tabulated in Table: 4. These complexes were chosen, and simulation studies of molecular dynamics were conducted on them. In order to double-check the IFD scoring function's precision in identifying the chosen natural ZINC subgroup inhibitors, a QM/MM based QPLD technique was also used. Similar QPLD ratings were used to rank the IFD hits. The top 5 leads from both IFD screenings were selected based on various interaction and Glide score systems. 3.3 Drug-likeness property analysis and ADMET Screening 3.3.1 Drug-likeness property analysis The lead zinc ligands, ZINC11911988 and ZINC04243820 were evaluated for their drug-likeness and physicochemical characteristics using the online server SwissADME and the results are tabulated in Table 5 . Molecular weight for ZINC11911988 and ZINC04243820 were found to be 242.25 g/mol and 220.227 g/mol respectively, aligning with Lipinski’s rule of five, which suggests a molecular weight limit of 500 g/mol. The lipophilicity (Log P) values, which measure the hydrophobicity of the compound were 2.85 for ZINC11911988 and 0.30 for ZINC04243820, indicating that ZINC11911988 is more hydrophobic. Both compounds exhibited acceptable hydrogen bond donor values of 2 for ZINC11911988 and 3 for ZINC04243820. The hydrogen bond acceptor count was also consistent for both compounds at 4, further contributing to their solubility and interaction potential. The molar refractivity which correlates with the volume and polarizability of the compounds was calculated as 102.57 for ZINC11911988 and 71.87 for ZINC04243820. Importantly, both ligands satisfied Lipinski’s rule of five with no violations, highlighting their potential as drug-like molecules. Table 5 Drug-likeness properties of lead molecules Zinc Natural Ligands Molecular Weight (g/mol) Lipophilicity (Log P) Hydrogen Bond Donors Hydrogen Bond Acceptors Molar Refractivity Drug likeness (Lipinski) ZINC11981988 242.25 2.85 2 4 102.57 Yes (0 violation) ZINC04243820 220.227 0.30 3 4 71.87 Yes (0 violation) 3.3.2 ADMET Screening The ADMET properties for the lead zinc ligands against Tip- \(\:\propto\:\) was predicted in-silico by utilizing qikprop module of Schrödinger suite and the results are tabulated in (Table: 6). Molecular weight for the compounds was in the range of 242.25 and 220.227 for ZINC11911988 and ZINC04243820 respectively. The evaluated numbers of hydrogen bond donors (HBD) by the solute to water atoms in the fluid arrangement of the complex were 2 and 3 respectively (ZINC11981988 had 2 and ZINC04243820) had 3 HBD. On the other hand, the evaluated numbers of hydrogen bonding acceptors detected by the solute from water particles in the fluid arrangement of the complex were 5.25 for ZINC11981988 and 5.2 for ZINC04243820. When comparing the two compounds, ZINC11981988 had the most elevated Lipophilicity, QplogPo/w value of 1.55 and 5.2 for the compound ZINC04243820. Another important physicochemical characteristic of a pharmacological molecule is lipophilicity, which affects its solubility in lipids, pharmacology effectiveness, penetration of membranes, and solubility and binding of plasma proteins. Hit compound Zinc11981988 and ZINC04243820 possess a possibility to cross the blood-brain barrier QP log BB for brain/blood of -1.237 and − 1.441 (recommended value range is -3.0 / 1.2), Cell permeability (QPPCaco) of 56.741 and 34.151 respectively. Further Human Oral Absorption of both hits was 3 which is a plausible range. The results of in silico ADMET screening of compound ZINC00225109 and ZINC44896875 possessed best pharmacokinetic properties to be considered as drug molecules. Table 6 ADMET scores of lead molecules Zinc Natural Ligands Molecular Weight 130.0/725 Molecular Volume 500.0/2000.0 Hydrogen Bond Donors 0.0/6.0 Hydrogen Bond Acceptors 2/20 Qplog Po/W -2.0/6.5 QPP Caco 500G QP logS -6.5/0.5 ZINC11981988 242.25 804.907 2 5.25 1.55 56.741 -2.749 ZINC04243820 220.227 732.821 3 5.2 0.838 34.151 -1.973 3.4 Binding Mode Analysis of ZINC 11981988 and ZINC04243820 with Tip -α The Induced fit analysis lead, ZINC11981988 with first set of active sites revealed, H-bonds, \(\:\pi\:\) bonds and minimum hydrophobic bonds. The 03 and O4 atom of the ZINC11981988 was found to interact with NH1 and HH11 of Arg 81. The side chain atoms NH1 and HH11 of Arg 81 donated H-bond with Zinc with a distance of interaction 3.14 and 3.07 respectively. Further, another H-bond was made by O4 of ZINC11981988 with side chain HE21 of Gln84. The interaction length was measured to 2.93Å. Pi-Pi stalking interactions were also made by the Quinolone moiety of ligand molecule with His88 of receptor protein. The solvent exposure is high revealing the minimal binding of ligand within the binding pocket. Further hydrophobic interactions were also minimal explaining the less availability of forming Van der Waals force of attraction. The only hydrophobic bond was limited with Ile52 (Figure: 2A) The functional group O3 of Zinc ZINC04243820 has found to donate an H bond with the NZ (nitrogen) atom of Lys91 side chain from Tip α which is residing around 4Å distance of ligand. This H- bond measures a length of 2.81Å. Another H- bond was formed by H11 atom of ligand with Oxygen atom in the side chain of Gly84 with a distance of 3.00. (Figure: 2B). A third H-bond was made by H10 of ZINC 04243820 with the Oxygen atom at the Proline 133 main chain. Apart from this Pi-Pi stalking interactions were made by the pentane and benzene moiety (adenyl group) of ligand with the side chains His 88 of the receptor protein respectively. These \(\:\pi\:\) bonds make electrons more available between nuclei. Hydrophobic interactions were made to maintain the binding in the specific region with several residues of active sites including Proline 133 and Val 87. Solvent Exposure of ligand ZINC04243820 is low which further confirms the binding of ligand to the surface pockets of receptor. Binding analysis of both leads ZINC 11981988 and ZINC04243820 on active site one and two revealed residues Gln and His are common in both interactions. Table 7 H-Bond and Hydrophobic interactions of hits from site 1 and 2 of Tip- α Compound ID π = π stalking Hydrophobic contacts H-bond - residue Atoms in H bond (Ligand-Receptor) Length in Angstrom(Å) Glide score (kcal/mol) ZINC04243820 His88 Val87, Pro133 N-H-O (Lys91) O3-N2 2.81 -8.975 N-H-O(Glyn84) O-N1 3.00 H-O-H (Pro133) O-H10 2.64 ZINC1198988 His88 Ile52 H-O-H Arg81 O1-NH1 2.078 -10.232 H-O-H(Gln84) H 19 -O 1.801 Pi-Pi(His80) N 1 -H 3.12 H-N-H(Arg77) O5-NH1 2.86 H-N-H(Gln84) O4-NE2 2.97 3.5 Molecular Dynamics To check the binding stability of the ligand during the protein dynamics MD simulation had been performed. ZINC04243820 with Tip-α, ZINC11981998 with Tip-α represented as complex 1 and complex 2 respectively. Close inspection of MD trajectories had been revealed that complex-2 binds with high stability than that of the complex-1. The Root Mean Square deviation (RMSD) and Root mean square fluctuation (RMSF) were calculated for protein-ligand complexes during 100ns of MD simulation, to check the ligand-induced conformational changes (Figure: 3). Results show that Tip- α protein is quite stable in its apo form as well as both the complexes as their amino acid residues shown very less fluctuations from their initial position as shown in Figure: 3(A), but the RMSD trends are different in both cases (Figure: 3A). In the case of complex 1, it was observed that the ligand-binding is poor, and it got fluctuated at its initial position for ~ 40 ns and after that, it left its binding cleft (Figure: 3B). The RMSD trend of the complex-1 supports this observation, as the RMSD value randomly gets increased after 40 ns of simulation time. Interestingly on the other side, in the case of complex-2, the thorough visualization of MD trajectory revealed that the ligand of complex-2 binds with the receptor more strongly. The smooth and linear trend of RMSD value further validate this observation. The radius of gyration (Rg) shows the compactness of the protein and protein-ligand system. Since the lower degree of fluctuation over time frame demonstrates better structural compactness. To check the compactness of the system we have calculated the radius of gyration of Cα-atoms of the backbone as shown in Figure: 3D. If Rg values of the apo and ligand bonded systems were compared, it has been observed that apoproteins considerably more compact. And binding of ligand into protein caused flexible changes into the protein structure. Among the two complexes, comparatively complex-2 is more compact, rigid, and stable. The superimposed snapshots (Figure: 4) that have been taken at different time frames clearly show the strong binding of the ligand in complex-2 as it remains at its initial position for the whole-time duration of MD simulation and the ligand in complex 1 fluctuates randomly at different-different positions. To justify this observation number of hydrogen between protein-ligand has also been calculated as shown in Fig. 3 C. We found that ligand-2 constantly has 2 or 3 hydrogen bonds throughout the simulation time which are responsible to holds the ligand-2 non covalently into the binding site. Whereas in the case of complex-1 initially the 2 hydrogen bonds were present but with the simulation time number of hydrogen bonds completely diminished (~ 40-50ns and ~ 60-90ns) which further supports the poorer binding of ligand-1 into the receptor protein. 3.5.1 MMGBSA The thermodynamic MMGBSA calculation supports our MD observation as the total binding free energy (ΔGbinding) is greater in complex-2 having a larger negative energy value of -32.36 kcal/mol than that of complex-1 i.e., -18.88 kcal/mol (Table 8 ). Since in both the case’s value of ΔGbinding is negative which indicates that ligand binding is possible in both the cases. But the high negative value of ligand binding in complex-2 is thermodynamically more favourable. Table 8 The energy components, involved in ligand binding. (A) For complex-1, and (B) for complex-2. (A)Energy Component Energy (kcal/mol) Std. Error ΔE VDW -21.061 0.2218 ΔE EEL -38.2449 1.4307 ΔE poalr 43.4969 1.1862 ΔE nonpolar -3.0802 0.0201 ΔG gas -59.3059 1.3913 ΔG solv 40.4167 1.1794 ΔG binding -18.8892 0.3892 (B) Energy Component Energy (kcal/mol) Std. Error ΔE VDW -28.3288 0.2292 ΔE EEL -69.7346 0.9443 ΔE poalr 69.6683 0.8475 ΔE nonpolar -3.972 0.016 ΔG gas -98.0633 0.9579 ΔG solv 65.6963 0.844 ΔG binding -32.367 0.3157 ΔE VDW = change in van der Waals interaction on ligand association. ΔE EEL = change in electrostatic interaction on ligand association. ΔE poalr = change in polar interaction on ligand association. ΔE nonpolar =change in nonpolar interaction on ligand binding. ΔG gas = ΔE VDW + ΔE EEL . ΔG solv = ΔE polar + ΔE nonpolar . ΔG binding = ΔG gas + ΔG solv . In both systems, the contribution of change in electrostatic energy is more dominant than the van der Waals contribution. The main reason behind this is found to be, the presence of a few polar amino acid residues which shows significant hydrogen bond interaction with the ligand. Gln95, Asp99, and Lys138, respectively, contribute − 5.7, -4.1, and − 7.0 kcal/mol to the overall energy decomposition in Complex-1. Similarly, in Complex-2 amino acid residues Gln26, Gln34, Arg61, and Asn65 contributes − 4.8, -5.9, -12.8, and − 3.8 kcal/mol respectively (Table: 6 (A,B) and 7 (A,B)). Also Figure: 5 shows the interacting residues at both sites of Tip- α with corresponding hits. Change in solvation-free energy is unfavourable in both cases. It was shown that residues Gn 92, Gln95, Lys138, Gly135, Pro96, Glu134, Ala131 and Asp99 are in close proximity with ligand ZINC11981988 and Arg 61, Asn65, Gln34, Tyr31, Val36, Gly35, Asn27, Leu121, Met24, Gln26, Trp23, Leu25 residues found to be interaction with ZINC04243820 (Figure: 5). To further emphasize the ligand-receptor interactions, we have calculated the residue interaction map as shown in Figure: 6. This residue interaction map clearly indicates that polar amino acid residues (Gln, Lys, Asp, Arg) show higher contributions in the ligand binding. 4. DISCUSSION The research offers a thorough computational method for finding natural inhibitors of Helicobacter pylori's (H. pylori) Tumour Necrosis Factor Alpha Inducing Protein (Tip-α). Tip-α is a potential therapeutic target because of its important role in gastric inflammation and carcinogenesis. The main conclusions, their ramifications, their shortcomings, and potential future study areas are all covered in detail in this conversation. Tip-α is a virulence factor that H. pylori produces. It causes the synthesis of TNF-α, which creates a pro-inflammatory environment that can lead to gastric cancer and chronic gastritis. When the protein interacts with nucleolin, nuclear factor-kappa B (NF-κB) signalling is triggered, leading to an increase in pro-inflammatory cytokines such as interleukin-8 (IL-8). Apoptosis of epithelial cells, immunological regulation, and ultimately injury to the stomach mucosa are all influenced by these cytokines. Finding strong inhibitors that can lessen Tip-α's actions is essential because of its importance in H. pylori-induced gastric disease. In order to screen natural inhibitors from the ZINC database and find possible binding sites on Tip-α, this study used molecular docking techniques with Glide. According to the docking tests, two main binding sites with a high affinity for particular natural chemicals were found. At -9.116 kcal/mol and − 10.455 kcal/mol, respectively, the two most promising inhibitors, ZINC11981988 and ZINC04243820, showed substantial interactions with Tip-α. These results imply that these substances may be able to effectively disrupt the function of Tip-α, hence lessening its harmful effects. The stability of the protein-ligand complexes over 100 nanoseconds was evaluated using molecular dynamics (MD) simulations, which showed that ZINC04243820 was more stable in the active site than ZINC11981988. The MM-GBSA free energy calculations showed that ZINC04243820 had a stronger binding affinity because of favourable electrostatic interactions, and the steady hydrogen bonding and lower RMSD fluctuations further support ZINC04243820's potential as a functional Tip-α inhibitor. Using ADMET screening, the pharmacokinetic characteristics of the chosen inhibitors were assessed. Lipinski's rule of five was followed by ZINC11981988 and ZINC04243820, suggesting good drug-likeness. Furthermore, they had favourable ADMET (absorption, distribution, metabolism, excretion, and toxicity) profiles, indicating that they could be used as lead agents in additional preclinical research. ZINC11981988 and ZINC04243820 emerged as the top candidates due to their substantial binding affinities and high glide scores, after the molecular docking research successfully identified two binding sites on Tip-α. Additional molecular dynamics simulations conducted on a timescale of 100 nanoseconds verified ZINC04243820's better stability within the binding pocket, indicating its increased potential as a therapeutic candidate. ZINC04243820 showed a more favourable binding affinity (-32.36 kcal/mol) than ZINC11981988 (-18.88 kcal/mol), according to the MM-GBSA free energy calculations, which further confirmed its stability. These findings provide vital information for upcoming drug design initiatives and highlight the significance of electrostatic interactions in maintaining the ligand-protein combination. According to Lipinski's Rule of Five, which was established by the ADMET study, ZINC04243820 and ZINC11981988 both exhibited drug-likeness and the potential for oral bioavailability. Additionally, their advantageous pharmacokinetic characteristics—such as high permeability, metabolic stability, and absorption—confirm their appropriateness for additional preclinical research. Interestingly, ZINC04243820 showed a more stable and compact interaction profile, indicating that it has better potential as a therapeutic agent. This work emphasises how important computational techniques are to early-stage drug development since they provide an economical and effective way to find promising therapeutic candidates. Multidrug resistance has emerged as a result of the heavy reliance on antibiotics in current therapies for H. pylori infections. The discovery of Tip-α-targeting non-antibiotic inhibitors offers a different approach to treatment. These chemicals have the potential to decrease inflammation and gastric carcinogenesis linked to chronic H. pylori infection by directly inhibiting Tip-α. The results also lend credence to the necessity of further refinement of these molecules through structure-based medication design. The potential of natural inhibitors to target Tip-α, a crucial virulence component of H. pylori, is demonstrated by this study. These inhibitors were found and validated using a computational method that offers a solid basis for further medication development. Since H. pylori is becoming more resistant to antibiotics, Tip-α inhibitors offer a viable substitute for treating stomach infections and lowering the risk of stomach cancer. Translating these findings into successful therapeutic strategies will require additional clinical research and experimental validation. 5. CONCLUSION A thorough computational method for finding natural inhibitors of Helicobacter pylori's Tumour Necrosis Factor-alpha Inducing Protein (Tip-α) is presented in this study's conclusion. Finding strong inhibitors is a promising step towards alternative treatment approaches because of the growing resistance of H. pylori to traditional antibiotics and the crucial function of Tip-α in gastric inflammation and carcinogenesis. Strong support for the potential of the discovered compounds, especially ZINC04243820, as potent Tip-α inhibitors is provided by the results of molecular docking, molecular dynamics simulations, and ADMET screening. Treatment options for H. pylori infections that do not involve antibiotics have expanded with the discovery of natural inhibitors that target Tip-α. The inflammatory and carcinogenic effects of chronic H. pylori infection may be lessened by these inhibitors, which particularly target Tip-α, lowering the incidence of gastric ulcers and stomach cancer. Future studies should concentrate on confirming these results in in vitro and in vivo tests to evaluate the safety and effectiveness of the discovered inhibitors in greater detail. The lead compounds' binding affinity and therapeutic potential may also be improved by structural optimisation. The discovery of new treatments for H. pylori-induced stomach disorders is greatly aided by our research, which moves these inhibitors closer to clinical evaluation. Declarations Acknowledgements The authors deeply express sincere thanks to Manonmaniam Sundaranar University, Tirunelveli, Tamil Nadu, India for their support and constant encouragement which makes this venture a success. Ethics approval Not applicable for the section. Consent to participate Not applicable for the section. Consent for publication Not applicable for the section. Conflicts of interests The authors declare that they have no conflicts of interest. Authors contribution: Divya Sunder Raj: Conceptualization, Methodology, data curation and Writing-original draft preparation, K. Chidhambara Priya Dharshini: Data curation and reviewing, Sukriti Singh: writing and editing, Shalini Yadav: Data curation and writing, Manish Dwivedi: Reviewing, editing and proofreading of the manuscript. References Hedayati, M., Tip-alpha gene expression of Helicobacter pylori using Real Time RT PCR and association with clinical outcomes. Int.J.Curr.Microbiol.App.Sci, 2014. Godlewska, R., et al., Tip-alpha (hp0596 gene product) is a highly immunogenic Helicobacter pylori protein involved in colonization of mouse gastric mucosa. Curr Microbiol, 2008. 56(3): p. 279-86. Suganuma, M., et al., TNF-alpha-inducing protein, a carcinogenic factor secreted from H. pylori, enters gastric cancer cells. International journal of cancer, 2008. 123(1): p. 117-122. Binfield, P., At PLoS ONE we're batty about bats , in PLoS: Public Library of Science . 2008. p. Web log message. Bird, C.D. and N.J. Emery, Insightful problem solving and creative tool modification by captive nontool-using rooks. Proceedings of the National Academy of Sciences of the United States of America, 2009. 106(25): p. 10370-10375. Ghorbani, A. and M. Esmaeilizadeh, Pharmacological properties of Salvia officinalis and its components. J Tradit Complement Med, 2017. 7(4): p. 433-440. Wajant, H., TRAIL and NFkappaB signaling--a complex relationship. Vitam Horm, 2004. 67: p. 101-32. Wu, Y.Y., et al., Helicobacter pylori enhances tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in human gastric epithelial cells. World J Gastroenterol, 2004. 10(16): p. 2334-9. Tsai, H.F. and P.N. Hsu, Modulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis by Helicobacter pylori in immune pathogenesis of gastric mucosal damage. J Microbiol Immunol Infect, 2017. 50(1): p. 4-9. Mizuno, T., Higashi, H., Tsutsumi, R., et al. (2005). Helicobacter pylori induces interleukin-8 production through a protein encoded by a pathogenicity island . Gastroenterology, 128(5), 1022-1034. Chaturvedi, R., Asim, M., Lewis, N. D., et al. (2014). Helicobacter pylori-induced gastric cancer: Molecular mechanisms and therapeutic strategies . World Journal of Gastroenterology , 20(18), 5564-5575. Watanabe, T., Higashi, H., Suda, K., et al. (2010). Nucleolin acts as a receptor for the Helicobacter pylori Tip-α cytotoxin . Journal of Experimental Medicine , 207(13), 2779-2793. Tang, C.L., et al., Helicobacter pylori tumor necrosis factor-α inducing protein promotes cytokine expression via nuclear factor-κB. World J Gastroenterol, 2013. 19(3): p. 399-403. Tosi, T., et al., Structures of the tumor necrosis factor alpha inducing protein Tipalpha: a novel virulence factor from Helicobacter pylori. FEBS Lett, 2009. 583(10): p. 1581-5. Kuzuhara, T., et al., Presence of a motif conserved between Helicobacter pylori TNF-alpha inducing protein (Tipalpha) and penicillin-binding proteins. Biol Pharm Bull, 2005. 28(11): p. 2133-7. Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE. The Protein Data Bank. Nucleic Acids Res. 2000 Jan 1;28(1):235-42. doi: 10.1093/nar/28.1.235. Aparna, V., et al., Identification of natural compound inhibitors for multidrug efflux pumps of Escherichia coli and Pseudomonas aeruginosa using in silico high-throughput virtual screening and in vitro validation. PLoS One, 2014. 9(7): p. e101840. Greener, J.G. and M.J.E. Sternberg, AlloPred: prediction of allosteric pockets on proteins using normal mode perturbation analysis. BMC Bioinformatics, 2015. 16(1): p. 335. Sankar K, Trainor K, Blazer LL, Adams JJ, Sidhu SS, Day T, Meiering E, Maier JKX. A Descriptor Set for Quantitative Structure-property Relationship Prediction in Biologics. Mol Inform. 2022 Sep;41(9):e2100240. doi: 10.1002/minf.202100240. Vasudevan, A., et al., In Silico and In Vitro Screening of Natural Compounds as Broad-Spectrum β-Lactamase Inhibitors against Acinetobacter baumannii New Delhi Metallo-β-lactamase-1 (NDM-1). BioMed Research International, 2022. 2022: p. 4230788. David, T.I., et al., Molecular docking analysis of phyto-constituents from Cannabis sativa with pfDHFR. Bioinformation, 2018. 14(9): p. 574-579. Daina A, Michielin O, Zoete V. SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Sci Rep. 2017 Mar 3;7:42717. doi: 10.1038/srep42717. Suganuma, M., et al., TNF-alpha-inducing protein, a carcinogenic factor secreted from H. pylori, enters gastric cancer cells. Int J Cancer, 2008. 123(1): p. 117-22. Mahant, S., et al., The Synergistic Role of Tip α, Nucleolin and Ras in Helicobacter pylori Infection Regulates the Cell Fate Towards Inflammation or Apoptosis. Curr Microbiol, 2021. 78(10): p. 3720-3732. Neu, B., et al., Expression of tumor necrosis factor- alpha -related apoptosis-inducing ligand and its proapoptotic receptors is down-regulated during gastric infection with virulent cagA+/vacAs1+ Helicobacter pylori strains. J Infect Dis, 2005. 191(4): p. 571-8. Friesner, R.A., et al., Extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes. J Med Chem, 2006. 49 (21): p. 6177-96. Dwivedi, M., Devi, S. S., Singh, S., Trivedi, M., Hussain, N., Yadav, S., & Dubey, K. D. (2024). Phytocompounds as versatile drug-leads targeting mProtease in the SARS-CoV-2 virus: insights from a molecular dynamics study. Journal of Biomaterials Science, Polymer Edition, 1–21. Dwivedi M, Jose S, Gupta M, Devi SS, Raj R, Kumar D. Copper transporter protein (MctB) as a therapeutic target to elicit antimycobacterial activity against tuberculosis. J Biomol Struct Dyn. 2024 Jul;42(10):5334-5348. Yadava A, Bhuyan MMR, Mukherjee D, Kumar D, Dwivedi M. Phytomolecules as potential candidates to intervene the function of E. coli sodium-proton antiporters; Ec-NhaA. J Biomol Struct Dyn. 2023 Mar 19:1-12. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5983215","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":413239147,"identity":"a9ce848b-5e8f-4183-baad-111fa0440b3c","order_by":0,"name":"Divya S Raj","email":"","orcid":"","institution":"Manonmaniam Sundaranar University","correspondingAuthor":false,"prefix":"","firstName":"Divya","middleName":"S","lastName":"Raj","suffix":""},{"id":413239148,"identity":"847463f8-02bc-4125-9793-52918b7a245b","order_by":1,"name":"K. Chidhambara Priyadarshini","email":"","orcid":"","institution":"Manonmaniam Sundaranar University","correspondingAuthor":false,"prefix":"","firstName":"K.","middleName":"Chidhambara","lastName":"Priyadarshini","suffix":""},{"id":413239149,"identity":"3c4fafea-5abf-45bd-92ee-e73a9f4f336a","order_by":2,"name":"Shalini Yadav","email":"","orcid":"","institution":"Shiv Nadar University","correspondingAuthor":false,"prefix":"","firstName":"Shalini","middleName":"","lastName":"Yadav","suffix":""},{"id":413239150,"identity":"4d9d8094-dd82-4c9b-bc4e-bd846aae159c","order_by":3,"name":"Sukriti Singh","email":"","orcid":"","institution":"Amity University Uttar Pradesh","correspondingAuthor":false,"prefix":"","firstName":"Sukriti","middleName":"","lastName":"Singh","suffix":""},{"id":413239151,"identity":"08da2d44-09aa-4054-8601-fe003da2100b","order_by":4,"name":"Manish Dwivedi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABE0lEQVRIie3PMUvEMBTA8ZRCbmnJJg8c+gmESKAUzg+TcOAtVRzdriDERb3Vw4/gclA4HFO6tnTN4XLuJ+gi10VNbu7VcxPMfwrh/XgJQi7XH015mY+wOYD4/JpM7RW/2Jdw7HuzzBL6wxpLbIAMmSt77CFH13VRtE9DcQejQq8C7LPmUbytKIrIgeokcXXOy7AaCwmno4RDgGP9moN52PHsgXcTldLSk+WZDKrYTEIQ63puCafPO0izpkW7Jc0HcGPYfZ1veolOqQotGdxg4IpTSm4X/Vv0mpahHE/kQLJEZIqDDheJ2bb7L03K3ls5ZNGV/7JsDSHTOl9uLk8icthNOoLtJOw7biPqN9Mul8v1D/oGfdRmRoNeIvIAAAAASUVORK5CYII=","orcid":"","institution":"Amity University Uttar Pradesh","correspondingAuthor":true,"prefix":"","firstName":"Manish","middleName":"","lastName":"Dwivedi","suffix":""}],"badges":[],"createdAt":"2025-02-07 17:53:18","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-5983215/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5983215/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":76010200,"identity":"372f62b5-3454-46aa-8726-d04a7f6d5347","added_by":"auto","created_at":"2025-02-11 12:06:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":256449,"visible":true,"origin":"","legend":"\u003cp\u003eBinding Cavity predicted from site map (a) Site 1, (b) Site 2\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5983215/v1/d3b6f0f37f0a201e82fd9786.png"},{"id":76010203,"identity":"ed071160-6cae-4a81-8614-3da3ceae2c82","added_by":"auto","created_at":"2025-02-11 12:06:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":381801,"visible":true,"origin":"","legend":"\u003cp\u003e(A) and (B) Shows binding Modes of ZINC11981988 and ZINC04243820 with binding site residues of Tip- α binding sites (Site 1 and Site 2) respectively in which (a) indicates interactions viewed on Pymol and (b) shows the 2D view of ligand interaction\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5983215/v1/734ce76733a819eb1014f11f.png"},{"id":76010199,"identity":"cd0d4e74-7930-41f1-b611-99fcb7a97044","added_by":"auto","created_at":"2025-02-11 12:06:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":254915,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003eRoot mean square deviation (RMSD) of the complexes during 100ns of MD simulation. \u003cstrong\u003e(B)\u003c/strong\u003e Root mean square fluctuations (RMSF) of protein residue. \u003cstrong\u003e(C) \u003c/strong\u003eThe number of hydrogen bonds between the ligand and amino acids of the surrounding protein.\u003cstrong\u003e (D) \u003c/strong\u003eThe radius of gyration of the apo-protein and complex molecule with respect to Cα atoms of the backbone of the protein\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5983215/v1/4667ab4b0d8dfa43268a2a7e.png"},{"id":76010201,"identity":"655eb5bd-ab30-44bd-ab4b-3b0ba7131da5","added_by":"auto","created_at":"2025-02-11 12:06:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":331404,"visible":true,"origin":"","legend":"\u003cp\u003esuperimposed snapshots taken during different time frames of MD simulation. \u003cstrong\u003e(A)\u003c/strong\u003e For complex 1. \u003cstrong\u003e(B)\u003c/strong\u003e For complex 2. Colour code is; green (0 ns), gray (50 ns), orange (100 ns).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5983215/v1/a76e3ee8e8d384dd3cee32db.png"},{"id":76010202,"identity":"35eb3a5b-9d55-4304-98e6-030ee66e2b18","added_by":"auto","created_at":"2025-02-11 12:06:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":704276,"visible":true,"origin":"","legend":"\u003cp\u003eA representative snapshot of complexes (A) complex 1 (B) complex 2, with the key interactions which are responsible for the ligand-binding observed in MMGBSA calculation.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-5983215/v1/f8fba1cf4947ba34926af937.png"},{"id":76010198,"identity":"36049c11-c5e7-4b1e-9dc8-39297695fca3","added_by":"auto","created_at":"2025-02-11 12:06:09","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":61027,"visible":true,"origin":"","legend":"\u003cp\u003eThe residue interaction map, for ligand binding (A) Complex-1, (B) Complex-2.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-5983215/v1/950f6f835e0c8fb5147970d2.png"},{"id":76011237,"identity":"31c71590-a000-46ef-a5cf-4f9fb277b19a","added_by":"auto","created_at":"2025-02-11 12:14:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3386303,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5983215/v1/e6446091-0a72-418d-a7dd-345681670655.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Dynamics based screening of Therapeutic compounds against Helicobacter pylori targeting TNF-α inducing protein","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eHelicobacter pylori (\u003cem\u003eH.pylori\u003c/em\u003e) is a Gram-negative microaerophilic bacterium that dwells in the human stomach and is responsible for 85 percent of gastric ulcers worldwide. Antibiotics have been used indefinitely to eradicate \u003cem\u003eH.pylori\u003c/em\u003e, which has raised the rate of multidrug resistance in them. This needs the development of a prospective pathogen-eradicating medication. Identification of a potential drug target is thus critical in the chemotherapeutics process. Tip- α is a virulence protein identified from \u003cem\u003eH. pylori\u003c/em\u003e which is immunogenic, and similar to other virulence factors (CagA, UreaseA, Vac A) of the bacterium [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It is produced by the HP0596 gene of the bacterium and is found to be unique to this bacterium [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Among the various virulence factors characterised in H. pylori, Tip-α has been characterised to be carcinogenic and found to induce potentially TNF-α and chemokine genes through the activation NF-Kapp B cells, where NF- kappa B (nuclear factor kappa B), the Rel family of proteins is made up of a collection of dimeric transcription factors that play an important role in inflammation and immune control [\u003cspan additionalcitationids=\"CR4 CR5 CR6\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Several studies have proved the apoptosis inducement of Tip- α on human gastric epithelial cell lines, where the protein modulates the sensitivity of cell lines\u0026rsquo; apoptosis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Tip- α is a newly identified gene in H. pylori. It is also known as H. pylori 0596 protein as it is found in the 0596 open reading frame of the H. pylori 26695 strain. It has a 519-bp open reading frame that contains 173 amino acids [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTip-α (Tip-alpha) is a protein that resembles tumour necrosis factor (TNF) and is mostly linked to Helicobacter pylori, a bacteria that is linked to gastritis, peptic ulcers, and stomach cancer. This protein helps H. pylori survive and remain in the stomach mucosa by influencing the host's immune system and encouraging inflammation.\u003c/p\u003e \u003cp\u003eThe cytokine-like action of tip-α, which is released by H. pylori, can trigger the production of chemokines and activate host cell nuclear factor-kappa B (NF-κB) pathways. These actions add to the inflammatory milieu that is a feature of an H. pylori infection. Tip-α increases the expression of pro-inflammatory cytokines including interleukin-8 (IL-8) by binding to nucleolin, a protein present on the surface of host cells, and promoting cellular signalling pathways (Mizuno et al., 2005). Increased IL-8 levels cause neutrophil recruitment, which exacerbates tissue damage and starts an inflammatory cycle.\u003c/p\u003e \u003cp\u003eThe advancement of gastric carcinogenesis is linked to Tip-α, which is interesting because it is not just an inflammatory mediator. Its capacity to trigger anti-apoptotic reactions, genetic instability, and cell proliferation highlights its carcinogenic potential (Chaturvedi et al., 2014). Furthermore, research indicates that Tip-α may decrease host T-cell responses to control immune evasion, which would enable H. pylori to colonise for a longer period of time (Watanabe et al., 2010).\u003c/p\u003e \u003cp\u003eTip-α has been shown to have a structure different from penicillin-binding proteins. It is composed of three closely linked domains that interact with other proteins and RNAs [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Tip- α protein exists in a sand-witched form of α-β, combined with helical bundles [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The topology includes β1, α1, α2, β2, β3 α3, α4 and a 310 helix at the N-terminal portion. α2, α3, and α4 form a helix bundle at the C-terminal and are connected by a loop of α1, α2 at the top of the helix bundle. Further, the N-terminal half of α1 was wrapped and covered by antiparallel β sheets 1, 2 and 3. A literature survey revealed Cys25-cys26, Arg81, and Leu50 as important residues for the binding of protein to DNA and dimeric formation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. There are limited studies on the \u003cem\u003eH. pylori\u003c/em\u003e Tip- α pockets and their inhibitor having efficient binding.\u003c/p\u003e \u003cp\u003eIn the present work, we have conducted structural and functional studies using bio-computational approaches including docking and molecular dynamics simulations to investigate the target protein and potential inhibitors of natural origin. Thereby, this analysis proposes that natural molecules as the probable drug lead having the affinity for Tip-α of \u003cem\u003eH. pylori\u003c/em\u003e. Previously, our group has conducted some similar studies (27, 28, 29).\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Retrieval of Target Protein and Preparation of Protein\u003c/h2\u003e \u003cp\u003eA repository of four crystal structures of Tip- α protein (HP0596) was deposited in PDB. 2WCQ crystal structure with a resolution of 1.9A\u0026ordm; was taken as reference molecule (Berman et al., 2000). As detailed study on the active binding site of this protein was absent, Glide was employed to identify the binding site information of Tip- α. To achieve this, the protein was prepared using protein preparation wizard of Glide During preparation, the Schrodinger prepares the protein by fixing major errors like incorporating missing residues near the active site, adjusting the ionization and tautomerization state of protein and further by refining the structure by relieving any strain from the adjustments [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Active Site Prediction\u003c/h2\u003e \u003cp\u003eWhen considering the drug discovery line, the identification of substrate active sites and inhibitor binding site residues are crucial as functional change is imposed by perturbing these sites on a molecule by the inhibitors/activators [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. As literature surveys were absent on active residue with binding potential, the Qsite tool of Glide was employed (Sankar et al., 2022). With the information on a series of physical parameters such as size, tightness, hydrophobic/hydrophilic character, degree of enclosure/exposure, and hydrogen bonding possibilities of Schrodinger, the sitemap assigns numerical descriptors to evaluate predicted binding site pockets. To prioritize possible binding sites, a weighted average of these measurements was then assigned.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.3. Ligand preparation\u003c/b\u003e: For the ligand selection and screening, we have utilized the Zinc Database was used. The ZINC database is a free, open-access collection of chemical compounds that are sold commercially and are selected for online screening investigations (Irwin et al., 2012). With millions of tiny compounds pre-filtered for drug-likeness, this database is a perfect tool for finding possible inhibitors of bacterial virulence factors like \u003cem\u003eHelicobacter pylori's\u003c/em\u003e Tumour Necrosis Factor Alpha Inducing Protein (Tip-α). Targeting Tip-α with natural inhibitors may offer an alternate therapeutic approach to treat \u003cem\u003eH. pylori\u003c/em\u003e infections, given its critical role in gastric inflammation and carcinogenesis. Utilising the ZINC Natural Products Subset, phytochemical compounds with structural characteristics that support Tip-α inhibition were recovered. These compounds' binding potential, bioavailability, and safety were evaluated by computational screening, which included molecular docking and ADMET analysis. Using the ZINC database to find natural inhibitors of Tip-α, presents a possible non-antibiotic strategy to treat \u003cem\u003eH. pylori\u003c/em\u003e-associated illnesses while addressing resistance issues.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Docking Analysis\u003c/h2\u003e \u003cp\u003e2WCQ was used to specify the grid. For Grid generation, active residues predicted from the q-site were given. Two grid files were generated for the sites one and two. All the docking calculations were performed using the HTVS, Standard Precision (SP) and \u0026lsquo;Extra Precision\u0026rsquo; (XP) mode of GLIDE program [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. After XP mode, IFD and QPLD were conducted to identify various interactions and bindings of hit molecules in the active site of Tip- alpha.\u003c/p\u003e \u003cp\u003eG-scores and Glide energy were used to rank the screened compounds in ascending order. The virtual screening technique penalises steric conflicts while recognising favourable hydrophobic, hydrogen-bonding, and metal-ligation interactions. Finally, Schr\u0026ouml;dinger's proprietary Glide Chem Scoring programme re-scored the minimised poses.\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\mathbf{G}\\mathbf{S}\\mathbf{c}\\mathbf{o}\\mathbf{r}\\mathbf{e}\\:=\\:0.065\\text{*}\\mathbf{v}\\mathbf{d}\\mathbf{W}\\:+\\:0.130\\text{*}\\mathbf{C}\\mathbf{o}\\mathbf{u}\\mathbf{l}\\:+\\:\\mathbf{L}\\mathbf{i}\\mathbf{p}\\mathbf{o}\\:+\\:\\mathbf{H}\\mathbf{b}\\mathbf{o}\\mathbf{n}\\mathbf{d}\\:+\\:\\mathbf{M}\\mathbf{e}\\mathbf{t}\\mathbf{a}\\mathbf{l}\\:+\\:\\mathbf{B}\\mathbf{u}\\mathbf{r}\\mathbf{y}\\mathbf{P}\\:+\\:\\mathbf{R}\\mathbf{o}\\mathbf{t}\\mathbf{B}\\:+\\:\\mathbf{e}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eWhere vdW denotes the energy of Vander Walls, Columbic energy is denoted by the term coul. Lipophilic is denoted as Lipo. The phrase \"hydrogen bonding\" is defined by Hbond. Metal is a term that refers to a metal-binding substance. BuryP is a programme that calculates a penalty for buried polar groups. Site represents active site polar interactions, while RotB is a penalty for freezing rotatable bonds. In this study, natural compounds from phase screens were virtually screened using HTVS and XP docking, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Drug-likeness Properties Analysis and ADMET Screening\u003c/h2\u003e \u003cp\u003eThe ADMET properties for the lead-zinc ligands against Tip- α was predicted in-silico by utilizing qikprop module of Schr\u0026ouml;dinger. Meanwhile their drug-likeness properties were analysed through SwissADME online server (Daina et al., 2017).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Molecular Dynamics\u003c/h2\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1 System Preparation\u003c/h2\u003e \u003cp\u003eMolecular dynamics simulation had been performed on the receptor-ligand complex obtained after docking. The lack of hydrogen atoms in the protein was fulfilled by the LEAP module of AMBER 20. Ligand molecules have been parametrized by the antechamber module employed with GAFF (generalized amber force field). Partial atomic charges on the ligand atom had been assigned by the semi empirical AM1-BCC charge method. An appropriate number of positively charged sodium (Na+) was added to the system to make the complex electrically neutral. The resulting complex system was solvated with a TIP3P water model, into a box having an octahedral shape stretched up to 10 \u0026Aring; from the protein boundary. The FF14SB force field was employed on the protein during the MD simulation.\u003c/p\u003e \u003cp\u003eFollowing system setup and parametrization of the two complexes, system geometries were minimized into two stages. i) First, all the solvent water molecules were minimized by keeping the protein fixed. ii) Second, the entire complex was minimized (5000 steps of steepest descent and 5000 steps of conjugate gradient) to get rid of bad contacts and to relax the system geometry. Under the NVT ensemble, system was gently annealed from 10 K to 300 K for 50 ps. To attain a uniform density, 1 ns of density equilibration was performed. During this, to maintain a constant temperature of 300K and the constant pressure of 1.0 atm, Langevin thermostat with a collision frequency of 2ps and Berendson barostat with a pressure relaxation time of 1ps were applied respectively. Afterwards, all the restraints had been removed and systems were further equilibrated for 3ns followed by 100ns of production MD. During MD simulation all the covalent bonds containing hydrogens were kept constrained using the SHAKE algorithm and a Particle Mesh Ewald (PME) was used to treat the long-range electrostatic interactions. We used an integration step of 2fs during the entire simulation. To get more accurate and reliable results we have performed two replicas of 100ns of both systems started from different initial velocities. All simulations had been performed by GPU version of AMBER 20 package. For the analyses purpose trajectory obtained by production run was used. CPPTRAJ module of AMBER 20 and the visualizing software VMD, Pymol were used for the analysis of the MD trajectories.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.5.2 MMGBSA\u003c/h2\u003e \u003cp\u003eTo quantify the observed binding stability of ligand in both the complexes, further, performed a thermodynamical free energy calculation i.e., MMGBSA (Molecular Mechanics Generalized Boltzmann Surface Area). This free energy calculation was carried over the 2000 frames selected from the trajectory in which RMSD of the complex was found well converged. Water molecules and all ions present in the system had been removed before preceding the MMPBSA analysis. For solute, the dielectric constant was 1 and for the surrounding solvent molecules, it had been kept at 80. MMGBSA calculation was carried out using the MMPBSA.py module of AMBER 20\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Active Site Identification\u003c/h2\u003e \u003cp\u003eTip- α appears to play a significant role in \u003cem\u003eH.pylori\u003c/em\u003e-induced inflammation and carcinogenesis, according to previous researches conducted so far [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. This is due to the increased expression of extra pro-inflammatory chemokines and the stimulation of TNF via Nuclear Factor kB (NFkB) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The unique presence and carcinogenic activity of this protein and the absence of homologous parts in any other gram negative pathogen other than \u003cem\u003eH.pylori\u003c/em\u003e, raises its important to be considered as a potential drug target\u003c/p\u003e \u003cp\u003eThe site map tool predicted three groups of sites with active binding residues in Tip- α. From these, two sites were selected based on the fitness score, as the third one was short, and the residues were the same as of the above two. First site included residues 47, 48, 49, 51, 52, 54, 55, 56, 81, 84, 85, 87, 88, 89, 91, 92, 111 of Chain a with a site score of 0.884 and second site included residues 90, 93, 94, 98, 99, 100, 101, 168, 169, 171, 172, 175, 133,134,135,136 with a site score of 0.823. Both the group was examined and employed further to analyse the binding scores (Table: 1). the residues at the pocket regions were visualised and characterised on Figure: 1\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBinding site residues with fitness score generated by sitemap.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS.No.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChain Site\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eResidues\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFitness score\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSite 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e47,48,49,51,52,54,55,56,81,84,85,87,88,89,91,92,111\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.884\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSite 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e90,93,94,98,99,100,101,168,169,171,172,175, 133,134,135,136\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.823\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Docking Analysis\u003c/h2\u003e \u003cp\u003eDocking analysis was performed using the Natural Compounds from the Zinc database using GLIDE. Initially HTVS (High Through put Virtual screening) was performed for 10,000,00 of naturals compounds towards both the selected sites. HTVS offers an accurate and selective filtration of ligands based on their efficiency to interact with the binding cavity residues [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. HTVS successfully retrieved compounds binding to the pockets. Best scoring hits among the database compounds were retained. Leads of site 1 produced maximum glide score of -8.05kcal/mol and site 2 of -8.25kcal/mol. Leads having glide score more than \u0026minus;\u0026thinsp;5.00 was taken for XP and Induced fit dockings (Table: 2 and Table: 3).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEnergy values of lead ZINC compounds obtained from docking on site 1\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eGlide Score\u003c/p\u003e \u003cp\u003e(kcal/mol)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eGlide Energy\u003c/p\u003e \u003cp\u003e(kcal/mol)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZinc Natural subset\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eXP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZINC11981988\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-10.232\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-9.065\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-50.236\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-52.134\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZINC 14455323\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-8.045\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-8.023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.-39.336\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-35.987\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZINC 04276931\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-6.496\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-6.215\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-24.456\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-23.655\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZINC 01985795\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-6.356\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.277\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-20.123\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-19.089\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZINC 14013067\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-6.247\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-6.138\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-19.234\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-19.232\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEnergy values of lead ZINC compounds obtained from docking on site 2\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eGlide Score\u003c/p\u003e \u003cp\u003e(kcal/mol)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eGlide Energy (kcal/mol)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZinc Natural subset\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eXP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZINC 04243820\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-7.120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-8.027\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-43.238\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-44.225\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZINC 19735228\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-6.445\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-9.287\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-55.347\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-40.899\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZINC 19735640\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-6.389\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-9.116\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-55.212\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-37.899\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZINC 18067210\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-5.114\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-7.876\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-30.149\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-27.090\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZINC 39925369\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-5.649\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-7.765\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-32.139\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-31.098\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1 Induced Fit Docking of Screened Compounds\u003c/h2\u003e \u003cp\u003eThe best fitness scored compounds from XP dock were subjected for IFD and QPLD on Schrodinger Glide tool. Hits from visual screening output were docked into the 2WCQ active site. Top twenty ligands with glide score \u0026gt; -6 from the XP dock output were selected. Among the top five ligands glide scores are tabulated on Table: 3. After IFD, top ten hits \u0026gt;-7 were selected. Among them from site 1, lead was ZINC11981988 which produced a high glide score of -10.347. From Site 2 docking, lead was ZINC 04243820 with a glide score of -8.975kcal/mol. The values are tabulated in Table: 4. These complexes were chosen, and simulation studies of molecular dynamics were conducted on them. In order to double-check the IFD scoring function's precision in identifying the chosen natural ZINC subgroup inhibitors, a QM/MM based QPLD technique was also used. Similar QPLD ratings were used to rank the IFD hits. The top 5 leads from both IFD screenings were selected based on various interaction and Glide score systems.\u003c/p\u003e \u003cp\u003e\u003cimg 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\" width=\"581\" height=\"563\"\u003e\u003c/p\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Drug-likeness property analysis and ADMET Screening\u003c/h2\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e3.3.1 Drug-likeness property analysis\u003c/h2\u003e \u003cp\u003eThe lead zinc ligands, ZINC11911988 and ZINC04243820 were evaluated for their drug-likeness and physicochemical characteristics using the online server SwissADME and the results are tabulated in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Molecular weight for ZINC11911988 and ZINC04243820 were found to be 242.25 g/mol and 220.227 g/mol respectively, aligning with Lipinski\u0026rsquo;s rule of five, which suggests a molecular weight limit of 500 g/mol. The lipophilicity (Log P) values, which measure the hydrophobicity of the compound were 2.85 for ZINC11911988 and 0.30 for ZINC04243820, indicating that ZINC11911988 is more hydrophobic. Both compounds exhibited acceptable hydrogen bond donor values of 2 for ZINC11911988 and 3 for ZINC04243820. The hydrogen bond acceptor count was also consistent for both compounds at 4, further contributing to their solubility and interaction potential. The molar refractivity which correlates with the volume and polarizability of the compounds was calculated as 102.57 for ZINC11911988 and 71.87 for ZINC04243820. Importantly, both ligands satisfied Lipinski\u0026rsquo;s rule of five with no violations, highlighting their potential as drug-like molecules.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDrug-likeness properties of lead molecules\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZinc Natural Ligands\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMolecular Weight (g/mol)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLipophilicity (Log P)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHydrogen Bond Donors\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHydrogen\u003c/p\u003e \u003cp\u003eBond Acceptors\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMolar Refractivity\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eDrug likeness (Lipinski)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZINC11981988\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e242.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e102.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eYes (0 violation)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZINC04243820\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e220.227\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e71.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eYes (0 violation)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e3.3.2 ADMET Screening\u003c/h2\u003e \u003cp\u003eThe ADMET properties for the lead zinc ligands against Tip- \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\propto\\:\\)\u003c/span\u003e\u003c/span\u003e was predicted in-silico by utilizing qikprop module of Schr\u0026ouml;dinger suite and the results are tabulated in (Table: 6). Molecular weight for the compounds was in the range of 242.25 and 220.227 for ZINC11911988 and ZINC04243820 respectively. The evaluated numbers of hydrogen bond donors (HBD) by the solute to water atoms in the fluid arrangement of the complex were 2 and 3 respectively (ZINC11981988 had 2 and ZINC04243820) had 3 HBD. On the other hand, the evaluated numbers of hydrogen bonding acceptors detected by the solute from water particles in the fluid arrangement of the complex were 5.25 for ZINC11981988 and 5.2 for ZINC04243820. When comparing the two compounds, ZINC11981988 had the most elevated Lipophilicity, QplogPo/w value of 1.55 and 5.2 for the compound ZINC04243820. Another important physicochemical characteristic of a pharmacological molecule is lipophilicity, which affects its solubility in lipids, pharmacology effectiveness, penetration of membranes, and solubility and binding of plasma proteins. Hit compound Zinc11981988 and ZINC04243820 possess a possibility to cross the blood-brain barrier QP log BB for brain/blood of -1.237 and \u0026minus;\u0026thinsp;1.441 (recommended value range is -3.0 / 1.2), Cell permeability (QPPCaco) of 56.741 and 34.151 respectively. Further Human Oral Absorption of both hits was 3 which is a plausible range. The results of in silico ADMET screening of compound ZINC00225109 and ZINC44896875 possessed best pharmacokinetic properties to be considered as drug molecules.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eADMET scores of lead molecules\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZinc Natural Ligands\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMolecular Weight\u003c/p\u003e \u003cp\u003e130.0/725\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMolecular\u003c/p\u003e \u003cp\u003eVolume\u003c/p\u003e \u003cp\u003e500.0/2000.0\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHydrogen Bond Donors\u003c/p\u003e \u003cp\u003e0.0/6.0\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHydrogen\u003c/p\u003e \u003cp\u003eBond Acceptors\u003c/p\u003e \u003cp\u003e2/20\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eQplog\u003c/p\u003e \u003cp\u003ePo/W\u003c/p\u003e \u003cp\u003e-2.0/6.5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eQPP\u003c/p\u003e \u003cp\u003eCaco\u003c/p\u003e \u003cp\u003e\u0026lt;\u0026thinsp;25P/\u0026gt;500G\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eQP\u003c/p\u003e \u003cp\u003elogS\u003c/p\u003e \u003cp\u003e-6.5/0.5\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZINC11981988\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e242.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e804.907\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e56.741\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-2.749\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZINC04243820\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e220.227\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e732.821\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.838\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e34.151\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-1.973\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Binding Mode Analysis of ZINC 11981988 and ZINC04243820 with Tip -α\u003c/h2\u003e \u003cp\u003eThe Induced fit analysis lead, ZINC11981988 with first set of active sites revealed, H-bonds, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pi\\:\\)\u003c/span\u003e\u003c/span\u003e bonds and minimum hydrophobic bonds. The 03 and O4 atom of the ZINC11981988 was found to interact with NH1 and HH11 of Arg 81. The side chain atoms NH1 and HH11 of Arg 81 donated H-bond with Zinc with a distance of interaction 3.14 and 3.07 respectively. Further, another H-bond was made by O4 of ZINC11981988 with side chain HE21 of Gln84. The interaction length was measured to 2.93\u0026Aring;. Pi-Pi stalking interactions were also made by the Quinolone moiety of ligand molecule with His88 of receptor protein. The solvent exposure is high revealing the minimal binding of ligand within the binding pocket. Further hydrophobic interactions were also minimal explaining the less availability of forming Van der Waals force of attraction. The only hydrophobic bond was limited with Ile52 (Figure: 2A)\u003c/p\u003e \u003cp\u003eThe functional group O3 of Zinc ZINC04243820 has found to donate an H bond with the NZ (nitrogen) atom of Lys91 side chain from Tip α which is residing around 4\u0026Aring; distance of ligand. This H- bond measures a length of 2.81\u0026Aring;. Another H- bond was formed by H11 atom of ligand with Oxygen atom in the side chain of Gly84 with a distance of 3.00. (Figure: 2B). A third H-bond was made by H10 of ZINC 04243820 with the Oxygen atom at the Proline 133 main chain. Apart from this Pi-Pi stalking interactions were made by the pentane and benzene moiety (adenyl group) of ligand with the side chains His 88 of the receptor protein respectively. These \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pi\\:\\)\u003c/span\u003e\u003c/span\u003e bonds make electrons more available between nuclei. Hydrophobic interactions were made to maintain the binding in the specific region with several residues of active sites including Proline 133 and Val 87. Solvent Exposure of ligand ZINC04243820 is low which further confirms the binding of ligand to the surface pockets of receptor. Binding analysis of both leads ZINC 11981988 and ZINC04243820 on active site one and two revealed residues Gln and His are common in both interactions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eH-Bond and Hydrophobic interactions of hits from site 1 and 2 of Tip- α\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompound ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eπ\u0026thinsp;=\u0026thinsp;π stalking\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHydrophobic contacts\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eH-bond - residue\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAtoms in H bond (Ligand-Receptor)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLength in Angstrom(\u0026Aring;)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eGlide score\u003c/p\u003e \u003cp\u003e(kcal/mol)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eZINC04243820\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eHis88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eVal87, Pro133\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN-H-O (Lys91)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eO3-N2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e-8.975\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN-H-O(Glyn84)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eO-N1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eH-O-H (Pro133)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eO-H10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2.64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eZINC1198988\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eHis88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eIle52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eH-O-H Arg81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eO1-NH1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2.078\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e-10.232\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eH-O-H(Gln84)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eH\u003csub\u003e19\u003c/sub\u003e-O\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.801\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePi-Pi(His80)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN\u003csub\u003e1\u003c/sub\u003e-H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eH-N-H(Arg77)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eO5-NH1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2.86\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eH-N-H(Gln84)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eO4-NE2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2.97\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e3.5 Molecular Dynamics\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eTo check the binding stability of the ligand during the protein dynamics MD simulation had been performed. ZINC04243820 with Tip-α, ZINC11981998 with Tip-α represented as complex 1 and complex 2 respectively. Close inspection of MD trajectories had been revealed that complex-2 binds with high stability than that of the complex-1.\u003c/p\u003e \u003cp\u003eThe Root Mean Square deviation (RMSD) and Root mean square fluctuation (RMSF) were calculated for protein-ligand complexes during 100ns of MD simulation, to check the ligand-induced conformational changes (Figure: 3). Results show that Tip- α protein is quite stable in its apo form as well as both the complexes as their amino acid residues shown very less fluctuations from their initial position as shown in Figure: 3(A), but the RMSD trends are different in both cases (Figure: 3A). In the case of complex 1, it was observed that the ligand-binding is poor, and it got fluctuated at its initial position for ~\u0026thinsp;40 ns and after that, it left its binding cleft (Figure: 3B). The RMSD trend of the complex-1 supports this observation, as the RMSD value randomly gets increased after 40 ns of simulation time. Interestingly on the other side, in the case of complex-2, the thorough visualization of MD trajectory revealed that the ligand of complex-2 binds with the receptor more strongly. The smooth and linear trend of RMSD value further validate this observation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe radius of gyration (Rg) shows the compactness of the protein and protein-ligand system. Since the lower degree of fluctuation over time frame demonstrates better structural compactness. To check the compactness of the system we have calculated the radius of gyration of Cα-atoms of the backbone as shown in Figure: 3D. If Rg values of the apo and ligand bonded systems were compared, it has been observed that apoproteins considerably more compact. And binding of ligand into protein caused flexible changes into the protein structure. Among the two complexes, comparatively complex-2 is more compact, rigid, and stable.\u003c/p\u003e \u003cp\u003eThe superimposed snapshots (Figure: 4) that have been taken at different time frames clearly show the strong binding of the ligand in complex-2 as it remains at its initial position for the whole-time duration of MD simulation and the ligand in complex 1 fluctuates randomly at different-different positions. To justify this observation number of hydrogen between protein-ligand has also been calculated as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC. We found that ligand-2 constantly has 2 or 3 hydrogen bonds throughout the simulation time which are responsible to holds the ligand-2 non covalently into the binding site. Whereas in the case of complex-1 initially the 2 hydrogen bonds were present but with the simulation time number of hydrogen bonds completely diminished (~\u0026thinsp;40-50ns and ~\u0026thinsp;60-90ns) which further supports the poorer binding of ligand-1 into the receptor protein.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e3.5.1 MMGBSA\u003c/h2\u003e \u003cp\u003eThe thermodynamic MMGBSA calculation supports our MD observation as the total binding free energy (ΔGbinding) is greater in complex-2 having a larger negative energy value of -32.36 kcal/mol than that of complex-1 i.e., -18.88 kcal/mol (Table\u0026nbsp;\u003cspan refid=\"Tab8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Since in both the case\u0026rsquo;s value of ΔGbinding is negative which indicates that ligand binding is possible in both the cases. But the high negative value of ligand binding in complex-2 is thermodynamically more favourable.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab8\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 8\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe energy components, involved in ligand binding. (A) For complex-1, and (B) for complex-2.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(A)Energy Component\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEnergy (kcal/mol)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStd. Error\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eΔE\u003csub\u003eVDW\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-21.061\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.2218\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eΔE\u003csub\u003eEEL\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-38.2449\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.4307\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eΔE\u003csub\u003epoalr\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e43.4969\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.1862\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eΔE\u003csub\u003enonpolar\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-3.0802\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0201\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eΔG\u003csub\u003egas\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-59.3059\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.3913\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eΔG\u003csub\u003esolv\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e40.4167\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.1794\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eΔG\u003c/b\u003e\u003csub\u003e\u003cb\u003ebinding\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-18.8892\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.3892\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(B) Energy Component\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEnergy (kcal/mol)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStd. Error\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eΔE\u003csub\u003eVDW\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-28.3288\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.2292\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eΔE\u003csub\u003eEEL\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-69.7346\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.9443\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eΔE\u003csub\u003epoalr\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e69.6683\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.8475\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eΔE\u003csub\u003enonpolar\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-3.972\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.016\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eΔG\u003csub\u003egas\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-98.0633\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.9579\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eΔG\u003csub\u003esolv\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e65.6963\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.844\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eΔG\u003c/b\u003e\u003csub\u003e\u003cb\u003ebinding\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-32.367\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.3157\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eΔE\u003csub\u003eVDW\u003c/sub\u003e = change in van der Waals interaction on ligand association. ΔE\u003csub\u003eEEL\u003c/sub\u003e = change in electrostatic interaction on ligand association. ΔE\u003csub\u003epoalr\u003c/sub\u003e = change in polar interaction on ligand association. ΔE\u003csub\u003enonpolar\u003c/sub\u003e =change in nonpolar interaction on ligand binding. \u003cb\u003eΔG\u003c/b\u003e\u003csub\u003e\u003cb\u003egas\u003c/b\u003e\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;ΔE\u003csub\u003eVDW\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;ΔE\u003csub\u003eEEL\u003c/sub\u003e. \u003cb\u003eΔG\u003c/b\u003e\u003csub\u003e\u003cb\u003esolv\u003c/b\u003e\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;ΔE\u003csub\u003epolar\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;ΔE\u003csub\u003enonpolar\u003c/sub\u003e. \u003cb\u003eΔG\u003c/b\u003e\u003csub\u003e\u003cb\u003ebinding\u003c/b\u003e\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;ΔG\u003csub\u003egas\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;ΔG\u003csub\u003esolv\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eIn both systems, the contribution of change in electrostatic energy is more dominant than the van der Waals contribution. The main reason behind this is found to be, the presence of a few polar amino acid residues which shows significant hydrogen bond interaction with the ligand. Gln95, Asp99, and Lys138, respectively, contribute \u0026minus;\u0026thinsp;5.7, -4.1, and \u0026minus;\u0026thinsp;7.0 kcal/mol to the overall energy decomposition in Complex-1. Similarly, in Complex-2 amino acid residues Gln26, Gln34, Arg61, and Asn65 contributes \u0026minus;\u0026thinsp;4.8, -5.9, -12.8, and \u0026minus;\u0026thinsp;3.8 kcal/mol respectively (Table: 6 (A,B) and 7 (A,B)). Also Figure: 5 shows the interacting residues at both sites of Tip- α with corresponding hits. Change in solvation-free energy is unfavourable in both cases. It was shown that residues Gn 92, Gln95, Lys138, Gly135, Pro96, Glu134, Ala131 and Asp99 are in close proximity with ligand ZINC11981988 and Arg 61, Asn65, Gln34, Tyr31, Val36, Gly35, Asn27, Leu121, Met24, Gln26, Trp23, Leu25 residues found to be interaction with ZINC04243820 (Figure: 5). To further emphasize the ligand-receptor interactions, we have calculated the residue interaction map as shown in Figure: 6. This residue interaction map clearly indicates that polar amino acid residues (Gln, Lys, Asp, Arg) show higher contributions in the ligand binding.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eThe research offers a thorough computational method for finding natural inhibitors of Helicobacter pylori's (H. pylori) Tumour Necrosis Factor Alpha Inducing Protein (Tip-α). Tip-α is a potential therapeutic target because of its important role in gastric inflammation and carcinogenesis. The main conclusions, their ramifications, their shortcomings, and potential future study areas are all covered in detail in this conversation. Tip-α is a virulence factor that H. pylori produces. It causes the synthesis of TNF-α, which creates a pro-inflammatory environment that can lead to gastric cancer and chronic gastritis. When the protein interacts with nucleolin, nuclear factor-kappa B (NF-κB) signalling is triggered, leading to an increase in pro-inflammatory cytokines such as interleukin-8 (IL-8). Apoptosis of epithelial cells, immunological regulation, and ultimately injury to the stomach mucosa are all influenced by these cytokines. Finding strong inhibitors that can lessen Tip-α's actions is essential because of its importance in H. pylori-induced gastric disease. In order to screen natural inhibitors from the ZINC database and find possible binding sites on Tip-α, this study used molecular docking techniques with Glide. According to the docking tests, two main binding sites with a high affinity for particular natural chemicals were found. At -9.116 kcal/mol and \u0026minus;\u0026thinsp;10.455 kcal/mol, respectively, the two most promising inhibitors, ZINC11981988 and ZINC04243820, showed substantial interactions with Tip-α. These results imply that these substances may be able to effectively disrupt the function of Tip-α, hence lessening its harmful effects. The stability of the protein-ligand complexes over 100 nanoseconds was evaluated using molecular dynamics (MD) simulations, which showed that ZINC04243820 was more stable in the active site than ZINC11981988. The MM-GBSA free energy calculations showed that ZINC04243820 had a stronger binding affinity because of favourable electrostatic interactions, and the steady hydrogen bonding and lower RMSD fluctuations further support ZINC04243820's potential as a functional Tip-α inhibitor. Using ADMET screening, the pharmacokinetic characteristics of the chosen inhibitors were assessed. Lipinski's rule of five was followed by ZINC11981988 and ZINC04243820, suggesting good drug-likeness. Furthermore, they had favourable ADMET (absorption, distribution, metabolism, excretion, and toxicity) profiles, indicating that they could be used as lead agents in additional preclinical research. ZINC11981988 and ZINC04243820 emerged as the top candidates due to their substantial binding affinities and high glide scores, after the molecular docking research successfully identified two binding sites on Tip-α. Additional molecular dynamics simulations conducted on a timescale of 100 nanoseconds verified ZINC04243820's better stability within the binding pocket, indicating its increased potential as a therapeutic candidate. ZINC04243820 showed a more favourable binding affinity (-32.36 kcal/mol) than ZINC11981988 (-18.88 kcal/mol), according to the MM-GBSA free energy calculations, which further confirmed its stability. These findings provide vital information for upcoming drug design initiatives and highlight the significance of electrostatic interactions in maintaining the ligand-protein combination.\u003c/p\u003e \u003cp\u003eAccording to Lipinski's Rule of Five, which was established by the ADMET study, ZINC04243820 and ZINC11981988 both exhibited drug-likeness and the potential for oral bioavailability. Additionally, their advantageous pharmacokinetic characteristics\u0026mdash;such as high permeability, metabolic stability, and absorption\u0026mdash;confirm their appropriateness for additional preclinical research. Interestingly, ZINC04243820 showed a more stable and compact interaction profile, indicating that it has better potential as a therapeutic agent. This work emphasises how important computational techniques are to early-stage drug development since they provide an economical and effective way to find promising therapeutic candidates.\u003c/p\u003e \u003cp\u003eMultidrug resistance has emerged as a result of the heavy reliance on antibiotics in current therapies for H. pylori infections. The discovery of Tip-α-targeting non-antibiotic inhibitors offers a different approach to treatment. These chemicals have the potential to decrease inflammation and gastric carcinogenesis linked to chronic H. pylori infection by directly inhibiting Tip-α. The results also lend credence to the necessity of further refinement of these molecules through structure-based medication design. The potential of natural inhibitors to target Tip-α, a crucial virulence component of H. pylori, is demonstrated by this study. These inhibitors were found and validated using a computational method that offers a solid basis for further medication development. Since H. pylori is becoming more resistant to antibiotics, Tip-α inhibitors offer a viable substitute for treating stomach infections and lowering the risk of stomach cancer. Translating these findings into successful therapeutic strategies will require additional clinical research and experimental validation.\u003c/p\u003e"},{"header":"5. CONCLUSION","content":"\u003cp\u003eA thorough computational method for finding natural inhibitors of Helicobacter pylori's Tumour Necrosis Factor-alpha Inducing Protein (Tip-α) is presented in this study's conclusion. Finding strong inhibitors is a promising step towards alternative treatment approaches because of the growing resistance of \u003cem\u003eH. pylori\u003c/em\u003e to traditional antibiotics and the crucial function of Tip-α in gastric inflammation and carcinogenesis. Strong support for the potential of the discovered compounds, especially ZINC04243820, as potent Tip-α inhibitors is provided by the results of molecular docking, molecular dynamics simulations, and ADMET screening. Treatment options for H. pylori infections that do not involve antibiotics have expanded with the discovery of natural inhibitors that target Tip-α. The inflammatory and carcinogenic effects of chronic H. pylori infection may be lessened by these inhibitors, which particularly target Tip-α, lowering the incidence of gastric ulcers and stomach cancer. Future studies should concentrate on confirming these results in in vitro and in vivo tests to evaluate the safety and effectiveness of the discovered inhibitors in greater detail. The lead compounds' binding affinity and therapeutic potential may also be improved by structural optimisation. The discovery of new treatments for H. pylori-induced stomach disorders is greatly aided by our research, which moves these inhibitors closer to clinical evaluation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors deeply express sincere thanks to Manonmaniam Sundaranar University, Tirunelveli, Tamil Nadu, India for their support and constant encouragement which makes this venture a success.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable for the section.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable for the section.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable for the section.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contribution:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDivya Sunder Raj: Conceptualization, Methodology, data curation and Writing-original draft preparation, K. Chidhambara Priya Dharshini: Data curation and reviewing, Sukriti Singh: writing and editing, Shalini Yadav: \u0026nbsp;Data curation and writing, Manish Dwivedi: Reviewing, editing and proofreading of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHedayati, M., \u003cem\u003eTip-alpha gene expression of Helicobacter pylori using Real Time RT PCR and association with clinical outcomes.\u003c/em\u003e Int.J.Curr.Microbiol.App.Sci, 2014.\u003c/li\u003e\n\u003cli\u003eGodlewska, R., et al., \u003cem\u003eTip-alpha (hp0596 gene product) is a highly immunogenic Helicobacter pylori protein involved in colonization of mouse gastric mucosa.\u003c/em\u003e Curr Microbiol, 2008. 56(3): p. 279-86.\u003c/li\u003e\n\u003cli\u003eSuganuma, M., et al., \u003cem\u003eTNF-alpha-inducing protein, a carcinogenic factor secreted from H. pylori, enters gastric cancer cells.\u003c/em\u003e International journal of cancer, 2008. 123(1): p. 117-122.\u003c/li\u003e\n\u003cli\u003eBinfield, P., \u003cem\u003eAt PLoS ONE we\u0026apos;re batty about bats\u003c/em\u003e, in \u003cem\u003ePLoS: Public Library of Science\u003c/em\u003e. 2008. p. 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Hsu, \u003cem\u003eModulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis by Helicobacter pylori in immune pathogenesis of gastric mucosal damage.\u003c/em\u003e J Microbiol Immunol Infect, 2017. 50(1): p. 4-9.\u003c/li\u003e\n\u003cli\u003eMizuno, T., Higashi, H., Tsutsumi, R., et al. (2005). \u003cem\u003eHelicobacter pylori induces interleukin-8 production through a protein encoded by a pathogenicity island\u003c/em\u003e. Gastroenterology, 128(5), 1022-1034.\u003c/li\u003e\n\u003cli\u003eChaturvedi, R., Asim, M., Lewis, N. D., et al. (2014). \u003cem\u003eHelicobacter pylori-induced gastric cancer: Molecular mechanisms and therapeutic strategies\u003c/em\u003e. \u003cem\u003eWorld Journal of Gastroenterology\u003c/em\u003e, 20(18), 5564-5575.\u003c/li\u003e\n\u003cli\u003eWatanabe, T., Higashi, H., Suda, K., et al. 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S., Singh, S., Trivedi, M., Hussain, N., Yadav, S., \u0026amp; Dubey, K. D. (2024). Phytocompounds as versatile drug-leads targeting mProtease in the SARS-CoV-2 virus: insights from a molecular dynamics study. Journal of Biomaterials Science, Polymer Edition, 1\u0026ndash;21.\u003c/li\u003e\n\u003cli\u003eDwivedi M, Jose S, Gupta M, Devi SS, Raj R, Kumar D. Copper transporter protein (MctB) as a therapeutic target to elicit antimycobacterial activity against tuberculosis. J Biomol Struct Dyn. 2024 Jul;42(10):5334-5348. \u003c/li\u003e\n\u003cli\u003eYadava A, Bhuyan MMR, Mukherjee D, Kumar D, Dwivedi M. Phytomolecules as potential candidates to intervene the function of \u003cem\u003eE. coli\u003c/em\u003e sodium-proton antiporters; Ec-NhaA. J Biomol Struct Dyn. 2023 Mar 19:1-12. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Helicobacter pylori, Gastric ulcer, Tumour Necrosis Factor Alpha, Inhibitors, Docking studies, Molecular dynamics","lastPublishedDoi":"10.21203/rs.3.rs-5983215/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5983215/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn about half of the world's population, \u003cem\u003eHelicobacter pylori\u003c/em\u003e (H. pylori) is the cause of stomach ulcers. Due to the extensive use of different drugs to treat H. pylori infections, multidrug resistance has developed, making treatment more difficult. Finding a viable therapeutic target becomes increasingly important as antibiotic resistance keeps increasing. A distinct protein of \u003cem\u003eH. pylori\u003c/em\u003e called Tumour Necrosis Factor-alpha Inducing Protein (Tip-α) is important in the development of gastric cancer, especially in patients with chronic ulcers. Because of its vital role, Tip-α may be a good target for medication. Using molecular docking experiments, two different binding sites on Tip-α were found, and possible inhibitors were assessed. ZINC11981988 and ZINC04243820, two ZINC database hits among the screened compounds, showed a significant binding affinity to Tip-α, with glide scores of -9.116 kcal/mol and -10.455 kcal/mol, respectively. A MM-GBSA analysis was conducted to have a better understanding of their binding mechanisms. The findings showed that the selectivity of Tip-α inhibitors was more influenced by electrostatic energy contributions than by van der Waals interactions. The stability of these ligands in their respective binding sites was evaluated using molecular dynamics (MD) simulations upto 100 ns. In contrast to ZINC11981988, ZINC04243820 showed a more stable binding association with Tip-α, according to the MD analysis. According to this, ZINC04243820 exhibits excellent binding stability and is therefore a promising candidate for additional research. Considering these results, ZINC04243820 could be proposed as potential lead to construct Tip-α inhibitors against \u003cem\u003eH. pylori which shows \u003c/em\u003efavourable interaction properties and persistent binding. Further, in vivo and in vitro investigations are \u0026nbsp;required to examine and validate its therapeutic potential.\u003c/p\u003e","manuscriptTitle":"Dynamics based screening of Therapeutic compounds against Helicobacter pylori targeting TNF-α inducing protein","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-11 12:06:05","doi":"10.21203/rs.3.rs-5983215/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9254aded-77c3-4b8a-a455-672275ea3985","owner":[],"postedDate":"February 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-02-11T12:06:05+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-11 12:06:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5983215","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5983215","identity":"rs-5983215","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}