Computational investigation of thiol-reductase inhibitors to overcome amebiasis using Docking and SMD-based simulation analysis

Computational investigation of thiol-reductase inhibitors to overcome amebiasis using Docking and SMD-based simulation analysis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Computational investigation of thiol-reductase inhibitors to overcome amebiasis using Docking and SMD-based simulation analysis Kalpana Singh, Akash Pratap Singh This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6461033/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 Amoebiasis is an infectious disease caused by an intestinal parasitic protozon, Entamoeba histolytica. It is well known for invading and destroying human tissues, leading to life-threatening abscesses. To treatment of amoebiasis, and reduce the parasitic infection thioredoxin reductase (EhTrR), a promising target. It catalyzes the NADP-dependent reduction of amoebic thioredoxins, which is essential for maintaining intracellular redox balance. Therefore, in these studies, we selected 1150 FDA compounds and docked them with thioredoxin reductase. The best docked-score compounds were Betrixaban (-9.9 kcal/mol), Netrasudil (-9.6 kcal/mol), and Novobiocin (-9.4 kcal/mol), as well as positive control NADPH and negative control Metroindazole. Netrasudil is the best drug candidate for future use among these three compounds, according to the RMSD value, steered molecular dynamics, and interaction pattern. Before clinical trials and in vitro investigations, these substances might be used as thioreductase inhibitors. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Entamoeba histolytica ( E. histolytica ) is a microaerobic intestinal parasite that causes human amebiasis, the fourth major cause of mortality and the third main cause of morbidity from parasitic infections. According to the WHO, Entamoeba histolytica deaths over 100,000 people a year and affects about 50 million people globally [ 1 ]. The parasite hinders the mental and physical development of children under the age of two by being one of the top 15 causes of diarrhoea in this age group [ 2 ]. Approximately 90% of those afflicted do not exhibit any symptoms. Although it is still unclear what causes the parasite to become virulent in the remaining people, gut microbiota has been linked to virulence [ 3 ]. It mostly affects places with poor water quality and inadequate sewage facilities [ 4 ]. Generally, symptoms of amoebiasis are large intestine inflammation and liver abscesses and Infection arises after consuming food infected with cysts. Trophozoites that arise from cysts move to the big intestine. Mild diarrhea, dysentery, invasive colitis, liver abscesses, and infrequent lung and/or brain abscesses are all signs of amebiasis [ 5 ]. E. histolytica, trophozoites thrive in the microaerophilic environment of the human colon, in which they can cause disease by infiltrating and damaging tissues [ 6 , 7 ].This microaerophilic parasite has a two-stage life cycle consisting of a non-invasive but infective cyst form that is dormant but highly resistant to harsh external environment conditions, and an invasive but non-infective trophozoite that is active inside the host but cannot survive in the external environment. [ 8 , 9 ]. Trophozoites inhabit the gut and endocytose both mucosal cells and commensal microorganisms. Amebiasis can be diagnosed via stool microscopy, ELISA, or PCR. The majority of infections (90%) are asymptomatic. Symptomatic infection is distinguished by bloody diarrhoea. E. histolytica can use oxygen; the pathogen's energy metabolism is thought to be solely fermentative, with phosphorylation occurring only at the substrate level and the transfer of electrons to molecular oxygen unlikely to be used for energetic purposes [ 10 – 12 ]. Anaerobic protozoa, such as pathogenic E. histolytica, which lack glutathione and have cysteine as their main low-molecular-mass thiol, depend on the thioredoxin system to maintain thiol homeostasis [ 13 , 14 ]. Thioredoxin reductase (TrxR) has developed into two primary classes, which are identified by their molecular weight (Mr), protein domain design, presence or absence of a second redox site that contains cysteine or selenocysteine, and, lastly, the disulfide/dithiol motif's position and structure [ 15 ]. E. histolytica. TrxR enzymes differ from bacterial and low-eukaryotic enzymes by having a smaller Mr due to missing the interface domain, the absence of the second redox site, and the C(X)2C structure of the disulfide/dithiol motif associated with the NADPH domain, where redox-active cysteines are spaced by two instead of four residues as in the high Mr TrxR C(X)4C motif associated with the FAD domain [ 16 , 17 ]. Metronidazole (MNZ) is currently used to treat invasive amoebiasis [ 18 ]. MNZ is converted inside the parasite by thioredoxin reductase (TrxR) to a nitro radical anion or nitroimidazole. This nitro group lowers O2, resulting in the production of lethal reactive oxygen species (ROS) within the parasite. Nitroimidazole can also alter cysteine-containing proteins such as thioredoxin (Trx), causing their deactivation [ 19 ]. These cytotoxic consequences that follow include breaking and instability of the DNA helix, leading to suppression of protein synthesis, which is lethal to the parasite [ 20 ]. There are several prevalent adverse effects associated with MNZ, including dizziness, heartburn, headaches, nausea, skin rashes, anorexia, ataxia, stomach cramps, difficulty sleeping, and weight loss, Along with the growing risk of resistance [ 20 – 23 ]. Therefore, it is necessary to look for novel therapeutic targets and alternative approaches to inhibit TrxR are urgent and unmet for the treatment of amebiasis. The application of modern computational techniques allows for the identification of therapeutic targets and the development of strategies that evaluate the advantages of the numerous accessible alternatives, thus minimizing both time and costs. The development of computer-assisted drug discovery approaches and molecular docking studies has aided the identification of novel therapeutic molecules in the present decade. Virtual screening (VS) has allowed researchers to quickly scan multiple compounds from specific databases in order to locate possible hit molecules [ 24 , 25 ]. Additionally, molecular dynamics simulation is a computational technique that integrates Newton's laws of motion to provide information about the protein structure of binding sites and provides pertinent and helpful information for comprehending and comparing various interactions with chemical compounds. In the present studies, we retrieved FDA-approved chemical compounds for the TrxR inhibitions. Materials and Methods The process for finding and validating drugs using computational methodologies is described in Fig. 1 . 3.1. Protein retrieval and active site prediction The TrxR protein's crystal structure was obtained from the Protein Data Bank (PDB ID: 4A5L) at a resolution of 1.66 Å. The removal of chain B done through the PyMol (The PyMOL Molecular Graphics System, Version 3.0 Schrödinger, LLC). Further, TrxR Energy minimization done through Obabel ( www.Obabel.com ). The FTSite server was used ( https://ftsite.bu.edu/ , visited on March 31, 2024) for prediction of ligand binding sites. 3.2: Ligand Preparation, Selection, and ADME Analysis We selected 1170 drugs approved by the FDA from the Pubchem database library ( https://pubchem.ncbi.nlm.nih.gov (accessed on 31 March 2024)) and stored them in structural data (.SDF) format (Supplementary Table-1). The ligands were converted into.pdbqt format and minimized energy using the Obabel program for docking experiments [ 26 ]. The Swiss ADME server ( http://www.swissadme.ch , accessed March 31, 2024) was used to analyze the most effective interaction molecules as drug likelihood. 3.3 Molecular Docking analysis The docking analysis of TrxR with the target proteins using AutoDock 4.2 ( https://autodock.scripps.edu (Accessed on June 15, 2024)) [ 27 ]. The binding conformation was supported the binding energies of the selected ligands. Lamarckian method was used to predict the free binding affinities, and the Root Means Square Deviation (RMSD) was examined. The TrxR was protonated using polar hydrogen that fixed Kollman charges. In the PDBQT received from TrxR, partial charges, atom kinds, and torsional degrees of freedom were all mentioned. However, TrxR, ligands’ torsional bonds, and side chains were flexible. With x, y, and z coordinates of TrxR active site 20.694, -5.361, and − 43.333, and a grid box with dimensions 86 Å × 70 Å × 66 Å was be created; all ligands was dock to the residue responsible for catalytic activity. 10 GA (genetic algorithm) runs was performed, each yielding 10 postures (source: https://vina.scripps.edu/manual/#linux (accessed on 31 June 2024)). The best stance from each run was captured in a cartoon picture. The Vina score of binding energy is based on the following equation: ΔG = ΔGvdw + ΔGhbond + ΔGdesolv + ΔGelect + ΔGtor ……………….(1) The docking score for each posture was calculated using the binding free energy (ΔG, Eq. 1), which includes vdW, H-bond interactions, desolvation energy, electrostatic energy, and torsional free energy [ 28 ]. In addition, 1170 FDA compounds were reused for docking and comprehensively analyzed from the binding posture. Molecular dynamic Simulation of TrxR with FDA The computational method known as molecular dynamics (MD) simulation makes use of Newton's equations of motion to examine the way atoms move within molecules. The top three ranked compounds based on docking energy, Betrixaban, Netrasudil, and Novobiocin, along with Positive Control (NADPH) and Negative Control (Metrotindazole), were then used in MD simulation studies to assess the binding mode of each compound with the TrxR active site and to demonstrate the interactions of ligand-protein in more depth. The simulation was executed with the Gromacs software package (2019.4), a widely used and well-known MD simulation program ( http://www.gromacs.org/ , observed on August 9, 2024). The protein's topology and force field parameters were generated with the CHARMM36 all-atom force field software (March 2019) (receptor). The ligand force field topology file were constructed using the default configurations on the CHARMM General Force Field (Cgenff) website ( https://cgenff.umary-land.edu/ (accessed on 31 August 2024). The target protein-ligand combination were placed in the center of a cube with 1.0 nm thick sides. The complex were dissolved utilizing the TIP3-transferable intermolecular potential with three points (TIP3P) water model. The complexes were electro-neutralized by adding the necessary amount of ions. The protein's poor connections and collisions were addressed using the 1000-step steepest descent algorithm. During energy minimization, all complexes went through two equilibration phases: 1 ns of NVT (Number, Volume, and Temperature) and 1 ns of NPT (Number, Pressure, and Temperature) equilibration. The GROMACS gmx create ndx module was used to index the system into non-water and water components, overcoming the cold solute-hot solvent problem by temperature coupling. A Berendsen thermostat were employed to maintain the system temperature at 310 K. A Parrinello-Rahman barostat was used to regulate the system's pressure. The LINCS approach was utilized to analyze the system's long-range interaction. MD simulations were ran for 100 ns, with coordinates saved every 1 ns [ 29 ]. The GROMACS package's several analysis modules (RMSD, RMSF and H-bond ) were employed to conduct structural and conformational analysis on all system. Steered Molecular Dynamic Simulation Steered MD simulations allow for the unbinding of ligands and conformational changes in proteins on timeframes suitable for simulations. A time-dependent external force is used to move the ligand from the bound state to the unbound state. During the transition, we may compute both the exerted force and external work performed on the system. To prevent protein distortions caused by tugging, the distance between the centre of mass of heavy atoms in a stable region of the protein and chosen ligand atoms was determined. The Pulling parameters were adopted from Justin et al [ 30 ]. Protein and ligand parameterised through CHARMM36 all-atom force field software (March 2019). Then Created a virtual box which dimensions were 6.560 X 4.362 X 12. Minimization was done as above mentioned methods. In particular, The spring constant was set to 100 kcal/mol·Å2, with a pulling velocity of 0.007 Å/ps. Further, 500 ps run was performed for SMD simulations. Results The virtual screening of compounds has emerged as a significant trend in the last ten years. It has been proven to be a reliable technique for finding strong inhibitors that may be applied to treat a variety of illnesses. Considering it is inexpensive and quick, it is widely used to create promising drug candidates for modulating essential enzymes involved in reduction processes. Inhibition of the thioreductase enzyme through metronidazole has been endorsed for amoebiasis diseases [ 31 ]. Molecular Docking and Interactions Analysis The FTSite server was used to anticipate TrxR active site. The selected FDA compounds and the key active site amino acids of TrxR exhibit a variety of interactions, including as hydrophobic and hydrogen bonding. We docked 1170 FDA compounds to the TrxR active site. We identified the top three FDA drugs along with positive control NADPH and Negative control Metronidazole from 1170 chemicals based on their unique binding patterns and energy (ΔG). Betrixaban (Compound ID: 10275777), Netrasudil (Compound ID: 66599893), Novobiocin (Compound ID: 15940185) with Positive Control NADPH (Compound ID: 5884) and Negative Control Metronidazole (Compound ID: 4173) were the FDA drug examined; their respective docking energy value were − 9.9 kcal/mol, -9.6 kcal/mol, -9.4 kcal/mol, -10.1 kcal/mol and − 4.5 kcal/mol respectively. During interactions analysis, Betrixaban showed Thr50 and Asp162 were involved in hbond while 2D figure exhibited Glu166 hydrophoic interations (Fig ). Netrasudil showed Thr51 were involved in hbond along with 2D image demonstrated Thr49, Ala163, Glu166, Leu169, Arg196 and His170 showed hydrophobic interaction. Novobiocin indicated two hbonds Thr51 and His170 along with Leu169 showed hydrophobic interaction in 2D image(Fig ). Positive Control NADPH interaction analysis were varied it indicated four hbond were participated Thr49,Thr50,Thr51,Asn55 with two hydrophobic interacting amino acids Leu47 and Thr50. Negative Control Metronidazole, Showed two hbonds Thr51, Glu167 along with 1 glu166 hydrophobic interacting residue(Fig. 2 ). Netrasudil was the best candidate among the FDA-approved drugs that docked with the TrxR active site; it showed a better binding profile than the other FDA medications, as well as positive and negative chemical compounds. As seen in the image, netrasudil (Docking Score − 9.4 kcal/mol) had a number of interacting hydrophobic residues while establishing a single hbond. In contrast, MZN (Docking Score − 4.5 kcal/mol ) had a lower interaction profile, whereas NADPH (-10.1 kcal/mol) had the highest binding patterns with TrxR. Molecular dynamic simulations The precise investigation of biological macromolecule dynamics in a regulated physiological environment is facilitated by the use of molecular dynamic simulations (MDS). During the simulation period, the flexibility and structural variations of docked complexes may be seen using MDS, a computational technique for examining the physical interactions in a biophysical system. Since the major goal of this MDS investigation was to determine the essential intermolecular interactions of the bound ligands and binding stability with the TrxR active site, the crystal structure of the protein–ligand complexes and the unbound TrxR active domain was simulated. (I) Protein alone (4a5L-apo),(ii) Protein bound to ligand 1 (betrixaban),(iii) Protein bound to ligand 2 (Netrasudil) (iv) Protein bound to Ligand 3(Novobiocin) (V) Protein bound to known inhibitor (Metronidazole-MZN) (VI) Protein bound to positive control (NADPH). Through MDS, we were able to evaluate the trajectories completed at a time function of 100 ns inside the solvated medium and infer important information about the dynamic activity of the drugs that were evaluated. Root mean square deviations (RMSDs) for all ligands and backbone atoms, root mean square fluctuations (RMSFs) for individual amino acids, gyration radius, and intermolecular hydrogen bond formation were among the parameters used to analyze the changes that occurred in protein-unbound and protein-bound ligand complexes with maintaining physiological conditions. Root Mean Square deviation All of the chosen docked complexes' minor structural and conformational changes were evaluated by comparing the RMSD of the protein backbone atoms to simulation time. Figure 3 a shows that, in relation to the RMSD from the Betrixaban, the RMSD values ascended at 22 ns, 39 ns, and 68 ns, reached RMSD 1.9 nm, and found equilibrium after a run of 70 ns. Over a 100 ns run, the average RMSD value was 2.4 ± 0.4 nm. Next, the Netrasudil trajectory revealed that RMSD increased at 20–25 ns, 55 ns, and 62–66 ns, with values of 8.9 ns. The average RMSD value was 8.9 ± 0.4 nm. The RMSD value for Novobiocin increased at 8–10 ns, 27–30 ns, and 32–57 ns after 75 ns to 100 ns, indicating that the chemical was very unstable. The average RMSD value was 7.6 ± 2.2 nm. MZN (negative control) demonstrated that after 37 ns of running, compounds were extremely unstable in their active site pockets. The average RMSD was 7.9 ± 1.3 nm. NADPH (Positive control) demonstrated that at 47 ns, the RMSD increased after reaching equilibrium. The average RMSD was 0.5 ± 0.3 nm. The crystal structure of RMSD were 0.3 ± 0.29 nm with no deviation in RMSD throughout 100 ns run. Root Mean square Fluctuation RMSF is defined as the displacement of an individual or group of amino acid atoms during ligand interactions (Fig. 3 b). The larger the variation, the stronger the interaction with the ligands. Betrixaban caused fluctuations in the Cα atoms at 100, 192–203, and 276. The average fluctuation measured 0.38 nm. Netrasudil's atoms (52–58 Cα, 203 Cα, and 267 Cα) varied. The average fluctuation was 0.44 nm. Novobiocin's Cα atoms (186, 213, and 271) varied. The average fluctuation was found at 0.41 nm. Metronidazole exhibited fluctuation in 43, 223 and 271 Cα atoms. The average fluctuation was 0.35 nm. NADPH levels (41–58 Cα, 110 Cα, 112 Cα, 142–149 Cα, 166–172 Cα, and 222 Cα) fluctuated. The average fluctuation was 0.43 nm. Hydrogen bond analysis Typically, hydrogen bonding stabilizes protein–ligand interactions. Monitoring the hydrogen bond formation during MDS allowed for the demonstration of the stability of TrxR-ligand complexes. A meticulous investigation of hydrogen bonds between each compound and the TrXR- active domain was predicted under the influence of the CHARMM force field. The TrXR- active domain H-Bond result of the complex with 4a5l-Betrixaban, 4a5L- Netrasudil, 4a5L- Novobiocin, 4a5L-Metroindazole, and 4a5L-NADPH is depicted in Fig. 4 . Before analyzing hbonds, an index group was generated using the gmx make_ndx command to select active site residues, which were GLN46, LEU47, THR49, THR50, THR51, ALA121, LYS122, ALA139,CYS140,ALA141,ILE142,CYS143,ALA163,GLU166,GLU167,HIS170,TYR244,ALA245,ILE246,GLY247,HIS248 for all complexes. Betrixaban demonstrated varying hbond interactions between 0–20 ns, 34 ns, and 39 ns. There were 34 donors and 81 acceptor atoms. There is an average of 0.127 hbond each time frame out of a total of 1040 hbonds in a 100 ns run. Netrasudil exhibited a range of hbond interactions during the course of a 100 ns run, with 31 donors and 63 acceptors and an average of 2.519 hbonds per time frame out of a potential 976.5 hbonds. Novobiocin tracked hbonds from 0 to 10 ns, after which no hbonds were found. There were 30 donors and 63 acceptors hbonds discovered with 0.140 potential time per frame out of 945 hbonds every 100ns run. During a 100-ns run in NADPH, hbonds were enhanced, resulting in 34 donors and 81 acceptor atoms with a potential duration per frame of 1.675 for 1377 hbonds. Steered Molecular dynamics simulation (SMD) A series of Pulling simulations (SMD) were run on all five inhibitors to establish their ability to detach from the TrXR receptor's pocket and their location. In Fig. 5 , high peaks were seen from 0 to 100 ps for all five inhibitors. In a thorough analysis, Betrixaban displayed average force from detaching active sites (~ 253 kj/mol) at 0-100 ps, followed by betrixaban out of pocket sites. Metroindazole was removed from the active site by applying a force (~ 240 kJ/mol) at 0-100 ps. The NADPH detachment force from TrXR's active site was approximately ~ 375 kJ/mol, with a temporal range of 0-300 ps. Netrasudil was detached with a force of ~ 550 kj/mol at 0-120 ps and then removed from the pocket. Novobiocin had a detached force of 350 kj/mol at 0-100 ps from the TrXR active site. In order to determine the pattern of interaction between TrXR and FDA compounds, Fig. 6 frame images were taken from a coordinated file containing three FDA compounds that were selected. Discussion Amoebiasis, the second most parasitic cause of mortality after malaria, is caused by the intestinal unicellular parasite E. histolytica [ 32 ]. E. histolytica is often found in the large intestine, although it can occasionally pass through the intestinal mucosa and spread to other organs [ 33 ]. Microorganisms' pathogenicity depends on their capacity to adapt to high oxygen pressures, ROS, and RNS concentrations [ 32 ]. Previous investigations demonstrated that E. histolytica possesses TRX systems that contribute to the parasite's redox metabolism [ 16 , 34 ]. The redox metabolic scenario in E. histolytica involves the involvement of EhTRXR and EhTRXs, which are crucial for preserving a redox balance in the parasite cytosol. As previously reported by Leitsch et al. and Debnath et al., reductase is an important enzyme for the parasite's viability [ 31 , 35 ]. TrxR's catalytic mechanism has been thoroughly studied in E. coli. The exceptionally high degree of NADPH oxidase activity displayed by EhTrxR protects against the molecular oxygen needed for these aerotolerant, anaerobic microbes to survive [ 36 ]. On the other hand, in highly reducing environments, large intracellular cysteine levels make up for the absence of glutathione, minimizing the auto-oxidation [ 37 ]. Remarkably, the E. histolytica thioredoxin-thioredoxin reductase system was mostly found at the plasma membrane, even though neither thioredoxin nor thioredoxin reductase variants have a transmembrane hydrophobic domain [ 38 ]. In addition to ROS, during tissue invasion, E. histolytica is exposed to significant levels of reactive nitrogen species (RNS), such as nitric oxide (NO), or S-nitrosothiols, such as GSNO and CyS9NO. E. histolytica can live and thrive during tissue invasion, even though high levels of these RNS may prevent the parasite's growth in vitro. This implies that the detoxification system of E. histolytica is adaptable enough to withstand harsh conditions. The E. histolytica Trx-TrxR system's capacity to lower RNS and employ an alternate electron donor, such as NADH, was a recent example of this adaptability [ 39 ]. In this study, we showed that increasing oxygen stress reduces pathogen survival rates. As a result of docking analysis, we predicted thioreductase inhibitors. In-depth betrixaban compounds had a binding energy of -9.9 kcal/mol, but their interaction pattern with active site residues was less. During simulation analysis, only 1040 hbonds were formed, with a significantly static and low RMSD pattern. Netrasudil had a binding score of -9.6 kcal/mol, which interacted with higher hydrophobic moieties at the active site. Simulation analysis revealed that the RMSD data was less flickering with 976.5 hbond interactions. Novobiocin's -9.4 kcal/mol binding energy with a single hydrophobic moiety demonstrated promising interactions. In the simulation analysis, 945 hydrogen bonds were formed. NADPH (positive control) showed a strong interaction pattern during docking (-10.1 kcal/mol), and the RMSD flickered very little, whereas Metronidazole (negative control) showed a -4.5 kcal/mol binding score, which was lower than the above-mentioned compounds, and their simulation RMSD and hbond were very low. Overall, we demonstrated that Netrasudil has a good hydrophobic moiety and is very stable in simulation throughout the 100 ns run. Additionally, SMD showed that the only compounds that detached from the active site with higher energy (~ 375 kJ/mol and ~ 550 kJ/mol, respectively) were NADPH and Netrasudil. Conclusion Every year, amoebiasis causes almost 100,000 deaths worldwide and 50 million cases in tropical areas. E. histolytica thioredoxin reductase (EhTrR) plays a key role in metabolic functions for maintaining intracellular redox balance. These homeostasis cause parasite proliferation into the host. Thus, thioredoxin reductase inhibition is essential and currently unmet. The only medication on the market that interferes with the parasite's redox balance cycle and prevents infections is metronidazole. However, resistance and adverse effects on human health cannot be disregarded. Thus, in this study, we predicted three compounds that interacted strongly at the thioredoxin reductase pocket site. Netrasudil may interact and inhibit the thioredoxin reductase the best out of the three. Clinical trials and in vitro testing may be required prior to use. Declarations Ethical Responsibilities : This study does not directly involve humans or other organisms. Consent for publication : All authors consent to submit the manuscript to the journal. Availability of data and material : All the data in supplementary material provided for extended validation or analysis produced during the docking phase can be made public after publication. Funding Declaration : No funding was available to carry out this study. Author contribution : Conceptualisation, data collection/curation, analysis, writing, reviewing the first draft, KS; Conceptualisation, data collection/curation, analysis, writing, reviewing and editing the first draft, APS. Acknowledgements : The author is greatly acknowledged and thankful to the Department of Zoology, RBS college, Agra and Maitreyi College, University of Delhi, New Delhi for providing the research facility and carrying out research work and other support. References Onursal, A. and B. 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Loftus, B., et al., The genome of the protist parasite Entamoeba histolytica. Nature, 2005. 433 (7028): p. 865-8. Arias, D.G., et al., Immunolocalization and enzymatic functional characterization of the thioredoxin system in Entamoeba histolytica. Free Radic Biol Med, 2008. 45 (1): p. 32-9. Debnath, A., et al., A high-throughput drug screen for Entamoeba histolytica identifies a new lead and target. Nat Med, 2012. 18 (6): p. 956-60. Bruchhaus, I., S. Richter, and E. Tannich, Recombinant expression and biochemical characterization of an NADPH:flavin oxidoreductase from Entamoeba histolytica. Biochem J, 1998. 330 ( Pt 3) (Pt 3): p. 1217-21. Nozaki, T., et al., Characterization of the gene encoding serine acetyltransferase, a regulated enzyme of cysteine biosynthesis from the protist parasites Entamoeba histolytica and Entamoeba dispar. Regulation and possible function of the cysteine biosynthetic pathway in Entamoeba. J Biol Chem, 1999. 274 (45): p. 32445-52. Andrade, R.M. and S.L. Reed, New drug target in protozoan parasites: the role of thioredoxin reductase. Front Microbiol, 2015. 6 : p. 975. Arias, D.G., et al., Entamoeba histolytica thioredoxin reductase: molecular and functional characterization of its atypical properties. Biochim Biophys Acta, 2012. 1820 (12): p. 1859-66. Additional Declarations No competing interests reported. Supplementary Files DockingScore.csv Supplementry Table no. 1 – FDA approved drug docking score with thioreductase SupplementryFigureno.1.docx 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-6461033","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":446693025,"identity":"8ede167f-fb95-4a97-826e-88909442fded","order_by":0,"name":"Kalpana Singh","email":"","orcid":"","institution":"Raja balwant singh College, Agra","correspondingAuthor":false,"prefix":"","firstName":"Kalpana","middleName":"","lastName":"Singh","suffix":""},{"id":446693026,"identity":"393b3640-98e7-4af5-976d-49b4a88a552f","order_by":1,"name":"Akash Pratap Singh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABE0lEQVRIie3Rv2vCQBTA8RcKulxwvSHov3BSSCkt+K9EHG5RcHQI5Y6CWaS79G/o0CVz4EGmgOuBDgbByaV0kWKLd/XH1AsdBe9LAnlwHx5HAFyuCy3TTxPAE2YgABEcP6rJrSZS/JsY1RWnNSdirfGalDj8WvC3BJPVaIQB1PsMtjEEdxZCFwXD6ct6kBZdKYsCCZAN8yY5kHthMaoP6E9wkGaayLEmVG/xBRCW/S1aii8N4eGs1OTnQLzvCsJUxJBsMQqV2SIO5KZqS9vcxRfYTlUppyLnpEbWQwxyaiXNebL6JDtshTO+/BDxQ6dR772Xm/ixYyNA9euNz+Mz1OD351LL+SOB3Xl8sp90uVyuq20PDOdmFjfk6A8AAAAASUVORK5CYII=","orcid":"","institution":"Department of Botany, Maitreyi College, University of Delhi","correspondingAuthor":true,"prefix":"","firstName":"Akash","middleName":"Pratap","lastName":"Singh","suffix":""}],"badges":[],"createdAt":"2025-04-16 08:08:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6461033/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6461033/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81269994,"identity":"be33ead8-c556-4fb2-a1cf-8d68932a5a06","added_by":"auto","created_at":"2025-04-24 08:20:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":701043,"visible":true,"origin":"","legend":"\u003cp\u003eShowing the Graphical Abstract for the method followed for complete analysis of thioreductase and their inhibitors.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6461033/v1/bac151aaf615feb6db1cd7f7.png"},{"id":81271383,"identity":"2d49074d-a0cc-4097-9638-ee4058992f56","added_by":"auto","created_at":"2025-04-24 08:36:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3024154,"visible":true,"origin":"","legend":"\u003cp\u003eDocking interaction analysis of thioreductase; A) NADPH (Positive control) is represented by purple colour and six hbonds with dotted lines and two hydrophobic amino acid moieties; B) Netrasudil is represented by cyan and three hbonds with seven hydrophobic amino acid moieties; C) Novobiocin is represented by green and four hbonds with one hydrophobic amino acid moiety; and D) Betrixaban is represented by navy blue and four hbonds with one hydrophobic amino acid moiety; E) Metronidazole (Negative Control) is represented in olive green, with three hbonds and one hydrophobic moiety.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6461033/v1/f4c2ad7232920debb7002da0.png"},{"id":81270001,"identity":"f9d57234-4ac8-4e8e-9659-7e2a59203fb0","added_by":"auto","created_at":"2025-04-24 08:20:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2574837,"visible":true,"origin":"","legend":"\u003cp\u003eMD simulation trajectory analysis following a 100 ns run; a) RMSD plot of drug candidates and the thioreductase protein backbone; b) RMSF plot for the thioreductase protein and drug ligands; fluctuation of residues indicated interaction with specific ligands.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6461033/v1/3f0416427140479fb1755bd6.png"},{"id":81271081,"identity":"fe985265-9bda-46e0-a1d6-1791e8f93a4a","added_by":"auto","created_at":"2025-04-24 08:28:18","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":682861,"visible":true,"origin":"","legend":"\u003cp\u003eSimulation-based H-bond analysis of complex; Novobiocin (blue); Betrixaban (red); Netrasudil (cyan), NADPH (green), and Metronidazole (black).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6461033/v1/8a2ce86c35d6a7765cbde0c3.png"},{"id":81269997,"identity":"8356aaef-edd4-412d-916b-b739915940bf","added_by":"auto","created_at":"2025-04-24 08:20:18","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1936373,"visible":true,"origin":"","legend":"\u003cp\u003eAverage pull force profile of inhibitors that exit the TrxR pocket site.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6461033/v1/8b9a9e53308dc32f1ad0ac0f.png"},{"id":81271083,"identity":"bbc635af-6a77-4e8d-a273-b282d80faffb","added_by":"auto","created_at":"2025-04-24 08:28:19","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3186305,"visible":true,"origin":"","legend":"\u003cp\u003eSteered molecular dynamic-based undocking process of FDA complexes with thioreductase; a) Netrasudil, b) Betrixaban, c) Novobiocin.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6461033/v1/d562811158f19eee8fcf4e79.png"},{"id":81807400,"identity":"1f598168-0763-4e78-9fb9-8da0b11c0ad7","added_by":"auto","created_at":"2025-05-02 07:47:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":16836440,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6461033/v1/441b9d38-3a60-42d1-a7e0-f2b2c18932d1.pdf"},{"id":81271080,"identity":"6bccb9c4-0e1e-45c5-8665-fc8807e835e8","added_by":"auto","created_at":"2025-04-24 08:28:18","extension":"csv","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":32410,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementry Table no. 1 – FDA approved drug docking score with thioreductase\u003c/p\u003e","description":"","filename":"DockingScore.csv","url":"https://assets-eu.researchsquare.com/files/rs-6461033/v1/aa05645642a120005dea5f74.csv"},{"id":81270003,"identity":"0e74e442-bde2-44e8-9e19-ece4b1e94e4e","added_by":"auto","created_at":"2025-04-24 08:20:19","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":12420663,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementryFigureno.1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6461033/v1/022e71d247c332efa2dcccce.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Computational investigation of thiol-reductase inhibitors to overcome amebiasis using Docking and SMD-based simulation analysis","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eEntamoeba histolytica\u003c/em\u003e (\u003cem\u003eE. histolytica\u003c/em\u003e) is a microaerobic intestinal parasite that causes human amebiasis, the fourth major cause of mortality and the third main cause of morbidity from parasitic infections. According to the WHO, Entamoeba histolytica deaths over 100,000 people a year and affects about 50\u0026nbsp;million people globally [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The parasite hinders the mental and physical development of children under the age of two by being one of the top 15 causes of diarrhoea in this age group [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Approximately 90% of those afflicted do not exhibit any symptoms. Although it is still unclear what causes the parasite to become virulent in the remaining people, gut microbiota has been linked to virulence [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. It mostly affects places with poor water quality and inadequate sewage facilities [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Generally, symptoms of amoebiasis are large intestine inflammation and liver abscesses and Infection arises after consuming food infected with cysts. Trophozoites that arise from cysts move to the big intestine.\u003c/p\u003e \u003cp\u003eMild diarrhea, dysentery, invasive colitis, liver abscesses, and infrequent lung and/or brain abscesses are all signs of amebiasis [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. E. histolytica, trophozoites thrive in the microaerophilic environment of the human colon, in which they can cause disease by infiltrating and damaging tissues [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].This microaerophilic parasite has a two-stage life cycle consisting of a non-invasive but infective cyst form that is dormant but highly resistant to harsh external environment conditions, and an invasive but non-infective trophozoite that is active inside the host but cannot survive in the external environment. [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Trophozoites inhabit the gut and endocytose both mucosal cells and commensal microorganisms. Amebiasis can be diagnosed via stool microscopy, ELISA, or PCR. The majority of infections (90%) are asymptomatic. Symptomatic infection is distinguished by bloody diarrhoea.\u003c/p\u003e \u003cp\u003eE. histolytica can use oxygen; the pathogen's energy metabolism is thought to be solely fermentative, with phosphorylation occurring only at the substrate level and the transfer of electrons to molecular oxygen unlikely to be used for energetic purposes [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e–\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Anaerobic protozoa, such as pathogenic E. histolytica, which lack glutathione and have cysteine as their main low-molecular-mass thiol, depend on the thioredoxin system to maintain thiol homeostasis [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Thioredoxin reductase (TrxR) has developed into two primary classes, which are identified by their molecular weight (Mr), protein domain design, presence or absence of a second redox site that contains cysteine or selenocysteine, and, lastly, the disulfide/dithiol motif's position and structure [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. E. histolytica. TrxR enzymes differ from bacterial and low-eukaryotic enzymes by having a smaller Mr due to missing the interface domain, the absence of the second redox site, and the C(X)2C structure of the disulfide/dithiol motif associated with the NADPH domain, where redox-active cysteines are spaced by two instead of four residues as in the high Mr TrxR C(X)4C motif associated with the FAD domain [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMetronidazole (MNZ) is currently used to treat invasive amoebiasis [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. MNZ is converted inside the parasite by thioredoxin reductase (TrxR) to a nitro radical anion or nitroimidazole.\u003c/p\u003e \u003cp\u003eThis nitro group lowers O2, resulting in the production of lethal reactive oxygen species (ROS) within the parasite. Nitroimidazole can also alter cysteine-containing proteins such as thioredoxin (Trx), causing their deactivation [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. These cytotoxic consequences that follow include breaking and instability of the DNA helix, leading to suppression of protein synthesis, which is lethal to the parasite [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. There are several prevalent adverse effects associated with MNZ, including dizziness, heartburn, headaches, nausea, skin rashes, anorexia, ataxia, stomach cramps, difficulty sleeping, and weight loss, Along with the growing risk of resistance [\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e–\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Therefore, it is necessary to look for novel therapeutic targets and alternative approaches to inhibit TrxR are urgent and unmet for the treatment of amebiasis.\u003c/p\u003e \u003cp\u003eThe application of modern computational techniques allows for the identification of therapeutic targets and the development of strategies that evaluate the advantages of the numerous accessible alternatives, thus minimizing both time and costs.\u003c/p\u003e \u003cp\u003eThe development of computer-assisted drug discovery approaches and molecular docking studies has aided the identification of novel therapeutic molecules in the present decade. Virtual screening (VS) has allowed researchers to quickly scan multiple compounds from specific databases in order to locate possible hit molecules [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Additionally, molecular dynamics simulation is a computational technique that integrates Newton's laws of motion to provide information about the protein structure of binding sites and provides pertinent and helpful information for comprehending and comparing various interactions with chemical compounds. In the present studies, we retrieved FDA-approved chemical compounds for the TrxR inhibitions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e "},{"header":"Materials and Methods","content":"\u003cp\u003eThe process for finding and validating drugs using computational methodologies is described in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003e3.1. Protein retrieval and active site prediction\u003c/p\u003e\n\u003cp\u003eThe TrxR protein\u0026apos;s crystal structure was obtained from the Protein Data Bank (PDB ID: 4A5L) at a resolution of 1.66 \u0026Aring;. The removal of chain B done through the PyMol (The PyMOL Molecular Graphics System, Version 3.0 Schr\u0026ouml;dinger, LLC). Further, TrxR Energy minimization done through Obabel (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.Obabel.com\u003c/span\u003e\u003c/span\u003e). The FTSite server was used (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ftsite.bu.edu/\u003c/span\u003e\u003c/span\u003e, visited on March 31, 2024) for prediction of ligand binding sites.\u003c/p\u003e\n\u003cp\u003e3.2: Ligand Preparation, Selection, and ADME Analysis\u003c/p\u003e\n\u003cp\u003eWe selected 1170 drugs approved by the FDA from the Pubchem database library (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov\u003c/span\u003e\u003c/span\u003e (accessed on 31 March 2024)) and stored them in structural data (.SDF) format (Supplementary Table-1). The ligands were converted into.pdbqt format and minimized energy using the Obabel program for docking experiments [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e]. The Swiss ADME server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.swissadme.ch\u003c/span\u003e\u003c/span\u003e, accessed March 31, 2024) was used to analyze the most effective interaction molecules as drug likelihood.\u003c/p\u003e\n\u003cp\u003e3.3 Molecular Docking analysis\u003c/p\u003e\n\u003cp\u003eThe docking analysis of TrxR with the target proteins using AutoDock 4.2 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://autodock.scripps.edu\u003c/span\u003e\u003c/span\u003e (Accessed on June 15, 2024)) [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]. The binding conformation was supported the binding energies of the selected ligands. Lamarckian method was used to predict the free binding affinities, and the Root Means Square Deviation (RMSD) was examined. The TrxR was protonated using polar hydrogen that fixed Kollman charges. In the PDBQT received from TrxR, partial charges, atom kinds, and torsional\u003c/p\u003e\n\u003cp\u003edegrees of freedom were all mentioned. However, TrxR, ligands\u0026rsquo; torsional bonds, and side chains were flexible. With x, y, and z coordinates of TrxR active site 20.694, -5.361, and \u0026minus;\u0026thinsp;43.333, and a grid box with dimensions 86 \u0026Aring; \u0026times; 70 \u0026Aring; \u0026times; 66 \u0026Aring; was be created; all ligands was dock to the residue responsible for catalytic activity. 10 GA (genetic algorithm) runs was performed, each yielding 10 postures (source: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://vina.scripps.edu/manual/#linux\u003c/span\u003e\u003c/span\u003e (accessed on 31 June 2024)). The best stance from each run was captured in a cartoon picture. The Vina score of binding energy is based on the following equation:\u003c/p\u003e\n\u003cp\u003e\u0026Delta;G\u0026thinsp;=\u0026thinsp;\u0026Delta;Gvdw\u0026thinsp;+\u0026thinsp;\u0026Delta;Ghbond\u0026thinsp;+\u0026thinsp;\u0026Delta;Gdesolv\u0026thinsp;+\u0026thinsp;\u0026Delta;Gelect\u0026thinsp;+\u0026thinsp;\u0026Delta;Gtor \u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;.(1)\u003c/p\u003e\n\u003cp\u003eThe docking score for each posture was calculated using the binding free energy (\u0026Delta;G, Eq.\u0026nbsp;1), which includes vdW, H-bond interactions, desolvation energy, electrostatic energy, and torsional free energy [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. In addition, 1170 FDA compounds were reused for docking and comprehensively analyzed from the binding posture.\u003c/p\u003e\n\u003cp\u003eMolecular dynamic Simulation of TrxR with FDA\u003c/p\u003e\n\u003cp\u003eThe computational method known as molecular dynamics (MD) simulation makes use of Newton\u0026apos;s equations of motion to examine the way atoms move within molecules. The top three ranked compounds based on docking energy, Betrixaban, Netrasudil, and Novobiocin, along with Positive Control (NADPH) and Negative Control (Metrotindazole), were then used in MD simulation studies to assess the binding mode of each compound with the TrxR active site and to demonstrate the interactions of ligand-protein in more depth. The simulation was executed with the Gromacs software package (2019.4), a widely used and well-known MD simulation program (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.gromacs.org/\u003c/span\u003e\u003c/span\u003e, observed on August 9, 2024). The protein\u0026apos;s topology and force field parameters were generated with the CHARMM36 all-atom force field software (March 2019) (receptor). The ligand force field topology file were constructed using the default configurations on the CHARMM General Force Field (Cgenff) website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cgenff.umary-land.edu/\u003c/span\u003e\u003c/span\u003e (accessed on 31 August 2024). The target protein-ligand combination were placed in the center of a cube with 1.0 nm thick sides. The complex were dissolved utilizing the TIP3-transferable intermolecular potential with three points (TIP3P) water model. The complexes were electro-neutralized by adding the necessary amount of ions. The protein\u0026apos;s poor connections and collisions were addressed using the 1000-step steepest descent algorithm. During energy minimization, all complexes went through two equilibration phases: 1 ns of NVT (Number, Volume, and Temperature) and 1 ns of NPT (Number, Pressure, and Temperature) equilibration. The GROMACS gmx create ndx module was used to index the system into non-water and water components, overcoming the cold solute-hot solvent problem by temperature coupling. A Berendsen thermostat were employed to maintain the system temperature at 310 K. A Parrinello-Rahman barostat was used to regulate the system\u0026apos;s pressure. The LINCS approach was utilized to analyze the system\u0026apos;s long-range interaction. MD simulations were ran for 100 ns, with coordinates saved every 1 ns [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]. The GROMACS package\u0026apos;s several analysis modules (RMSD, RMSF and H-bond ) were employed to conduct structural and conformational analysis on all system.\u003c/p\u003e\n\u003cp\u003eSteered Molecular Dynamic Simulation\u003c/p\u003e\n\u003cp\u003eSteered MD simulations allow for the unbinding of ligands and conformational changes in proteins on timeframes suitable for simulations. A time-dependent external force is used to move the ligand from the bound state to the unbound state. During the transition, we may compute both the exerted force and external work performed on the system. To prevent protein distortions caused by tugging, the distance between the centre of mass of heavy atoms in a stable region of the protein and chosen ligand atoms was determined. The Pulling parameters were adopted from Justin et al [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e]. Protein and ligand parameterised through CHARMM36 all-atom force field software (March 2019). Then Created a virtual box which dimensions were 6.560 X 4.362 X 12. Minimization was done as above mentioned methods. In particular, The spring constant was set to 100 kcal/mol\u0026middot;\u0026Aring;2, with a pulling velocity of 0.007 \u0026Aring;/ps. Further, 500 ps run was performed for SMD simulations.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe virtual screening of compounds has emerged as a significant trend in the last ten years. It has been proven to be a reliable technique for finding strong inhibitors that may be applied to treat a variety of illnesses. Considering it is inexpensive and quick, it is widely used to create promising drug candidates for modulating essential enzymes involved in reduction processes.\u003c/p\u003e \u003cp\u003eInhibition of the thioreductase enzyme through metronidazole has been endorsed for amoebiasis diseases [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMolecular Docking and Interactions Analysis\u003c/p\u003e \u003cp\u003eThe FTSite server was used to anticipate TrxR active site. The selected FDA compounds and the key active site amino acids of TrxR exhibit a variety of interactions, including as hydrophobic and hydrogen bonding. We docked 1170 FDA compounds to the TrxR active site. We identified the top three FDA drugs along with positive control NADPH and Negative control Metronidazole from 1170 chemicals based on their unique binding patterns and energy (ΔG). Betrixaban (Compound ID: 10275777), Netrasudil (Compound ID: 66599893), Novobiocin (Compound ID: 15940185) with Positive Control NADPH (Compound ID: 5884) and Negative Control Metronidazole (Compound ID: 4173) were the FDA drug examined; their respective docking energy value were \u0026minus;\u0026thinsp;9.9 kcal/mol, -9.6 kcal/mol, -9.4 kcal/mol, -10.1 kcal/mol and \u0026minus;\u0026thinsp;4.5 kcal/mol respectively. During interactions analysis, Betrixaban showed Thr50 and Asp162 were involved in hbond while 2D figure exhibited Glu166 hydrophoic interations (Fig ). Netrasudil showed Thr51 were involved in hbond along with 2D image demonstrated Thr49, Ala163, Glu166, Leu169, Arg196 and His170 showed hydrophobic interaction. Novobiocin indicated two hbonds Thr51 and His170 along with Leu169 showed hydrophobic interaction in 2D image(Fig ). Positive Control NADPH interaction analysis were varied it indicated four hbond were participated Thr49,Thr50,Thr51,Asn55 with two hydrophobic interacting amino acids Leu47 and Thr50. Negative Control Metronidazole, Showed two hbonds Thr51, Glu167 along with 1 glu166 hydrophobic interacting residue(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Netrasudil was the best candidate among the FDA-approved drugs that docked with the TrxR active site; it showed a better binding profile than the other FDA medications, as well as positive and negative chemical compounds. As seen in the image, netrasudil (Docking Score \u0026minus;\u0026thinsp;9.4 kcal/mol) had a number of interacting hydrophobic residues while establishing a single hbond. In contrast, MZN (Docking Score \u0026minus;\u0026thinsp;4.5 kcal/mol ) had a lower interaction profile, whereas NADPH (-10.1 kcal/mol) had the highest binding patterns with TrxR.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMolecular dynamic simulations\u003c/p\u003e \u003cp\u003eThe precise investigation of biological macromolecule dynamics in a regulated physiological environment is facilitated by the use of molecular dynamic simulations (MDS). During the simulation period, the flexibility and structural variations of docked complexes may be seen using MDS, a computational technique for examining the physical interactions in a biophysical system. Since the major goal of this MDS investigation was to determine the essential intermolecular interactions of the bound ligands and binding stability with the TrxR active site, the crystal structure of the protein\u0026ndash;ligand complexes and the unbound TrxR active domain was simulated. (I) Protein alone (4a5L-apo),(ii) Protein bound to ligand 1 (betrixaban),(iii) Protein bound to ligand 2 (Netrasudil) (iv) Protein bound to Ligand 3(Novobiocin) (V) Protein bound to known inhibitor (Metronidazole-MZN) (VI) Protein bound to positive control (NADPH). Through MDS, we were able to evaluate the trajectories completed at a time function of 100 ns inside the solvated medium and infer important information about the dynamic activity of the drugs that were evaluated. Root mean square deviations (RMSDs) for all ligands and backbone atoms, root mean square fluctuations (RMSFs) for individual amino acids, gyration radius, and intermolecular hydrogen bond formation were among the parameters used to analyze the changes that occurred in protein-unbound and protein-bound ligand complexes with maintaining physiological conditions.\u003c/p\u003e \u003cp\u003eRoot Mean Square deviation\u003c/p\u003e \u003cp\u003eAll of the chosen docked complexes' minor structural and conformational changes were evaluated by comparing the RMSD of the protein backbone atoms to simulation time. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea shows that, in relation to the RMSD from the Betrixaban, the RMSD values ascended at 22 ns, 39 ns, and 68 ns, reached RMSD 1.9 nm, and found equilibrium after a run of 70 ns. Over a 100 ns run, the average RMSD value was 2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 nm. Next, the Netrasudil trajectory revealed that RMSD increased at 20\u0026ndash;25 ns, 55 ns, and 62\u0026ndash;66 ns, with values of 8.9 ns. The average RMSD value was 8.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 nm. The RMSD value for Novobiocin increased at 8\u0026ndash;10 ns, 27\u0026ndash;30 ns, and 32\u0026ndash;57 ns after 75 ns to 100 ns, indicating that the chemical was very unstable. The average RMSD value was 7.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2 nm. MZN (negative control) demonstrated that after 37 ns of running, compounds were extremely unstable in their active site pockets. The average RMSD was 7.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 nm. NADPH (Positive control) demonstrated that at 47 ns, the RMSD increased after reaching equilibrium. The average RMSD was 0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 nm. The crystal structure of RMSD were 0.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29 nm with no deviation in RMSD throughout 100 ns run.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRoot Mean square Fluctuation\u003c/p\u003e \u003cp\u003eRMSF is defined as the displacement of an individual or group of amino acid atoms during ligand interactions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). The larger the variation, the stronger the interaction with the ligands. Betrixaban caused fluctuations in the Cα atoms at 100, 192\u0026ndash;203, and 276. The average fluctuation measured 0.38 nm. Netrasudil's atoms (52\u0026ndash;58 Cα, 203 Cα, and 267 Cα) varied. The average fluctuation was 0.44 nm. Novobiocin's Cα atoms (186, 213, and 271) varied. The average fluctuation was found at 0.41 nm. Metronidazole exhibited fluctuation in 43, 223 and 271 Cα atoms. The average fluctuation was 0.35 nm. NADPH levels (41\u0026ndash;58 Cα, 110 Cα, 112 Cα, 142\u0026ndash;149 Cα, 166\u0026ndash;172 Cα, and 222 Cα) fluctuated. The average fluctuation was 0.43 nm.\u003c/p\u003e \u003cp\u003eHydrogen bond analysis\u003c/p\u003e \u003cp\u003eTypically, hydrogen bonding stabilizes protein\u0026ndash;ligand interactions. Monitoring the hydrogen bond formation during MDS allowed for the demonstration of the stability of TrxR-ligand complexes. A meticulous investigation of hydrogen bonds between each compound and the TrXR- active domain was predicted under the influence of the CHARMM force field. The TrXR- active domain H-Bond result of the complex with 4a5l-Betrixaban, 4a5L- Netrasudil, 4a5L- Novobiocin, 4a5L-Metroindazole, and 4a5L-NADPH is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Before analyzing hbonds, an index group was generated using the gmx make_ndx command to select active site residues, which were GLN46, LEU47, THR49, THR50, THR51, ALA121, LYS122, ALA139,CYS140,ALA141,ILE142,CYS143,ALA163,GLU166,GLU167,HIS170,TYR244,ALA245,ILE246,GLY247,HIS248 for all complexes. Betrixaban demonstrated varying hbond interactions between 0\u0026ndash;20 ns, 34 ns, and 39 ns. There were 34 donors and 81 acceptor atoms. There is an average of 0.127 hbond each time frame out of a total of 1040 hbonds in a 100 ns run. Netrasudil exhibited a range of hbond interactions during the course of a 100 ns run, with 31 donors and 63 acceptors and an average of 2.519 hbonds per time frame out of a potential 976.5 hbonds. Novobiocin tracked hbonds from 0 to 10 ns, after which no hbonds were found.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThere were 30 donors and 63 acceptors hbonds discovered with 0.140 potential time per frame out of 945 hbonds every 100ns run. During a 100-ns run in NADPH, hbonds were enhanced, resulting in 34 donors and 81 acceptor atoms with a potential duration per frame of 1.675 for 1377 hbonds.\u003c/p\u003e \u003cp\u003eSteered Molecular dynamics simulation (SMD)\u003c/p\u003e \u003cp\u003eA series of Pulling simulations (SMD) were run on all five inhibitors to establish their ability to detach from the TrXR receptor's pocket and their location. In Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, high peaks were seen from 0 to 100 ps for all five inhibitors. In a thorough analysis, Betrixaban displayed average force from detaching active sites (~\u0026thinsp;253 kj/mol) at 0-100 ps, followed by betrixaban out of pocket sites. Metroindazole was removed from the active site by applying a force (~\u0026thinsp;240 kJ/mol) at 0-100 ps. The NADPH detachment force from TrXR's active site was approximately\u0026thinsp;~\u0026thinsp;375 kJ/mol, with a temporal range of 0-300 ps.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNetrasudil was detached with a force of ~\u0026thinsp;550 kj/mol at 0-120 ps and then removed from the pocket. Novobiocin had a detached force of 350 kj/mol at 0-100 ps from the TrXR active site. In order to determine the pattern of interaction between TrXR and FDA compounds, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e frame images were taken from a coordinated file containing three FDA compounds that were selected.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAmoebiasis, the second most parasitic cause of mortality after malaria, is caused by the intestinal unicellular parasite E. histolytica [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. E. histolytica is often found in the large intestine, although it can occasionally pass through the intestinal mucosa and spread to other organs [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Microorganisms' pathogenicity depends on their capacity to adapt to high oxygen pressures, ROS, and RNS concentrations [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Previous investigations demonstrated that E. histolytica possesses TRX systems that contribute to the parasite's redox metabolism [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The redox metabolic scenario in E. histolytica involves the involvement of EhTRXR and EhTRXs, which are crucial for preserving a redox balance in the parasite cytosol. As previously reported by Leitsch et al. and Debnath et al., reductase is an important enzyme for the parasite's viability [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. TrxR's catalytic mechanism has been thoroughly studied in E. coli. The exceptionally high degree of NADPH oxidase activity displayed by EhTrxR protects against the molecular oxygen needed for these aerotolerant, anaerobic microbes to survive [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. On the other hand, in highly reducing environments, large intracellular cysteine levels make up for the absence of glutathione, minimizing the auto-oxidation [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Remarkably, the E. histolytica thioredoxin-thioredoxin reductase system was mostly found at the plasma membrane, even though neither thioredoxin nor thioredoxin reductase variants have a transmembrane hydrophobic domain [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In addition to ROS, during tissue invasion, E. histolytica is exposed to significant levels of reactive nitrogen species (RNS), such as nitric oxide (NO), or S-nitrosothiols, such as GSNO and CyS9NO. E. histolytica can live and thrive during tissue invasion, even though high levels of these RNS may prevent the parasite's growth in vitro. This implies that the detoxification system of E. histolytica is adaptable enough to withstand harsh conditions. The E. histolytica Trx-TrxR system's capacity to lower RNS and employ an alternate electron donor, such as NADH, was a recent example of this adaptability [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we showed that increasing oxygen stress reduces pathogen survival rates. As a result of docking analysis, we predicted thioreductase inhibitors. In-depth betrixaban compounds had a binding energy of -9.9 kcal/mol, but their interaction pattern with active site residues was less. During simulation analysis, only 1040 hbonds were formed, with a significantly static and low RMSD pattern. Netrasudil had a binding score of -9.6 kcal/mol, which interacted with higher hydrophobic moieties at the active site. Simulation analysis revealed that the RMSD data was less flickering with 976.5 hbond interactions.\u003c/p\u003e \u003cp\u003eNovobiocin's -9.4 kcal/mol binding energy with a single hydrophobic moiety demonstrated promising interactions. In the simulation analysis, 945 hydrogen bonds were formed. NADPH (positive control) showed a strong interaction pattern during docking (-10.1 kcal/mol), and the RMSD flickered very little, whereas Metronidazole (negative control) showed a -4.5 kcal/mol binding score, which was lower than the above-mentioned compounds, and their simulation RMSD and hbond were very low. Overall, we demonstrated that Netrasudil has a good hydrophobic moiety and is very stable in simulation throughout the 100 ns run. Additionally, SMD showed that the only compounds that detached from the active site with higher energy (~\u0026thinsp;375 kJ/mol and ~\u0026thinsp;550 kJ/mol, respectively) were NADPH and Netrasudil.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eEvery year, amoebiasis causes almost 100,000 deaths worldwide and 50\u0026nbsp;million cases in tropical areas. E. histolytica thioredoxin reductase (EhTrR) plays a key role in metabolic functions for maintaining intracellular redox balance. These homeostasis cause parasite proliferation into the host. Thus, thioredoxin reductase inhibition is essential and currently unmet. The only medication on the market that interferes with the parasite's redox balance cycle and prevents infections is metronidazole. However, resistance and adverse effects on human health cannot be disregarded. Thus, in this study, we predicted three compounds that interacted strongly at the thioredoxin reductase pocket site. Netrasudil may interact and inhibit the thioredoxin reductase the best out of the three. Clinical trials and in vitro testing may be required prior to use.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Responsibilities\u003c/strong\u003e: This study does not directly involve humans or other organisms.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e: All authors consent to submit the manuscript to the journal.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e: All the data in supplementary material provided for extended validation or analysis produced during the docking phase can be made public after publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e: No funding was available to carry out this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e: Conceptualisation, data collection/curation, analysis, writing, reviewing the first draft, KS; Conceptualisation, data collection/curation, analysis, writing, reviewing and editing the first draft, APS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e: The author is greatly acknowledged and thankful to the Department of Zoology, RBS college, Agra and Maitreyi College, University of Delhi, New Delhi for providing the research facility and carrying out research work and other support.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eOnursal, A. and B. 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Reed, \u003cem\u003eNew drug target in protozoan parasites: the role of thioredoxin reductase.\u003c/em\u003e Front Microbiol, 2015. \u003cstrong\u003e6\u003c/strong\u003e: p. 975.\u003c/li\u003e\n\u003cli\u003eArias, D.G., et al., \u003cem\u003eEntamoeba histolytica thioredoxin reductase: molecular and functional characterization of its atypical properties.\u003c/em\u003e Biochim Biophys Acta, 2012. \u003cstrong\u003e1820\u003c/strong\u003e(12): p. 1859-66.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"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":"","lastPublishedDoi":"10.21203/rs.3.rs-6461033/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6461033/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAmoebiasis is an infectious disease caused by an intestinal parasitic protozon, Entamoeba histolytica. It is well known for invading and destroying human tissues, leading to life-threatening abscesses. To treatment of amoebiasis, and reduce the parasitic infection thioredoxin reductase (EhTrR), a promising target. It catalyzes the NADP-dependent reduction of amoebic thioredoxins, which is essential for maintaining intracellular redox balance. Therefore, in these studies, we selected 1150 FDA compounds and docked them with thioredoxin reductase. The best docked-score compounds were Betrixaban (-9.9 kcal/mol), Netrasudil (-9.6 kcal/mol), and Novobiocin (-9.4 kcal/mol), as well as positive control NADPH and negative control Metroindazole. Netrasudil is the best drug candidate for future use among these three compounds, according to the RMSD value, steered molecular dynamics, and interaction pattern. Before clinical trials and in vitro investigations, these substances might be used as thioreductase inhibitors.\u003c/p\u003e","manuscriptTitle":"Computational investigation of thiol-reductase inhibitors to overcome amebiasis using Docking and SMD-based simulation analysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-24 08:20:13","doi":"10.21203/rs.3.rs-6461033/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":"3e1f88d5-5e1b-4004-a656-4cce87410a08","owner":[],"postedDate":"April 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-22T21:53:16+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-24 08:20:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6461033","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6461033","identity":"rs-6461033","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}