Molecular Docking and Dynamic Simulation Studies of PHD2 Interactions with Gut Siderophores: Implications for HIF-1α Stabilization

Molecular Docking and Dynamic Simulation Studies of PHD2 Interactions with Gut Siderophores: Implications for HIF-1α Stabilization | 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 Article Molecular Docking and Dynamic Simulation Studies of PHD2 Interactions with Gut Siderophores: Implications for HIF-1α Stabilization Jainabbi Irshad Ahamed Patel, Namrata Manjunath Kurdekar, Apeksha Padakannaya, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3714812/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract In oxygen-deprived conditions cells respond by activating adaptive mechanisms to bolster their survival and protect tissue integrity. A key player in this process is the HIF-1α signaling cascade, meticulously regulated by Prolyl Hydroxylase Domain 2 (PHD2), which plays a central role in orchestrating cellular responses to fluctuating oxygen levels. The primary aim of this investigation is to explore potential PHD2 inhibitors using in-silico methods by employing molecular docking and dynamic simulation techniques. Low molecular-weight secondary metabolites known as Siderophores secreted from gut microbiota were selected and subjected for stringent assessment against PHD2. Molecular docking analysis revealed that Salmochelin SX (-9.527 Kcal/mol), Mycobactin (-9.166 Kcal/mol), Staphyloferrin A (-7.819 Kcal/mol), and Enterobactin (-7.302 Kcal/mol) displayed substantial affinities for the protein, suggesting them as potential inhibitors. Furthermore, Molecular Dynamic Simulation studies unveiled a noteworthy interaction between the metal ion, Fe 2+ , and the ligand molecules, indicating enhanced stability in these interactions. The iron-chelating property is a salient characteristic of many siderophores, and in the same lines our in-silico investigations have yielded promising results. However, it is essential to underscore that the validation of this study remains a critical step to substantiate our hypothesis. Further research and experimental investigations are necessary to confirm the practical implications of these findings and to assess the potential of the identified compounds as effective PHD2 inhibitors. Biological sciences/Computational biology and bioinformatics Biological sciences/Drug discovery Ischemia Hypoxia HIF-1α signaling Prolyl Hydroxylase Domain 2 Gut siderophores Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction In response to hypoxic microenvironments induced by ischemia, cells activate intricate adaptive pathways to cope with the limited oxygen availability. Among these pathways, the Hypoxia-Inducible Factor − 1 alpha (HIF-1) signaling cascade plays a pivotal role in mediating cellular responses to oxygen fluctuations during ischemia 1 . HIF-1 is a transcription factor composed of HIF-1α and HIF-1β subunits, whose stabilization and activation are regulated by Prolyl Hydroxylase Domain 2 (PHD2). Under normoxic conditions, PHD2 hydroxylates HIF-1α, marking it for proteasomal degradation and preventing the formation of the active HIF-1 complex 2 . However, PHD2 activity is reduced under hypoxic conditions leading to HIF-1α stabilization and translocation to the nucleus. Subsequently, HIF-1α dimerizes with HIF-1β, forming the active HIF-1 complex that initiates the transcription of various hypoxia-responsive genes 3 . PHD2 is a crucial enzyme that plays a pivotal role in regulating cellular responses to oxygen levels, a process known as oxygen sensing 4 . PHD2 belongs to the family of prolyl hydroxylases, which are essential components of cellular oxygen-sensing machinery. These enzymes are key players in the hypoxia-inducible factor (HIF) pathway, a signaling cascade that orchestrates adaptive responses to low oxygen conditions 4 , 5 . PHD2 specifically targets the HIF transcription factor, facilitating its degradation under normoxic (normal oxygen) conditions by hydroxylating specific proline residues. This hydroxylation marks HIF for recognition by the von Hippel-Lindau (VHL) protein, leading to its subsequent degradation by the ubiquitin-proteasome system 6 . The intricate interplay between PHD2, HIF, and VHL underscores the enzyme's significance in finely tuning cellular responses to oxygen availability. Understanding the molecular intricacies of PHD2 not only enhances our comprehension of fundamental cellular processes but also holds potential therapeutic implications, making it a promising target for the development of drugs aimed at modulating oxygen-sensing pathways in various physiological and pathological contexts 7 . The inhibition of Prolyl Hydroxylase Domain 2 (PHD2) has emerged as a compelling therapeutic strategy in various diseases characterized by aberrant oxygen sensing and HIF-mediated responses. Notably, in cancer, where the tumor microenvironment often experiences hypoxia, inhibiting PHD2 can stabilize HIF and promote the expression of genes involved in angiogenesis, glycolysis, and cell survival 8 . This adaptive response aids tumor progression and metastasis, making PHD2 an attractive target for anti-cancer therapies 9 . Additionally, in ischemic conditions such as myocardial infarction and stroke, PHD2 inhibition has shown promise in promoting angiogenesis and tissue repair by enhancing HIF-dependent pathways 10 . Moreover, in certain inflammatory and autoimmune disorders, PHD2 inhibition has demonstrated potential in modulating immune responses and mitigating tissue damage 11 . The versatility of PHD2 inhibitors across diverse pathological contexts underscores their therapeutic potential and positions them as promising candidates for the development of targeted interventions aimed at manipulating oxygen-sensing pathways to treat a spectrum of diseases. Nevertheless, the complexity of these pathways necessitates careful consideration of potential side effects and thorough understanding of the context-specific roles of PHD2 in different disease settings 12 . Ongoing research in this area holds the promise of uncovering new therapeutic avenues and refining strategies for PHD2 inhibition in the pursuit of improved patient outcomes. There are few natural regulators of PHD2, such as low oxygen concentration 13 , iron chelator 14 , and metabolite from TCA cycle 15 . The regulation of the PHD2 is necessary in a variety of physiological conditions, such as stem cell niche 16 and ischemic 17 , where HIF-1α stability is very essential to cellular adaptation to low oxygen conditions. Iron chelators have emerged as intriguing agents in the potential inhibition of PHD2 18 , primarily due to the role of iron as a crucial cofactor for the enzymatic activity of Prolyl Hydroxylases, including PHD2. These chelators function by sequestering intracellular iron, thereby limiting its availability for PHD2 catalysis. The interaction between iron and PHD2 is pivotal in the hydroxylation of proline residues on hypoxia-inducible factor (HIF), marking it for subsequent degradation under normoxic conditions 14 . In conditions where iron chelators are employed, such as in the treatment of certain iron overload disorders or as experimental tools in research, the reduction of available iron can lead to the stabilization of HIF, mimicking a hypoxic cellular response 19 . This stabilization occurs because iron chelation inhibits PHD2 activity, preventing the hydroxylation of proline residues on HIF. Consequently, the stabilized HIF can translocate to the nucleus and activate genes associated with adaptive responses to low oxygen levels 20 . Siderophores are one such iron chelators secreted by gut microbiota in human. Certain groups siderophores are having capacity to chelates the iron 21 . Siderophores are a fascinating class of small molecules produced by microorganisms, plants, and some fungi to facilitate the acquisition of essential iron from the environment 22 . Iron is a crucial micronutrient for various biological processes, including cellular respiration, DNA synthesis, and immune response. However, in many environments, iron is often present in insoluble and poorly bioavailable forms. Siderophores act as high-affinity iron chelators, binding to ferric iron (Fe 3+ ) with exceptional specificity and solubilizing it, making it accessible for cellular uptake 21 . In the context of PHD2 inhibition, siderophores could sequester extracellular iron, thereby limiting its availability for PHD2 catalysis. Since PHD2 requires iron for the hydroxylation of specific proline residues on hypoxia-inducible factor (HIF), a reduction in accessible iron could impede PHD2-mediated degradation of HIF under normoxic conditions. As a consequence, HIF stabilization would occur, mimicking a cellular response to low oxygen levels 23 – 25 . The potential use of siderophores as PHD2 inhibitors opens up new possibilities in therapeutic interventions, particularly in diseases where dysregulation of the hypoxia-inducible factor pathway plays a pivotal role. Given the intricate link between iron homeostasis and oxygen sensing, siderophores may offer a targeted approach to modulate PHD2 activity, influencing downstream cellular responses associated with HIF-mediated gene expression 26 , 27 . During ischemic conditions stable HIF-1α expression is necessary. The downstream targets of HIF-1α encompass a wide array of genes involved in vital physiological processes, including angiogenesis, glycolysis, erythropoiesis, and cell survival. HIF-1α-induced angiogenesis is crucial for promoting the formation of new blood vessels, enhancing oxygen supply to ischemic tissues, and facilitating tissue repair 28 , 29 . Additionally, HIF-1-driven glycolysis enables cells to adapt their metabolic pathways to generate energy efficiently even in oxygen-deprived conditions, supporting cellular survival 30 . Furthermore, HIF-1α stimulates erythropoiesis through the production of erythropoietin, which increases red blood cell production and improves oxygen-carrying capacity 3 , 31 . Given the central role of HIF-1 in cellular adaptation to ischemic conditions, the PHD2-HIF-1α axis emerges as a potential therapeutic target 3 , 32 . Modulating PHD2 activity could stabilize HIF-1α and enhance adaptive cellular responses during ischemia 33 . Our hypothesis revolves around the prospect of harnessing the capabilities of gut siderophores as inhibitors of Prolyl Hydroxylase Domain 2 (PHD2) (Fig. 1 ) in order to stabilize HIF-1α during ischemic conditions 17 . Through a comprehensive in-silico screening process and Molecular simulations, we anticipate the identification and modulation of potential PHD2 inhibitors that can facilitate the preservation of HIF-1α, thereby activating essential adaptive pathways crucial for cellular responses to changes in oxygen levels 34 , 35 . Methodology Protein Preparation The crystal structure of PHD2 (PDB ID: 4BQY) was retrieved from Protein Data Bank ( http://www.rcsb.org/pdb/home/home.do ) 36 . Water molecules and heteroatoms were removed from the active site for docking various compounds of interest. Schrodinger's Protein Preparation Wizard function was utilized to optimize and minimize protein structure using the OPLS3 forcefield 37 . The Protein Preparation Wizard fixes the protein and makes it suitable for docking. It corrects the incorrect bond orders, charge states, orientations of different amide and hydroxyl, and aromatic groups within a protein structure. The X-ray structure cannot determine due to limited resolution. Energy minimization was done by molecular mechanics calculation using OPLS3 available in the Protein Preparation tool to minimize the strains and steric collisions in protein. Ligand Preparation Schrodinger's LigPrep module was used to create a library of siderophore and other gut microbial metabolites. The compound was processed with Glide's LigPrep tool 38 , 39 , which generates accurate, and energy minimized 3-dimensional structures and applied sophisticated rules to correct the Lewis structure and eliminates mistakes in the ligand structures. Active site and Grid generation The active site was generated using the receptor grid generation module of Schrodinger. The grid enclosed the active site of the PHD2, which includes key factors such as ARG322 and Fe 2+ . The grid size was about 20X20X20 Å which can accommodate the active site of the protein to allow each ligand to search for the potential binding. Virtual Screening, Molecular Docking, and MM/GBSA calculations Protein-ligand docking studies were conducted using the optimized structure of Protein. To evaluate docking efficiency, we employed Molecular Mechanics-Generalized Born Surface Area. (MM/GBSA) study 40 – 42 . The Prime MM/GBSA calculates the binding free energy (G bind ) of the siderophores, protein, and complex with Protein using the MM/GBSA method. Virtual Screening Workflow performs docking of an extensive collection of compounds against one target. The docking interactions were compared with the standard drug. Molecular Dynamic Simulations The best-ranked siderophores were selected for Molecular dynamics simulation using Desmond 2020.1 by D. E. Shaw Research 43 . In total, four siderophores, along with the enzyme, were simulated. System setup includes TIP3P 44 , three site water model with a cubic box with a 10Å buffer region between enzyme-ligand complex, and a system neutralized using appropriate numbers of counter ions (Na + and Cl − ) with a fixed salt concentration of 0.15 M. OPLS3e force field was employed to simulate computations. Isothermal-isobaric (NPT) ensemble was used with temperature and pressure adjusted to 300K and 1.01325 bar, respectively. A simulation time of 200ns was adjusted, whereas trajectories were saved at every 100ps. Simulation event analysis, simulation quality analysis, and simulation interaction diagram protocols of the Desmond package were exercised to analyze the trajectory files. Results PHD2, also known as prolyl hydroxylase domain 2, plays a crucial role in both normal and disease physiology. In normal physiology, PHD2 is involved in the regulation of cellular responses to oxygen levels, primarily through its interaction with hypoxia-inducible factors (HIFs) 45 . By hydroxylating specific proline residues on HIFs, PHD2 targets them for degradation under normoxic conditions, preventing their accumulation and subsequent activation of hypoxia-responsive genes 2 . This process ensures proper oxygen sensing and adaptation in various tissues and is vital for maintaining homeostasis 9 . Intermolecular interactions are essential in drug discovery as they govern the binding affinity and specificity of drug compounds to their target proteins. These interactions include hydrogen bonding, pi-pi cation, pi-pi stacking, and van der Waals forces of attraction. Hydrogen bonding stabilizes drug-protein complexes, while pi-pi cation and pi-pi stacking interactions contribute to altered pharmacological effects 46 . Van der Waals forces, though weak individually, collectively play a significant role in the overall binding process. Understanding these interactions aids in rational drug design, identifying new drug targets, and predicting pharmacokinetics and safety 47 . Molecular Docking Studies To identify suitable lead molecules, docking studies were performed with a natural product library, using Glide to validate the hypothesis. The HIF-1α -binding site of PHD2 contains key residues, including ARG322 and Fe 2+ , which play an active role in substrate binding through hydrogen bonding interactions. Specifically, the positively charged amino acid ARG322 interacts with PRO564 of the HIF-1α protein within pocket one of PHD2, making it a critical residue in the enzyme's active site and facilitating the interaction with the HIF-1α hydroxylation site, PRO564 48 . Table 1 presents the binding scores and affinities of compounds Salmochelin SX, Mycobactin, Staphyloferrin A, and Enterobactin towards PHD2. Among these compounds, SALMOCHELIN SX exhibits the highest affinity with a binding score of -9.527 Kcal/mol (MM/GBSA score: -42.58 Kcal/mol). It engages in crucial interactions with the target protein, forming hydrogen bonds with ASP254, TYR310, and ASP315, while accepting hydrogen bonds from TYR303 and ARG322. Additionally, Salmochelin SX coordinates with Fe 2+ , contributing significantly to its interaction with the key residue ARG322 and the co-factor Fe 2+ . This coordination of Fe 2+ is vital for stabilizing the ligand-protein complex and plays a pivotal role in determining Salmochelin SX's strong affinity towards PHD2. Table 1 Prioritized Compounds List Compound ID Docking score (Kcal/mol) MM/GBSA (Kcal/mol) Interacting Amino acid residue and cofactor (Fe 2+ ) Interacting Amino Acids residue and co-factors during MD Simulation Amino Acids bridged to Ferrous ion during simulation Salmochelin SX -9.527 -42.58 ASP254, TYR303, TYR310 , ASP315, ARG322 , Fe 2+ ASP254 , ARG383 HIS313, ASP315, HIS374 Mycobactin -9.166 -34.25 VAL314, ARG322 , Fe 2+ ASP254 , THR387 HIS313, ASP315, HIS374 Staphyloferrin A -7.819 -25.74 TYR310 , ASP315, ARG322 , Fe 2+ ASP254 , ARG383, THR387 HIS313, ASP315, HIS374 Enterobactin -7.302 -38.16 TYR310, ARG322 ASP254 , TYR310, ASP320, TRP389 HIS313, ASP315, HIS374 Salmochelin SX showed interaction with ASP315, ASP254, TYR303 and ARG322 residue. The carboxyl group of compound forms hydrogen bonds with a distance of 1.910 Å with TYR310 respectively. The oxan ring of 3,4,5-trihydroxy-6-(hydroxymethyl) oxan-2-yl] phenyl} formamido) accepts the hydrogen bond from TYR303 (2.178 Å). And the hydroxypropionic group donates hydrogen bond to ASP315 and accepts hydrogen bond from key residue ARG322 with bond distance (2.028 Å) (Table 2 ). The negatively charged ASP254 forms a two-hydrogen bond (1.985 Å and 2.392 Å) with the hydroxyl group of the phenyl ring and Fe 2+ interaction with the OH group. In the same way, we tested the interactions of other lead molecules (Fig. 2 (a)). Table 2 Hydrogen Bond distance of Salmochelin SX Interaction H Bond distance (Å) PHD2 TYR303: HH - D1:O1 2.178 PHD2 ARG322:HH12 - D1:O9 2.028 PHD2 ARG322:HH22 - D1:O9 2.237 PHD2: ARG383:HH12 - D1:O5 1.190 D1:H44 - PHD2: ASP254:OD2 1.985 D1:H45 - PHD2: ASP254:OD2 2.392 D1:H49 - PHD2: TYR310: OH 1.910 Next, Mycobactin shows an affinity towards PHD2 with a binding score of -9.166 Kcal/mol. Like Salmochelin SX, it forms multiple interactions with the target protein, donating a hydrogen bond to ARG322 (Fig. 3 (a)) and VAL314 donating and accepting hydrogen bonds from the compound. Furthermore, Mycobactin coordinates with Fe 2+ , which is crucial for its favorable association with the key residue ARG322 and the co-factor Fe 2+ . The coordination of Fe 2+ enhances Mycobactin 's binding to PHD2, contributing to its significant affinity. Table 3 Hydrogen Bond distance of Aerobactin Interaction H Bond distance (Å) PHD2:VAL314:H - Mycobactin: O10 1.972 PHD2:ARG322: HH12 - Mycobactin: O6 1.860 Mycobactin:H75 - PHD2:VAL314:O 1.794 The two carboxyl groups of Staphyloferrin A form hydrogen bonds with ASP315 with distances (2.242 Å and 1.480 Å) and one of them, accepts hydrogen bond from key residue ARG322 (2.089Å). Similarly, at 2.182 Å TYR310 (Fig. 4 (a) forms an interaction with the hydroxyl group of Staphyloferrin A (Table 4 ). There is also metal coordination with the carboxyl group. Finally, Enterobactin exhibits an affinity towards PHD2 with a binding score of -7.302 Kcal/mol. It forms bonds with the target protein by donating a hydrogen bond (Table 5 ) to TYR310 and engaging in a pi-cation, noncovalent interaction with the key residue ARG322(Fig. 5 (a)). Table 4 H Bond distance of Staphyloferrin A Interaction H Bond distance (Å) PHD2: TYR310: HH - Staphyloferrin A: O3 2.182 PHD2: ARG322:HH12 - Staphyloferrin A: O9 2.089 Staphyloferrin A:H51 - PHD2: TYR310: OH 1.878 Staphyloferrin A:H54 - PHD2: ASP315:O 2.242 Staphyloferrin A:H56 - PHD2: ASP315:OD1 1.480 Table 5 Hydrogen Bond distance of Enterobactin Interaction H Bond distance (Å) PHD2: TYR310: HH - Enterobactin:O11 2.174 Enterobactin:H70 - PHD2: TYR310: OH 1.699 Enterobactin:H73 - PHD2: TYR310: OH 2.170 The interaction analysis revealed that Salmochelin SX forms a variety of bonds with the target protein, including donating hydrogen bonds to ASP254, TYR310, and ASP315, while accepting hydrogen bonds from TYR303 and ARG322. Moreover, Salmochelin SX coordinates with Fe 2+ . These intricate bonds are crucial as they play a vital role in the compound's association with the key residue ARG322 and the co-factor Fe 2+ within PHD2's active site. This strong and specific binding can modulate PHD2's enzymatic activity, potentially leading to therapeutic effects in the context of diseases associated with oxygen sensing and HIF regulation. Comparing the results to the Salmochelin SX, Aerobactin, Staphyloferrin A, and Enterobactin also exhibited significant affinities towards PHD2, albeit with slightly lower binding scores compared to Salmochelin SX. Aerobactin, like Salmochelin SX, forms multiple hydrogen bonds (Tables 2 & 3 ) with the target protein, while Staphyloferrin A (Table 4 ) and Enterobactin (Table 5 ) donate hydrogen bonds (Table 3 ) and engage in pi-cation, non-covalent interactions, respectively, with key residue ARG322. Molecular Dynamic Simulation The MD simulation revealed stable conformational changes in the target proteins upon ligand binding (Fig. 2 (b) and (c). During the MD simulation of Salmochelin SX and PHD2 complex, the RMSD of alpha carbon and Salmochelin SX initially fluctuated but stabilized after 30 ns, with a final RMSD change of less than 3 Å. The Root Mean Square Fluctuation (RMSF) of the protein-ligand complex showed fluctuations only in the loop regions, a common occurrence during MD simulations (Fig. 2 (d)). Ligand contact had no significant impact on protein RMSF, even in the beta-pleated sheets. The simulation interaction fraction graph represented the strength of bonded interactions, with 40 bonds observed. Only a few bonds, like ASP254, HIS313, ASP315, HIS374, and ARG383 (Fig. 2 (e)), remained in contact for more than 30% of the simulation time. ARG254 had the highest interaction, forming hydrogen bonds and water bridges, while ARG383 had similar interactions. The PHD2 enzyme's co-factor, Fe 2+ , interacted with histidine and aspartate, specifically HIS313, ASP315, and HIS374. This interaction was not present during molecular docking but formed during MD simulations. This interaction is crucial for inhibiting PHD2, as the ferrous ion plays a pivotal role in oxygen-mediated oxidoreductase reactions. Mycobactin displayed similar results to the Salmochelin SX, with changed interactions (Fig. 3 (b) compared to its docking results (Fig. 3 (a)). RMSD values for the alpha carbon and ligands staying below 3 Å (Fig. 3 (c). During the simulation, RMSF for the protein primarily fluctuated in the loop regions, as is typically observed in loop regions (Fig. 3 (d)). Ligand interactions with the protein did not induce significant fluctuations. The Ligand contact graph, showing interaction fractions, revealed numerous interactions (Fig. 3 (e)). Some of these interactions persisted with the protein for over 30% of the simulation time. In the two-dimensional pose view, ASP254, TYR310, and ARG383 formed hydrogen bonds with the protein-ligand, which had the highest interaction fraction scores (Fig. 3 (e)). The ferrous ion's connection with HIS313, ASP315, and HIS374 played a substantial role in the ligand interaction (Fig. 3 (b). These ionic interactions were nearly as significant as hydrogen bonding in terms of interaction strength. This interaction is crucial, and inhibiting the ferrous ion may reduce PHD2 enzyme activity. Furthermore, a few water bridges formed during the simulation, involving THR236 and THR387. The few interactions found during the docking studies were missing and additional interactions were observed. This may be due to the structural changes undergoes during MD simulation. Further, Staphyloferrin A and Enterobactin have similar results like Salmochelin SX and Mycobactin in the case of protein and ligand RMSD (Figs. 4 & 5 (c) and RMSF (Fig. 4 & 5 (d). The post MD simulation interaction pose (Fig. 4 (b) & 5(b)) showed changes in interaction compared to docking pose (Fig. 4 (a) & 5(a)). RMSF results both siderophores (Staphyloferrin A and Enterobactin) was found changes in only loop regions of the PHD2(Fig. 4 (d) & 5(d)). In Staphyloferrin A and PHD2 complex, ARG254, ARG383, and THR387 were formed hydrogen bonds (Fig. 4 (e)). Ferrous ion showed the same interaction with Staphyloferrin A (Fig. 4 (b)) as Salmochelin SX and Mycobactin. In this case water bridge observed with ASP254 and ASP325(Fig. 4 (b)&(e)). Enterobactin was found stable throughout the simulations. The interaction of residues was changed during simulation compared to docked pose (Fig. 5 (b)). Further, RMSD (Fig. 5 (c)), RMSF (Fig. 5 (d) were found stable and there are no changes were observed. Enterobactin showed ASP254, TYR310, ASP320 hydrogen bonded (Fig. 5 (b)(e)) to the protein for more than 30% of simulation time and TYR389 showed pi-pi cationic interaction. In a molecular dynamic simulation, we studied four compounds interacting with the PHD2 protein. These simulations showed that the protein and compounds changed their shapes but eventually stabilized. The compounds interacted with the protein without causing major fluctuations. We found specific interactions, like hydrogen bonds, that were crucial for inhibiting PHD2. These results differed from initial predictions using molecular docking, highlighting the importance of dynamic simulations in understanding how drugs interact with proteins. Discussion Human organs undergo various physiological conditions such as ischemia, where flow of blood is reduced and subsequently lacks oxygen supply to tissues or organs 49 , 50 . In this context, this study was conducted to identify potential inhibitors of PHD2, an enzyme involved in the regulation of Hypoxia-Inducible Factor (HIF), could be a potential strategy to mitigate the effects of ischemia 51 , 52 . HIF is a transcription factor that plays a key role in cellular responses to low oxygen levels, and its stabilization can promote cellular adaptation and survival under hypoxic conditions 28 . An in-silico analysis of selected gut microbial siderophores was done to screen and identify molecules that may have the ability to inhibit PHD2. Results showed that, among 103 molecules, four lead molecules had interactions with key residues. First, Salmochelin SX exhibits the highest affinity with a binding score of -9.527 Kcal/mol. It engages in crucial interactions with the target protein, forming hydrogen bonds with ASP254, TYR310, and ASP315, while accepting hydrogen bonds from TYR303 and ARG322. Additionally, Salmochelin SX coordinates with Fe 2+ , contributing significantly to its interaction with the key residue ARG322 and the co-factor Fe 2+ . This coordination of Fe 2+ is vital for stabilizing the ligand-protein complex and plays a pivotal role in determining Salmochelin SX's strong affinity towards PHD2. Salmochelin SX, secreted by Salmonella enterica characterized as a catecholate-type siderophore 53 , exhibits promising attributes that may contribute to mitigating oxidative stress during ischemic reperfusion. In the intricate milieu of cellular responses to oxidative challenges, particularly in the context of the Fenton reaction where hydrogen peroxide (H2O2) interacts with intracellular iron sulfur clusters, leading to the generation of harmful free radicals like hydroxyl radicals, Salmochelin SX emerges as a potential protective agent 54 . During ischemic reperfusion, when tissues experience a restoration of blood flow, the potential for reactive oxygen species (ROS) generation is heightened 50 . Salmochelin SX, with its catecholate nature, could play a pivotal role in reducing ROS production. Recent findings have highlighted the capacity of catecholate siderophores, including Salmochelin SX, to act as defenders against oxidative stress, offering a positive perspective on its potential applications. Even though, Salmochelin SX part of pathogenic microbial strain, it may have therapeutic application where proper balance of gut microbiota can regulate the level of the pathogenicity of the certain bacterial strains 55 , 56 . The catecholate-type siderophore Enterobactin, previously examined for its anticancer properties, showcased cytotoxic effects on two monocyte tumor cells while leaving bone marrow–derived macrophages unharmed in a study by Saha et al. (2019) 57 . The investigation unveiled a significant increase in the intracellular labile iron pool in both cell lines, indicative of a disruption in iron homeostasis induced by the siderophore. Additionally, Enterobactin demonstrated a dose-dependent inhibition of the generation of reactive oxygen species (ROS) by mitochondria. These combined effects, involving alterations in iron levels and the attenuation of ROS production, have the potential to induce apoptosis in cancer cells 57 . Mycobacterium smegmatis produces the mycobactin, which has strong antiproliferative action against leukemia, breast, and liver cancer cell lines 58 . Mycobactin was showing interaction with TYR310, ASP254, ARG322, ASN318 residues. The hydroxyl group of compound accepts hydrogen bond from TYR310 with bond distance (2.010 Å) and simultaneously it donates hydrogen bond to ASP254. carbonyl group of compound forms hydrogen bond interaction with ASN318 (2.031 Å). And carboxyl group of Mycobactin accepts hydrogen bond from key residue ARG322 with a bond distance of 2.037Å. And also forms metal coordination Fe 2+ with carboxyl and hydroxyl groups. Similarly, Staphyloferrin A demonstrates an affinity towards PHD2 with a binding score of -7.819 Kcal/mol. It establishes bonds with the target protein, donating a hydrogen bond to TYR310 and ASP315, while accepting hydrogen bonds from TYR310 and ARG322. Like Salmochelin SX and Mycobactin, Staphyloferrin A also coordinates with Fe 2+ , and this coordination significantly influences its interaction with the key residue ARG322 and the co-factor Fe 2+ . The coordination with Fe 2+ is a critical determinant in Staphyloferrin A's affinity towards PHD2. Similar to the other compounds, Enterobactin also coordinates with Fe 2+ , contributing significantly to its interaction with ARG322 and Fe 2+ . The coordination of Fe 2+ plays a vital role in stabilizing Enterobactin 's binding to PHD2. In Enterobactin, the hydroxyl group of the benzene ring forms a hydrogen bond interaction with TYR310, with a bond distance of 1.699 Å. It also has a Pi-cation interaction, i.e., a noncovalent bond with key residue ARG322. In this study, ARG322 most often interacts with the carboxyl group of lead molecules. This kind of observation in reported studies, where small molecules are able to inhibit the activity of PHD2 48 . The coordination and interaction between ARG322 and Fe 2+ within the substrate-binding site are crucial for PHD2's activity and its role in the oxygen-sensing pathway 59 . Understanding the molecular details of these interactions can provide valuable insights for the development of potential therapeutic strategies targeting PHD2 to modulate cellular responses under hypoxic conditions. By manipulating PHD2's function, researchers may pave the way for novel approaches in treating various diseases related to oxygen sensing and HIF regulation. Exposure to iron chelators or cobalt ions can mimic the effects of hypoxia on HIF, which is easily explained by the known characteristics of 2-oxoglutarate-dependent oxygenases 60 . For these observations, there have previously been two proposed interpretations. First, it has been suggested that cobalt ions could take the place of ferrous ions at an iron core that senses oxygen. It was suggested that such a protein must rotate quickly because the majority of iron centers (such as heme and the vast majority of iron-sulfur clusters) do not swap in this manner. Second, it has been suggested that cobaltous ions and iron chelators affect signaling by reactive oxygen species and Fenton chemistry in nonenzymatic "metal catalyzed oxidation" systems 61 . In all cases, the coordination of Fe 2+ is significant for the interactions between the lead compounds and PHD2. This coordination contributes significantly to the binding of each compound with the key residue ARG322 and the co-factor Fe 2+ . These interactions play a crucial role in stabilizing the ligand-protein complexes and influencing the compounds' overall affinities towards PHD2. Furthermore, the coordination with Fe 2+ is known to be important in regulating the enzymatic activity of PHD2 62 , making it a crucial aspect to consider in the design of potential therapeutic agents targeting PHD2 and related pathways involved in oxygen sensing and HIF regulation 63 , 64 . The interactions observed in all four compounds highlight the importance of hydrogen bonds and metal coordination in mediating their binding to PHD2. These interactions are well-known determinants of ligand-protein binding specificity and play a crucial role in the selectivity of lead compounds in drug discovery. Conclusion By inhibiting PHD2, our goal is to increase HIF stabilization, activating genes that promote cell survival, angiogenesis, and metabolism under low oxygen conditions. While in silico screening can narrow down potential candidates, further experimental validation is necessary. This involves in vitro assays and cellular studies to confirm the inhibitory activity and specificity of identified compounds against PHD2. The study's findings show significant promise for developing therapeutic agents targeting the PHD2-HIF-1α axis, enhancing adaptive responses during ischemic events. Understanding HIF-1 and PHD2 interactions in various ischemic diseases provides valuable insights for designing pharmacological interventions. This could mitigate detrimental effects of oxygen deprivation and improve patient outcomes, addressing unmet clinical needs in managing ischemic diseases. This approach, integrating computational screening with experimental validation, underscores the potential translational impact in developing effective therapies for ischemic conditions. Declarations Author Contribution JIP: Methodology, formal validation, initial manuscript draft, NK: Visualization, AP: Visualization, English editing, PKJ: manuscript corrections, ASK: support in draft preparation, CGJ: supervision, review and editing, RKM: review and editing, KM: software, writing, JP: Conceptualization, Methodology, writing-review and editing, project administration, supervision, and editing manuscript. Acknowledgment The author expresses gratitude to Google Inc. for generously providing Google Research Cloud Credit (EDU Credit - wilsonjessica − 209474439) and to the SDM Research Institute for Biomedical Sciences and Shri Dharmasthala Manjunatheshwara University, and Department of Biochemistry and Industrial Chemistry, Mangalore University for their facility. Data Availability All data generated or analysed during this study are included in this manuscript. References Majmundar, A. J., Wong, W. J. & Simon, M. C. Hypoxia-Inducible Factors and the Response to Hypoxic Stress. Molecular Cell 40, 294–309 (2010). Weidemann, A. & Johnson, R. S. Biology of HIF-1a. Cell Death and Differentiation 15, 621–627 (2008). Luo, Z. et al. Hypoxia signaling in human health and diseases: implications and prospects for therapeutics. Sig Transduct Target Ther 7, 218 (2022). McDonough, M. A. et al. Cellular oxygen sensing: Crystal structure of hypoxia-inducible factor prolyl hydroxylase (PHD2). Proc Natl Acad Sci U S A 103, 9814–9819 (2006). Semenza, G. L. Oxygen sensing, hypoxia-inducible factors, and disease pathophysiology. Annu Rev Pathol 9, 47–71 (2014). Groulx, I. & Lee, S. Oxygen-dependent ubiquitination and degradation of hypoxia-inducible factor requires nuclear-cytoplasmic trafficking of the von Hippel-Lindau tumor suppressor protein. Mol Cell Biol 22, 5319–5336 (2002). Zhao, Y. et al. Hypoxia-induced signaling in the cardiovascular system: pathogenesis and therapeutic targets. Sig Transduct Target Ther 8, 431 (2023). Berra, E., Roux, D., Richard, D. E. & Pouysségur, J. Hypoxia-inducible factor-1α (HIF-1α) escapes O(2)-driven proteasomal degradation irrespective of its subcellular localization: nucleus or cytoplasm. EMBO Reports 2, 615–620 (2001). Poon, E., Harris, A. L. & Ashcroft, M. Targeting the hypoxia-inducible factor (HIF) pathway in cancer. Expert Reviews in Molecular Medicine 11, null-null (2009). Bereczki, D., Balla, J. & Bereczki, D. Heme Oxygenase-1: Clinical Relevance in Ischemic Stroke. Curr Pharm Des 24, 2229–2235 (2018). Pålsson-McDermott, E. M. & O’Neill, L. A. J. Targeting immunometabolism as an anti-inflammatory strategy. Cell Res 30, 300–314 (2020). Ogle, M. E., Gu, X., Espinera, A. R. & Wei, L. Inhibition of prolyl hydroxylases by dimethyloxaloylglycine after stroke reduces ischemic brain injury and requires hypoxia inducible factor-1α. Neurobiology of Disease 45, 733–742 (2012). Suhara, T. et al. Inhibition of the oxygen sensor PHD2 in the liver improves survival in lactic acidosis by activating the Cori cycle. Proc. Natl. Acad. Sci. U.S.A. 112, 11642–11647 (2015). Flagg, S. C., Martin, C. B., Taabazuing, C. Y., Holmes, B. E. & Knapp, M. J. Screening chelating inhibitors of HIF-prolyl hydroxylase domain 2 (PHD2) and factor inhibiting HIF (FIH). Journal of Inorganic Biochemistry 113, 25–30 (2012). Abboud, M. I. et al. 2-Oxoglutarate regulates binding of hydroxylated hypoxia-inducible factor to prolyl hydroxylase domain 2. Chem Commun (Camb) 54, 3130–3133 (2018). Foulks, J. M. et al. PAF-acetylhydrolase expressed during megakaryocyte differentiation inactivates PAF-like lipids. Blood 113, 6699–6706 (2009). DeSai, C. & Hays Shapshak, A. Cerebral Ischemia. in StatPearls (StatPearls Publishing, 2022). Speer, R. E. et al. Hypoxia-inducible factor prolyl hydroxylases as targets for neuroprotection by “antioxidant” metal chelators: From ferroptosis to stroke. Free Radical Biology and Medicine 62, 26–36 (2013). Bento, C. F. & Pereira, P. Regulation of hypoxia-inducible factor 1 and the loss of the cellular response to hypoxia in diabetes. Diabetologia 54, 1946–1956 (2011). Haase, V. H. The VHL tumor suppressor: master regulator of HIF. Curr Pharm Des 15, 3895–3903 (2009). Ellermann, M. & Arthur, J. C. Siderophore-mediated iron acquisition and modulation of host-bacterial interactions. Free Radic Biol Med 105, 68–78 (2017). Khasheii, B., Mahmoodi, P. & Mohammadzadeh, A. Siderophores: Importance in bacterial pathogenesis and applications in medicine and industry. Microbiological Research 250, 126790 (2021). Siegert, I. et al. Ferritin-Mediated Iron Sequestration Stabilizes Hypoxia-Inducible Factor-1α upon LPS Activation in the Presence of Ample Oxygen. Cell Reports 13, 2048–2055 (2015). Dev, S. et al. Role of extracellular Hydrogen peroxide in regulation of iron homeostasis genes in neuronal cells: Implication in iron accumulation. Free Radical Biology and Medicine 86, 78–89 (2015). Minervini, G., Quaglia, F. & Tosatto, S. C. E. Insights into the proline hydroxylase (PHD) family, molecular evolution and its impact on human health. Biochimie 116, 114–124 (2015). Chen, Z., Han, F., Du, Y., Shi, H. & Zhou, W. Hypoxic microenvironment in cancer: molecular mechanisms and therapeutic interventions. Sig Transduct Target Ther 8, 70 (2023). Barthels, D. & Das, H. Current advances in ischemic stroke research and therapies. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease 1866, 165260 (2020). Semenza, G. L. HIF-1: mediator of physiological and pathophysiological responses to hypoxia. Journal of Applied Physiology 88, 1474 (2000). Rajendran, G. et al. Inhibition of Endothelial PHD2 Suppresses Post-Ischemic Kidney Inflammation through Hypoxia-Inducible Factor-1. JASN 31, 501–516 (2020). Semenza, G. L. Hypoxia-Inducible Factors in Physiology and Medicine. Cell 148, 399–408 (2012). Avivi, A., Resnick, M. B., Nevo, E., Joel, A. & Levy, A. P. Adaptive hypoxic tolerance in the subterranean mole rat Spalax ehrenbergi: the role of vascular endothelial growth factor. FEBS Letters 452, 133–140. Poyya, J., Joshi, C. G., Kumar, D. J. & Nagendra, H. Sequence Analysis and Phylogenetic Studies of Hypoxia-Inducible Factor-1α. Cancer Informatics 16, 117693511771224 (2017). Leite de Oliveira, R. et al. Gene-Targeting of Phd2 Improves Tumor Response to Chemotherapy and Prevents Side-Toxicity. Cancer Cell 22, 263–277 (2012). Ziello, J. E., Jovin, I. S. & Huang, Y. Hypoxia-Inducible Factor (HIF)-1 regulatory pathway and its potential for therapeutic intervention in malignancy and ischemia. Yale J Biol Med 80, 51–60 (2007). Watts, E. R. & Walmsley, S. R. Inflammation and Hypoxia: HIF and PHD Isoform Selectivity. Trends in Molecular Medicine 25, 33–46 (2019). Berman, H. M. The Protein Data Bank. Nucleic Acids Research 28, 235–242 (2000). Harder, E. et al. OPLS3: A Force Field Providing Broad Coverage of Drug-like Small Molecules and Proteins. Journal of Chemical Theory and Computation 12, 281–296 (2016). Friesner, R. A. et al. Extra Precision Glide: Docking and Scoring Incorporating a Model of Hydrophobic Enclosure for Protein – Ligand Complexes. Journal of Medicinal Chemistry 49, 6177–6196 (2006). Halgren, T. A. et al. Glide: A New Approach for Rapid, Accurate Docking and Scoring. 2. Enrichment Factors in Database Screening. Journal of Medicinal Chemistry 47, 1750–1759 (2004). Godschalk, F., Genheden, S., Söderhjelm, P. & Ryde, U. Comparison of MM/GBSA calculations based on explicit and implicit solvent simulations. Phys. Chem. Chem. Phys. 15, 7731 (2013). Hou, T., Wang, J., Li, Y. & Wang, W. Assessing the performance of the molecular mechanics/Poisson Boltzmann surface area and molecular mechanics/generalized Born surface area methods. II. The accuracy of ranking poses generated from docking. J Comput Chem 32, 866–877 (2011). Poyya, J. et al. Receptor based virtual screening of potential novel inhibitors of tigar [TP53 (tumour protein 53)-induced glycolysis and apoptosis regulator. Medical Hypotheses 110683 (2021) doi: 10.1016/j.mehy.2021.110683 . Shaw, D. E. Desmond Maestro 2020 by DE Shaw Research. (2021). Mark, P. & Nilsson, L. Structure and Dynamics of the TIP3P, SPC, and SPC/E Water Models at 298 K. J. Phys. Chem. A 105, 9954–9960 (2001). Fong, G.-H. & Takeda, K. Role and regulation of prolyl hydroxylase domain proteins. Cell Death Differ 15, 635–641 (2008). Liang, Z. & Li, Q. X. π-Cation Interactions in Molecular Recognition: Perspectives on Pharmaceuticals and Pesticides. J Agric Food Chem 66, 3315–3323 (2018). Bissantz, C., Kuhn, B. & Stahl, M. A medicinal chemist’s guide to molecular interactions. J Med Chem 53, 5061–5084 (2010). Chowdhury, R. et al. Selective Small Molecule Probes for the Hypoxia Inducible Factor (HIF) Prolyl Hydroxylases. ACS Chem. Biol. 8, 1488–1496 (2013). Flanigan, W. R. & Jain, I. H. The Goldilocks Oxygen Principle: not too little and not too much. Nat Cardiovasc Res 1, 1101–1103 (2022). Kalogeris, T., Baines, C. P., Krenz, M. & Korthuis, R. J. Cell Biology of Ischemia/Reperfusion Injury. in International Review of Cell and Molecular Biology vol. 298 229–317 (Elsevier, 2012). Loor, G. & Schumacker, P. T. Role of hypoxia-inducible factor in cell survival during myocardial ischemia–reperfusion. Cell Death Differ 15, 686–690 (2008). Conde, E. et al. HIF-1α induction during reperfusion avoids maladaptive repair after renal ischemia/reperfusion involving miR127-3p. Sci Rep 7, 41099 (2017). Mohsen, Y. et al. The Different Types of Metallophores Produced by Salmonella enterica: A Review. Microbiology Research 14, 1457–1469 (2023). Winterbourn, C. C., Kettle, A. J. & Hampton, M. B. Reactive Oxygen Species and Neutrophil Function. Annu. Rev. Biochem. 85, 765–792 (2016). Hou, K. et al. Microbiota in health and diseases. Sig Transduct Target Ther 7, 135 (2022). Rolhion, N. & Chassaing, B. When pathogenic bacteria meet the intestinal microbiota. Philos Trans R Soc Lond B Biol Sci 371, 20150504 (2016). Saha, P. et al. Enterobactin, an iron chelating bacterial siderophore, arrests cancer cell proliferation. Biochemical Pharmacology 168, 71–81 (2019). Gokarn, K., Sarangdhar, V. & Pal, R. B. Effect of microbial siderophores on mammalian non-malignant and malignant cell lines. BMC Complement Altern Med 17, 145 (2017). Koivunen, P., Hirsilä, M., Günzler, V., Kivirikko, K. I. & Myllyharju, J. Catalytic properties of the asparaginyl hydroxylase (FIH) in the oxygen sensing pathway are distinct from those of its prolyl 4-hydroxylases. J Biol Chem 279, 9899–9904 (2004). Tian, Y.-M. et al. Differential sensitivity of hypoxia inducible factor hydroxylation sites to hypoxia and hydroxylase inhibitors. J Biol Chem 286, 13041–13051 (2011). Jaakkola, P. et al. Targeting of HIF-α to the von Hippel-Lindau Ubiquitylation Complex by O 2 -Regulated Prolyl Hydroxylation. Science 292, 468–472 (2001). Chowdhury, R. et al. Structural basis for oxygen degradation domain selectivity of the HIF prolyl hydroxylases. Nat Commun 7, 12673 (2016). Schofield, C. J. & Ratcliffe, P. J. Oxygen sensing by HIF hydroxylases. Nat Rev Mol Cell Biol 5, 343–354 (2004). Eltzschig, H. K., Bratton, D. L. & Colgan, S. P. Targeting hypoxia signalling for the treatment of ischaemic and inflammatory diseases. Nat Rev Drug Discov 13, 852–869 (2014). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 21 Dec, 2023 Reviewers invited by journal 21 Dec, 2023 Editor assigned by journal 21 Dec, 2023 Editor invited by journal 11 Dec, 2023 Submission checks completed at journal 11 Dec, 2023 First submitted to journal 06 Dec, 2023 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-3714812","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":262877980,"identity":"3d718c71-864c-49f4-8ae6-b587dda89d71","order_by":0,"name":"Jainabbi Irshad Ahamed Patel","email":"","orcid":"","institution":"SDM Research Institute for Biomedical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jainabbi","middleName":"Irshad Ahamed","lastName":"Patel","suffix":""},{"id":262877981,"identity":"466d3d36-14e6-4af0-b70d-b53025e081b0","order_by":1,"name":"Namrata Manjunath Kurdekar","email":"","orcid":"","institution":"SDM Research Institute for Biomedical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Namrata","middleName":"Manjunath","lastName":"Kurdekar","suffix":""},{"id":262877982,"identity":"d4e7a7fa-0ce1-4298-8e43-0dbe9b893d01","order_by":2,"name":"Apeksha Padakannaya","email":"","orcid":"","institution":"SDM Research Institute for Biomedical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Apeksha","middleName":"","lastName":"Padakannaya","suffix":""},{"id":262877984,"identity":"78ae498a-e5f3-4fbf-830e-8fb0822e94cb","order_by":3,"name":"Palaksha Kanive Javaregowda","email":"","orcid":"","institution":"SDM Research Institute for Biomedical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Palaksha","middleName":"Kanive","lastName":"Javaregowda","suffix":""},{"id":262877985,"identity":"a04106d3-205e-47c9-af55-2972c2283f88","order_by":4,"name":"Ajay Sathayanarayan Khandagale","email":"","orcid":"","institution":"SDM Research Institute for Biomedical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ajay","middleName":"Sathayanarayan","lastName":"Khandagale","suffix":""},{"id":262877986,"identity":"7cd75ea4-ffcd-4158-a14a-a30fe1e35838","order_by":5,"name":"Chandrashekhar Gajana Joshi","email":"","orcid":"","institution":"Mangalore University","correspondingAuthor":false,"prefix":"","firstName":"Chandrashekhar","middleName":"Gajana","lastName":"Joshi","suffix":""},{"id":262877989,"identity":"26aabf06-6784-4387-84de-8c07c8d289ee","order_by":6,"name":"Renukaradhya K Math","email":"","orcid":"","institution":"SDM Research Institute for Biomedical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Renukaradhya","middleName":"K","lastName":"Math","suffix":""},{"id":262877990,"identity":"edfdfa6a-2d3a-4798-a99b-46ec475d5574","order_by":7,"name":"Krishnakishore Majalakere","email":"","orcid":"","institution":"Vivekananda College of Arts, Science, and Commerce","correspondingAuthor":false,"prefix":"","firstName":"Krishnakishore","middleName":"","lastName":"Majalakere","suffix":""},{"id":262877995,"identity":"f6403687-ad5b-43ec-b85c-448dea83c1d9","order_by":8,"name":"Jagadeesha Poyya","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEUlEQVRIiWNgGAWjYNACHhCRwMDA2MAgB2IeeECKFmOwlgTirIJoSWyAsnECc/YeM4kfMnZ2/e3JBx/+3GGXPj/s8EOgLXZyug3YtVj2nDGT7OFJTp5x5lmyMe+Z5NyNt9MMgFqSjc0OYNdicCPHTIKHhzmZAciQZmxjzt04OwGk5UDiNjxaJP/w1CfL38j//vNnW3264ez0DwS1SPPwHLYDMtgYeNsOJ8hL5+C3xbLnWLG1DM/xBMMzz4yleduOG26Qzik4kGCA2y/m7M0bb77tqbaXO5788OPPtmp5+dnpmz98qLCTw+l9Bg4TCcYeaHSARQ5AxHECAwb2xx8YfjDYw0XkG3CrHgWjYBSMgpEJABxkZqJ6V0WTAAAAAElFTkSuQmCC","orcid":"","institution":"SDM Research Institute for Biomedical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Jagadeesha","middleName":"","lastName":"Poyya","suffix":""}],"badges":[],"createdAt":"2023-12-06 11:59:57","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3714812/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3714812/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":62154135,"identity":"526e522f-fb08-46fc-9e5b-fb02bf0ee881","added_by":"auto","created_at":"2024-08-09 20:56:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":90503,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIllustrates the GUT-PHD2-HIF1α axis, which is modulated by gut microbial siderophores. \u003c/strong\u003eUnder normoxic conditions, PHD2 hydroxylates the HIF-1A at proline residues at positions 402 and 564. These hydroxylated proline residues are subsequently recognized by vHL and undergo proteasomal-mediated degradation. Given PHD2's role as a heme oxygenase, its catalytic activity relies on iron availability. Notably, a limited number of siderophores secreted by gut microbiota possess the ability to chelate iron, thereby inhibiting PHD2 activity. Consequently, our hypothesis posits that siderophores are equipped to impede PHD2 activity, leading to the activation of the HIF-1α signaling pathway. This activation, in turn, stimulates the expression of genes associated with hypoxia responses.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3714812/v1/9b5c47c96e197e14f4580a06.png"},{"id":62154136,"identity":"7190a079-1482-43df-a948-538c712ef487","added_by":"auto","created_at":"2024-08-09 20:56:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":60059,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe Moleuclar Docking and Dynamic Simulation data for Salmochelin SX and PHD2 complex.\u003c/strong\u003e (a) Docked pose for compound D1 and PHD2. The key amino acid ARG322, which invoves in interactions with HFI-1α being interacts with compound 1 by donating hydrogen bond. (b) post MD simulation pose showing changes in active site residue which invovled in interaction with PHD2. (c) RMSD graph shows the stability of alpha carbon and ligand during simulation period. (d)RMSF observed unchanged for protein helix and beta pleate sheets. (e) The interaction fraction graph represents the stability of the interaction fraction.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3714812/v1/cfe12eeae96ed4270b610650.png"},{"id":62154139,"identity":"5c241c0a-cc4f-473a-8a86-b07278012084","added_by":"auto","created_at":"2024-08-09 20:56:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":114447,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIllustration of the Molecular Docking and Dynamic Simulation data concerning the complex formed by Mycobactin and PHD2.\u003c/strong\u003e (a) The docked pose reveals the interaction between Mycobactin and PHD2, with the key residues, playing a role in interactions with HFI-1α, forming a hydrogen bond with compound 1. (b) The post MD simulation pose highlights alterations in active site residues that engage in interactions with PHD2. (c) The RMSD graph depicts the stability of the alpha carbon and ligand throughout the simulation period. (d) RMSF indicates the unchanged nature of protein helix and beta pleat sheets. (e) The interaction fraction graph illustrates the stability of the interaction fraction.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3714812/v1/5f055361723ac957255e227e.png"},{"id":62154141,"identity":"441578b3-40af-42f7-9b87-25ff4242d8b2","added_by":"auto","created_at":"2024-08-09 20:56:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":63422,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe molecular docking and dynamic simulation results showing affinity of Staphyloferrin A with PHD2.\u003c/strong\u003e Staphyloferrin A and PHD2 engage as shown by the docked pose (a), with Staphyloferrin A and the important amino acids forms bonds during docking studies. Changes in active site residues that interact with PHD2 are highlighted in the post MD simulation pose (b). (c) The alpha carbon and ligand stability during the course of the simulation is shown by the RMSD graph. The protein helix and beta pleat sheets remain intact, as indicated by (d) RMSF. (e) An illustration of the interaction fraction's stability is provided by the graph.\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-3714812/v1/c3597f049af42bf97e5dd4cc.png"},{"id":62154137,"identity":"67443610-10b2-4b59-a076-2cc5148bd6e7","added_by":"auto","created_at":"2024-08-09 20:56:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":59066,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe complex formed by Enterobactin and PHD2, along with the outcomes of molecular docking and dynamic simulation.\u003c/strong\u003e As indicated by the docked position (a), Enterobactin and PHD2. In the post MD simulation pose (b), changes in active site residues that interact with PHD2 are noted. (c) The RMSD graph displays the stability of the ligand and alpha carbon throughout the simulation. (d) RMSF shows that the beta pleat sheets and protein helix are still intact. (e) The graph presents a picture of the stability of the interaction fraction.\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-3714812/v1/2a05f46521b68e34e8c18cb0.png"},{"id":62155419,"identity":"76a3bfd6-0442-4845-9813-75059fd066fc","added_by":"auto","created_at":"2024-08-09 21:04:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1210042,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3714812/v1/5da58ba1-8ad0-4e65-840e-b38f6ba4dfb5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Molecular Docking and Dynamic Simulation Studies of PHD2 Interactions with Gut Siderophores: Implications for HIF-1α Stabilization","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn response to hypoxic microenvironments induced by ischemia, cells activate intricate adaptive pathways to cope with the limited oxygen availability. Among these pathways, the Hypoxia-Inducible Factor \u0026minus;\u0026thinsp;1 alpha (HIF-1) signaling cascade plays a pivotal role in mediating cellular responses to oxygen fluctuations during ischemia\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. HIF-1 is a transcription factor composed of HIF-1α and HIF-1β subunits, whose stabilization and activation are regulated by Prolyl Hydroxylase Domain 2 (PHD2). Under normoxic conditions, PHD2 hydroxylates HIF-1α, marking it for proteasomal degradation and preventing the formation of the active HIF-1 complex\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. However, PHD2 activity is reduced under hypoxic conditions leading to HIF-1α stabilization and translocation to the nucleus. Subsequently, HIF-1α dimerizes with HIF-1β, forming the active HIF-1 complex that initiates the transcription of various hypoxia-responsive genes\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePHD2 is a crucial enzyme that plays a pivotal role in regulating cellular responses to oxygen levels, a process known as oxygen sensing\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. PHD2 belongs to the family of prolyl hydroxylases, which are essential components of cellular oxygen-sensing machinery. These enzymes are key players in the hypoxia-inducible factor (HIF) pathway, a signaling cascade that orchestrates adaptive responses to low oxygen conditions\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. PHD2 specifically targets the HIF transcription factor, facilitating its degradation under normoxic (normal oxygen) conditions by hydroxylating specific proline residues. This hydroxylation marks HIF for recognition by the von Hippel-Lindau (VHL) protein, leading to its subsequent degradation by the ubiquitin-proteasome system\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The intricate interplay between PHD2, HIF, and VHL underscores the enzyme's significance in finely tuning cellular responses to oxygen availability. Understanding the molecular intricacies of PHD2 not only enhances our comprehension of fundamental cellular processes but also holds potential therapeutic implications, making it a promising target for the development of drugs aimed at modulating oxygen-sensing pathways in various physiological and pathological contexts\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe inhibition of Prolyl Hydroxylase Domain 2 (PHD2) has emerged as a compelling therapeutic strategy in various diseases characterized by aberrant oxygen sensing and HIF-mediated responses. Notably, in cancer, where the tumor microenvironment often experiences hypoxia, inhibiting PHD2 can stabilize HIF and promote the expression of genes involved in angiogenesis, glycolysis, and cell survival\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. This adaptive response aids tumor progression and metastasis, making PHD2 an attractive target for anti-cancer therapies\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Additionally, in ischemic conditions such as myocardial infarction and stroke, PHD2 inhibition has shown promise in promoting angiogenesis and tissue repair by enhancing HIF-dependent pathways\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Moreover, in certain inflammatory and autoimmune disorders, PHD2 inhibition has demonstrated potential in modulating immune responses and mitigating tissue damage\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The versatility of PHD2 inhibitors across diverse pathological contexts underscores their therapeutic potential and positions them as promising candidates for the development of targeted interventions aimed at manipulating oxygen-sensing pathways to treat a spectrum of diseases. Nevertheless, the complexity of these pathways necessitates careful consideration of potential side effects and thorough understanding of the context-specific roles of PHD2 in different disease settings\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Ongoing research in this area holds the promise of uncovering new therapeutic avenues and refining strategies for PHD2 inhibition in the pursuit of improved patient outcomes.\u003c/p\u003e \u003cp\u003eThere are few natural regulators of PHD2, such as low oxygen concentration\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, iron chelator\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, and metabolite from TCA cycle\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. The regulation of the PHD2 is necessary in a variety of physiological conditions, such as stem cell niche\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e and ischemic\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, where HIF-1α stability is very essential to cellular adaptation to low oxygen conditions. Iron chelators have emerged as intriguing agents in the potential inhibition of PHD2\u003csup\u003e18\u003c/sup\u003e, primarily due to the role of iron as a crucial cofactor for the enzymatic activity of Prolyl Hydroxylases, including PHD2. These chelators function by sequestering intracellular iron, thereby limiting its availability for PHD2 catalysis. The interaction between iron and PHD2 is pivotal in the hydroxylation of proline residues on hypoxia-inducible factor (HIF), marking it for subsequent degradation under normoxic conditions\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn conditions where iron chelators are employed, such as in the treatment of certain iron overload disorders or as experimental tools in research, the reduction of available iron can lead to the stabilization of HIF, mimicking a hypoxic cellular response\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. This stabilization occurs because iron chelation inhibits PHD2 activity, preventing the hydroxylation of proline residues on HIF. Consequently, the stabilized HIF can translocate to the nucleus and activate genes associated with adaptive responses to low oxygen levels\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSiderophores are one such iron chelators secreted by gut microbiota in human. Certain groups siderophores are having capacity to chelates the iron\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Siderophores are a fascinating class of small molecules produced by microorganisms, plants, and some fungi to facilitate the acquisition of essential iron from the environment\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Iron is a crucial micronutrient for various biological processes, including cellular respiration, DNA synthesis, and immune response. However, in many environments, iron is often present in insoluble and poorly bioavailable forms. Siderophores act as high-affinity iron chelators, binding to ferric iron (Fe\u003csup\u003e3+\u003c/sup\u003e) with exceptional specificity and solubilizing it, making it accessible for cellular uptake\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn the context of PHD2 inhibition, siderophores could sequester extracellular iron, thereby limiting its availability for PHD2 catalysis. Since PHD2 requires iron for the hydroxylation of specific proline residues on hypoxia-inducible factor (HIF), a reduction in accessible iron could impede PHD2-mediated degradation of HIF under normoxic conditions. As a consequence, HIF stabilization would occur, mimicking a cellular response to low oxygen levels\u003csup\u003e\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe potential use of siderophores as PHD2 inhibitors opens up new possibilities in therapeutic interventions, particularly in diseases where dysregulation of the hypoxia-inducible factor pathway plays a pivotal role. Given the intricate link between iron homeostasis and oxygen sensing, siderophores may offer a targeted approach to modulate PHD2 activity, influencing downstream cellular responses associated with HIF-mediated gene expression\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDuring ischemic conditions stable HIF-1α expression is necessary. The downstream targets of HIF-1α encompass a wide array of genes involved in vital physiological processes, including angiogenesis, glycolysis, erythropoiesis, and cell survival. HIF-1α-induced angiogenesis is crucial for promoting the formation of new blood vessels, enhancing oxygen supply to ischemic tissues, and facilitating tissue repair\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Additionally, HIF-1-driven glycolysis enables cells to adapt their metabolic pathways to generate energy efficiently even in oxygen-deprived conditions, supporting cellular survival\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Furthermore, HIF-1α stimulates erythropoiesis through the production of erythropoietin, which increases red blood cell production and improves oxygen-carrying capacity\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Given the central role of HIF-1 in cellular adaptation to ischemic conditions, the PHD2-HIF-1α axis emerges as a potential therapeutic target\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Modulating PHD2 activity could stabilize HIF-1α and enhance adaptive cellular responses during ischemia\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur hypothesis revolves around the prospect of harnessing the capabilities of gut siderophores as inhibitors of Prolyl Hydroxylase Domain 2 (PHD2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) in order to stabilize HIF-1α during ischemic conditions\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Through a comprehensive \u003cem\u003ein-silico\u003c/em\u003e screening process and Molecular simulations, we anticipate the identification and modulation of potential PHD2 inhibitors that can facilitate the preservation of HIF-1α, thereby activating essential adaptive pathways crucial for cellular responses to changes in oxygen levels\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Methodology","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eProtein Preparation\u003c/h2\u003e \u003cp\u003eThe crystal structure of PHD2 (PDB ID: 4BQY) was retrieved from Protein Data Bank (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.rcsb.org/pdb/home/home.do\u003c/span\u003e\u003cspan address=\"http://www.rcsb.org/pdb/home/home.do\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003csup\u003e36\u003c/sup\u003e. Water molecules and heteroatoms were removed from the active site for docking various compounds of interest. Schrodinger's Protein Preparation Wizard function was utilized to optimize and minimize protein structure using the OPLS3 forcefield\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe Protein Preparation Wizard fixes the protein and makes it suitable for docking. It corrects the incorrect bond orders, charge states, orientations of different amide and hydroxyl, and aromatic groups within a protein structure. The X-ray structure cannot determine due to limited resolution. Energy minimization was done by molecular mechanics calculation using OPLS3 available in the Protein Preparation tool to minimize the strains and steric collisions in protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eLigand Preparation\u003c/h2\u003e \u003cp\u003eSchrodinger's LigPrep module was used to create a library of siderophore and other gut microbial metabolites. The compound was processed with Glide's LigPrep tool\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, which generates accurate, and energy minimized 3-dimensional structures and applied sophisticated rules to correct the Lewis structure and eliminates mistakes in the ligand structures.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eActive site and Grid generation\u003c/h2\u003e \u003cp\u003eThe active site was generated using the receptor grid generation module of Schrodinger. The grid enclosed the active site of the PHD2, which includes key factors such as ARG322 and Fe\u003csup\u003e2+\u003c/sup\u003e. The grid size was about 20X20X20 \u0026Aring; which can accommodate the active site of the protein to allow each ligand to search for the potential binding.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eVirtual Screening, Molecular Docking, and MM/GBSA calculations\u003c/h2\u003e \u003cp\u003eProtein-ligand docking studies were conducted using the optimized structure of Protein. To evaluate docking efficiency, we employed Molecular Mechanics-Generalized Born Surface Area. (MM/GBSA) study\u003csup\u003e\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. The Prime MM/GBSA calculates the binding free energy (G\u003cem\u003ebind\u003c/em\u003e) of the siderophores, protein, and complex with Protein using the MM/GBSA method.\u003c/p\u003e \u003cp\u003eVirtual Screening Workflow performs docking of an extensive collection of compounds against one target. The docking interactions were compared with the standard drug.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMolecular Dynamic Simulations\u003c/h2\u003e \u003cp\u003eThe best-ranked siderophores were selected for Molecular dynamics simulation using Desmond 2020.1 by D. E. Shaw Research\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. In total, four siderophores, along with the enzyme, were simulated. System setup includes TIP3P\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, three site water model with a cubic box with a 10\u0026Aring; buffer region between enzyme-ligand complex, and a system neutralized using appropriate numbers of counter ions (Na\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e) with a fixed salt concentration of 0.15 M. OPLS3e force field was employed to simulate computations. Isothermal-isobaric (NPT) ensemble was used with temperature and pressure adjusted to 300K and 1.01325 bar, respectively. A simulation time of 200ns was adjusted, whereas trajectories were saved at every 100ps. Simulation event analysis, simulation quality analysis, and simulation interaction diagram protocols of the Desmond package were exercised to analyze the trajectory files.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003ePHD2, also known as prolyl hydroxylase domain 2, plays a crucial role in both normal and disease physiology. In normal physiology, PHD2 is involved in the regulation of cellular responses to oxygen levels, primarily through its interaction with hypoxia-inducible factors (HIFs)\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. By hydroxylating specific proline residues on HIFs, PHD2 targets them for degradation under normoxic conditions, preventing their accumulation and subsequent activation of hypoxia-responsive genes\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. This process ensures proper oxygen sensing and adaptation in various tissues and is vital for maintaining homeostasis\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIntermolecular interactions are essential in drug discovery as they govern the binding affinity and specificity of drug compounds to their target proteins. These interactions include hydrogen bonding, pi-pi cation, pi-pi stacking, and van der Waals forces of attraction. Hydrogen bonding stabilizes drug-protein complexes, while pi-pi cation and pi-pi stacking interactions contribute to altered pharmacological effects\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Van der Waals forces, though weak individually, collectively play a significant role in the overall binding process. Understanding these interactions aids in rational drug design, identifying new drug targets, and predicting pharmacokinetics and safety\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eMolecular Docking Studies\u003c/h2\u003e \u003cp\u003eTo identify suitable lead molecules, docking studies were performed with a natural product library, using Glide to validate the hypothesis. The HIF-1α -binding site of PHD2 contains key residues, including ARG322 and Fe\u003csup\u003e2+\u003c/sup\u003e, which play an active role in substrate binding through hydrogen bonding interactions. Specifically, the positively charged amino acid ARG322 interacts with PRO564 of the HIF-1α protein within pocket one of PHD2, making it a critical residue in the enzyme's active site and facilitating the interaction with the HIF-1α hydroxylation site, PRO564\u003csup\u003e48\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e presents the binding scores and affinities of compounds Salmochelin SX, Mycobactin, Staphyloferrin A, and Enterobactin towards PHD2. Among these compounds, SALMOCHELIN SX exhibits the highest affinity with a binding score of -9.527 Kcal/mol (MM/GBSA score: -42.58 Kcal/mol). It engages in crucial interactions with the target protein, forming hydrogen bonds with ASP254, TYR310, and ASP315, while accepting hydrogen bonds from TYR303 and ARG322. Additionally, Salmochelin SX coordinates with Fe\u003csup\u003e2+\u003c/sup\u003e, contributing significantly to its interaction with the key residue ARG322 and the co-factor Fe\u003csup\u003e2+\u003c/sup\u003e. This coordination of Fe\u003csup\u003e2+\u003c/sup\u003e is vital for stabilizing the ligand-protein complex and plays a pivotal role in determining Salmochelin SX's strong affinity towards PHD2.\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\u003ePrioritized Compounds List\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\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 \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 \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\u003eDocking score (Kcal/mol)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMM/GBSA\u003c/p\u003e \u003cp\u003e(Kcal/mol)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInteracting Amino acid residue and cofactor (Fe\u003csup\u003e2+\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eInteracting Amino Acids residue and co-factors during MD Simulation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAmino Acids bridged to Ferrous ion during simulation\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSalmochelin SX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-9.527\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-42.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eASP254, TYR303, \u003cb\u003eTYR310\u003c/b\u003e, ASP315, \u003cb\u003eARG322\u003c/b\u003e, Fe\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eASP254\u003c/b\u003e, ARG383\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHIS313, ASP315, HIS374\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMycobactin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-9.166\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-34.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVAL314, \u003cb\u003eARG322\u003c/b\u003e, Fe\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eASP254\u003c/b\u003e, THR387\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHIS313, ASP315, HIS374\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStaphyloferrin A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-7.819\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-25.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eTYR310\u003c/b\u003e, ASP315, \u003cb\u003eARG322\u003c/b\u003e, Fe\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eASP254\u003c/b\u003e, ARG383, THR387\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHIS313, ASP315, HIS374\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEnterobactin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-7.302\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-38.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eTYR310, ARG322\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eASP254\u003c/b\u003e, TYR310, ASP320, TRP389\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHIS313, ASP315, HIS374\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\u003eSalmochelin SX showed interaction with ASP315, ASP254, TYR303 and ARG322 residue. The carboxyl group of compound forms hydrogen bonds with a distance of 1.910 \u0026Aring; with TYR310 respectively. The oxan ring of 3,4,5-trihydroxy-6-(hydroxymethyl) oxan-2-yl] phenyl} formamido) accepts the hydrogen bond from TYR303 (2.178 \u0026Aring;). And the hydroxypropionic group donates hydrogen bond to ASP315 and accepts hydrogen bond from key residue ARG322 with bond distance (2.028 \u0026Aring;) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The negatively charged ASP254 forms a two-hydrogen bond (1.985 \u0026Aring; and 2.392 \u0026Aring;) with the hydroxyl group of the phenyl ring and Fe\u003csup\u003e2+\u003c/sup\u003e interaction with the OH group. In the same way, we tested the interactions of other lead molecules (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(a)).\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\u003eHydrogen Bond distance of Salmochelin SX\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInteraction\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH Bond distance (\u0026Aring;)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePHD2 TYR303: HH - D1:O1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.178\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePHD2 ARG322:HH12 - D1:O9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.028\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePHD2 ARG322:HH22 - D1:O9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.237\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePHD2: ARG383:HH12 - D1:O5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.190\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD1:H44 - PHD2: ASP254:OD2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.985\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD1:H45 - PHD2: ASP254:OD2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.392\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD1:H49 - PHD2: TYR310: OH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.910\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 \u003cp\u003eNext, Mycobactin shows an affinity towards PHD2 with a binding score of -9.166 Kcal/mol. Like Salmochelin SX, it forms multiple interactions with the target protein, donating a hydrogen bond to ARG322 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(a)) and VAL314 donating and accepting hydrogen bonds from the compound. Furthermore, Mycobactin coordinates with Fe\u003csup\u003e2+\u003c/sup\u003e, which is crucial for its favorable association with the key residue ARG322 and the co-factor Fe\u003csup\u003e2+\u003c/sup\u003e. The coordination of Fe\u003csup\u003e2+\u003c/sup\u003e enhances Mycobactin 's binding to PHD2, contributing to its significant affinity.\u003c/p\u003e \u003cp\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\u003eHydrogen Bond distance of Aerobactin\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInteraction\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH Bond distance (\u0026Aring;)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePHD2:VAL314:H - Mycobactin: O10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.972\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePHD2:ARG322: HH12 - Mycobactin: O6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.860\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMycobactin:H75 - PHD2:VAL314:O\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.794\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\u003eThe two carboxyl groups of Staphyloferrin A form hydrogen bonds with ASP315 with distances (2.242 \u0026Aring; and 1.480 \u0026Aring;) and one of them, accepts hydrogen bond from key residue ARG322 (2.089\u0026Aring;). Similarly, at 2.182 \u0026Aring; TYR310 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(a) forms an interaction with the hydroxyl group of Staphyloferrin A (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). There is also metal coordination with the carboxyl group. Finally, Enterobactin exhibits an affinity towards PHD2 with a binding score of -7.302 Kcal/mol. It forms bonds with the target protein by donating a hydrogen bond (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) to TYR310 and engaging in a pi-cation, noncovalent interaction with the key residue ARG322(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(a)).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eH Bond distance of Staphyloferrin A\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInteraction\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH Bond distance (\u0026Aring;)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePHD2: TYR310: HH - Staphyloferrin A: O3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.182\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePHD2: ARG322:HH12 - Staphyloferrin A: O9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.089\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStaphyloferrin A:H51 - PHD2: TYR310: OH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.878\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStaphyloferrin A:H54 - PHD2: ASP315:O\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.242\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStaphyloferrin A:H56 - PHD2: ASP315:OD1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.480\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=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHydrogen Bond distance of Enterobactin\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInteraction\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH Bond distance (\u0026Aring;)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePHD2: TYR310: HH - Enterobactin:O11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.174\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEnterobactin:H70 - PHD2: TYR310: OH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.699\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEnterobactin:H73 - PHD2: TYR310: OH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.170\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\u003eThe interaction analysis revealed that Salmochelin SX forms a variety of bonds with the target protein, including donating hydrogen bonds to ASP254, TYR310, and ASP315, while accepting hydrogen bonds from TYR303 and ARG322. Moreover, Salmochelin SX coordinates with Fe\u003csup\u003e2+\u003c/sup\u003e. These intricate bonds are crucial as they play a vital role in the compound's association with the key residue ARG322 and the co-factor Fe\u003csup\u003e2+\u003c/sup\u003e within PHD2's active site. This strong and specific binding can modulate PHD2's enzymatic activity, potentially leading to therapeutic effects in the context of diseases associated with oxygen sensing and HIF regulation. Comparing the results to the Salmochelin SX, Aerobactin, Staphyloferrin A, and Enterobactin also exhibited significant affinities towards PHD2, albeit with slightly lower binding scores compared to Salmochelin SX. Aerobactin, like Salmochelin SX, forms multiple hydrogen bonds (Tables\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e \u0026amp; \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) with the target protein, while Staphyloferrin A (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) and Enterobactin (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) donate hydrogen bonds (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) and engage in pi-cation, non-covalent interactions, respectively, with key residue ARG322.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMolecular Dynamic Simulation\u003c/h3\u003e\n\u003cp\u003eThe MD simulation revealed stable conformational changes in the target proteins upon ligand binding (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(b) and (c). During the MD simulation of Salmochelin SX and PHD2 complex, the RMSD of alpha carbon and Salmochelin SX initially fluctuated but stabilized after 30 ns, with a final RMSD change of less than 3 \u0026Aring;. The Root Mean Square Fluctuation (RMSF) of the protein-ligand complex showed fluctuations only in the loop regions, a common occurrence during MD simulations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(d)). Ligand contact had no significant impact on protein RMSF, even in the beta-pleated sheets.\u003c/p\u003e \u003cp\u003eThe simulation interaction fraction graph represented the strength of bonded interactions, with 40 bonds observed. Only a few bonds, like ASP254, HIS313, ASP315, HIS374, and ARG383 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(e)), remained in contact for more than 30% of the simulation time. ARG254 had the highest interaction, forming hydrogen bonds and water bridges, while ARG383 had similar interactions. The PHD2 enzyme's co-factor, Fe\u003csup\u003e2+\u003c/sup\u003e, interacted with histidine and aspartate, specifically HIS313, ASP315, and HIS374. This interaction was not present during molecular docking but formed during MD simulations. This interaction is crucial for inhibiting PHD2, as the ferrous ion plays a pivotal role in oxygen-mediated oxidoreductase reactions.\u003c/p\u003e \u003cp\u003eMycobactin displayed similar results to the Salmochelin SX, with changed interactions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(b) compared to its docking results (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(a)). RMSD values for the alpha carbon and ligands staying below 3 \u0026Aring; (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(c). During the simulation, RMSF for the protein primarily fluctuated in the loop regions, as is typically observed in loop regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(d)). Ligand interactions with the protein did not induce significant fluctuations. The Ligand contact graph, showing interaction fractions, revealed numerous interactions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(e)). Some of these interactions persisted with the protein for over 30% of the simulation time. In the two-dimensional pose view, ASP254, TYR310, and ARG383 formed hydrogen bonds with the protein-ligand, which had the highest interaction fraction scores (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(e)). The ferrous ion's connection with HIS313, ASP315, and HIS374 played a substantial role in the ligand interaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(b). These ionic interactions were nearly as significant as hydrogen bonding in terms of interaction strength. This interaction is crucial, and inhibiting the ferrous ion may reduce PHD2 enzyme activity. Furthermore, a few water bridges formed during the simulation, involving THR236 and THR387. The few interactions found during the docking studies were missing and additional interactions were observed. This may be due to the structural changes undergoes during MD simulation.\u003c/p\u003e \u003cp\u003eFurther, Staphyloferrin A and Enterobactin have similar results like Salmochelin SX and Mycobactin in the case of protein and ligand RMSD (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e \u0026amp; \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(c) and RMSF (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u0026amp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(d). The post MD simulation interaction pose (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(b) \u0026amp; 5(b)) showed changes in interaction compared to docking pose (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(a) \u0026amp; 5(a)). RMSF results both siderophores (Staphyloferrin A and Enterobactin) was found changes in only loop regions of the PHD2(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(d) \u0026amp; 5(d)). In Staphyloferrin A and PHD2 complex, ARG254, ARG383, and THR387 were formed hydrogen bonds (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(e)). Ferrous ion showed the same interaction with Staphyloferrin A (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(b)) as Salmochelin SX and Mycobactin. In this case water bridge observed with ASP254 and ASP325(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(b)\u0026amp;(e)). Enterobactin was found stable throughout the simulations. The interaction of residues was changed during simulation compared to docked pose (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(b)). Further, RMSD (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(c)), RMSF (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(d) were found stable and there are no changes were observed. Enterobactin showed ASP254, TYR310, ASP320 hydrogen bonded (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(b)(e)) to the protein for more than 30% of simulation time and TYR389 showed pi-pi cationic interaction.\u003c/p\u003e \u003cp\u003eIn a molecular dynamic simulation, we studied four compounds interacting with the PHD2 protein. These simulations showed that the protein and compounds changed their shapes but eventually stabilized. The compounds interacted with the protein without causing major fluctuations. We found specific interactions, like hydrogen bonds, that were crucial for inhibiting PHD2. These results differed from initial predictions using molecular docking, highlighting the importance of dynamic simulations in understanding how drugs interact with proteins.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHuman organs undergo various physiological conditions such as ischemia, where flow of blood is reduced and subsequently lacks oxygen supply to tissues or organs\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. In this context, this study was conducted to identify potential inhibitors of PHD2, an enzyme involved in the regulation of Hypoxia-Inducible Factor (HIF), could be a potential strategy to mitigate the effects of ischemia\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. HIF is a transcription factor that plays a key role in cellular responses to low oxygen levels, and its stabilization can promote cellular adaptation and survival under hypoxic conditions\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAn \u003cem\u003ein-silico\u003c/em\u003e analysis of selected gut microbial siderophores was done to screen and identify molecules that may have the ability to inhibit PHD2. Results showed that, among 103 molecules, four lead molecules had interactions with key residues. First, Salmochelin SX exhibits the highest affinity with a binding score of -9.527 Kcal/mol. It engages in crucial interactions with the target protein, forming hydrogen bonds with ASP254, TYR310, and ASP315, while accepting hydrogen bonds from TYR303 and ARG322. Additionally, Salmochelin SX coordinates with Fe\u003csup\u003e2+\u003c/sup\u003e, contributing significantly to its interaction with the key residue ARG322 and the co-factor Fe\u003csup\u003e2+\u003c/sup\u003e. This coordination of Fe\u003csup\u003e2+\u003c/sup\u003e is vital for stabilizing the ligand-protein complex and plays a pivotal role in determining Salmochelin SX's strong affinity towards PHD2.\u003c/p\u003e \u003cp\u003eSalmochelin SX, secreted by \u003cem\u003eSalmonella enterica\u003c/em\u003e characterized as a catecholate-type siderophore\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e, exhibits promising attributes that may contribute to mitigating oxidative stress during ischemic reperfusion. In the intricate milieu of cellular responses to oxidative challenges, particularly in the context of the Fenton reaction where hydrogen peroxide (H2O2) interacts with intracellular iron sulfur clusters, leading to the generation of harmful free radicals like hydroxyl radicals, Salmochelin SX emerges as a potential protective agent\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDuring ischemic reperfusion, when tissues experience a restoration of blood flow, the potential for reactive oxygen species (ROS) generation is heightened\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Salmochelin SX, with its catecholate nature, could play a pivotal role in reducing ROS production. Recent findings have highlighted the capacity of catecholate siderophores, including Salmochelin SX, to act as defenders against oxidative stress, offering a positive perspective on its potential applications. Even though, Salmochelin SX part of pathogenic microbial strain, it may have therapeutic application where proper balance of gut microbiota can regulate the level of the pathogenicity of the certain bacterial strains\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe catecholate-type siderophore Enterobactin, previously examined for its anticancer properties, showcased cytotoxic effects on two monocyte tumor cells while leaving bone marrow\u0026ndash;derived macrophages unharmed in a study by Saha et al. (2019)\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. The investigation unveiled a significant increase in the intracellular labile iron pool in both cell lines, indicative of a disruption in iron homeostasis induced by the siderophore. Additionally, Enterobactin demonstrated a dose-dependent inhibition of the generation of reactive oxygen species (ROS) by mitochondria. These combined effects, involving alterations in iron levels and the attenuation of ROS production, have the potential to induce apoptosis in cancer cells\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eMycobacterium smegmatis\u003c/em\u003e produces the mycobactin, which has strong antiproliferative action against leukemia, breast, and liver cancer cell lines\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Mycobactin was showing interaction with TYR310, ASP254, ARG322, ASN318 residues. The hydroxyl group of compound accepts hydrogen bond from TYR310 with bond distance (2.010 \u0026Aring;) and simultaneously it donates hydrogen bond to ASP254. carbonyl group of compound forms hydrogen bond interaction with ASN318 (2.031 \u0026Aring;). And carboxyl group of Mycobactin accepts hydrogen bond from key residue ARG322 with a bond distance of 2.037\u0026Aring;. And also forms metal coordination Fe\u003csup\u003e2+\u003c/sup\u003e with carboxyl and hydroxyl groups. Similarly, Staphyloferrin A demonstrates an affinity towards PHD2 with a binding score of -7.819 Kcal/mol. It establishes bonds with the target protein, donating a hydrogen bond to TYR310 and ASP315, while accepting hydrogen bonds from TYR310 and ARG322. Like Salmochelin SX and Mycobactin, Staphyloferrin A also coordinates with Fe\u003csup\u003e2+\u003c/sup\u003e, and this coordination significantly influences its interaction with the key residue ARG322 and the co-factor Fe\u003csup\u003e2+\u003c/sup\u003e. The coordination with Fe\u003csup\u003e2+\u003c/sup\u003e is a critical determinant in Staphyloferrin A's affinity towards PHD2. Similar to the other compounds, Enterobactin also coordinates with Fe\u003csup\u003e2+\u003c/sup\u003e, contributing significantly to its interaction with ARG322 and Fe\u003csup\u003e2+\u003c/sup\u003e. The coordination of Fe\u003csup\u003e2+\u003c/sup\u003e plays a vital role in stabilizing Enterobactin 's binding to PHD2. In Enterobactin, the hydroxyl group of the benzene ring forms a hydrogen bond interaction with TYR310, with a bond distance of 1.699 \u0026Aring;. It also has a Pi-cation interaction, i.e., a noncovalent bond with key residue ARG322. In this study, ARG322 most often interacts with the carboxyl group of lead molecules. This kind of observation in reported studies, where small molecules are able to inhibit the activity of PHD2\u003csup\u003e48\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe coordination and interaction between ARG322 and Fe\u003csup\u003e2+\u003c/sup\u003e within the substrate-binding site are crucial for PHD2's activity and its role in the oxygen-sensing pathway\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Understanding the molecular details of these interactions can provide valuable insights for the development of potential therapeutic strategies targeting PHD2 to modulate cellular responses under hypoxic conditions. By manipulating PHD2's function, researchers may pave the way for novel approaches in treating various diseases related to oxygen sensing and HIF regulation.\u003c/p\u003e \u003cp\u003eExposure to iron chelators or cobalt ions can mimic the effects of hypoxia on HIF, which is easily explained by the known characteristics of 2-oxoglutarate-dependent oxygenases\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. For these observations, there have previously been two proposed interpretations. First, it has been suggested that cobalt ions could take the place of ferrous ions at an iron core that senses oxygen. It was suggested that such a protein must rotate quickly because the majority of iron centers (such as heme and the vast majority of iron-sulfur clusters) do not swap in this manner. Second, it has been suggested that cobaltous ions and iron chelators affect signaling by reactive oxygen species and Fenton chemistry in nonenzymatic \"metal catalyzed oxidation\" systems\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn all cases, the coordination of Fe\u003csup\u003e2+\u003c/sup\u003e is significant for the interactions between the lead compounds and PHD2. This coordination contributes significantly to the binding of each compound with the key residue ARG322 and the co-factor Fe\u003csup\u003e2+\u003c/sup\u003e. These interactions play a crucial role in stabilizing the ligand-protein complexes and influencing the compounds' overall affinities towards PHD2. Furthermore, the coordination with Fe\u003csup\u003e2+\u003c/sup\u003e is known to be important in regulating the enzymatic activity of PHD2\u003csup\u003e62\u003c/sup\u003e, making it a crucial aspect to consider in the design of potential therapeutic agents targeting PHD2 and related pathways involved in oxygen sensing and HIF regulation\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e,\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe interactions observed in all four compounds highlight the importance of hydrogen bonds and metal coordination in mediating their binding to PHD2. These interactions are well-known determinants of ligand-protein binding specificity and play a crucial role in the selectivity of lead compounds in drug discovery.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eBy inhibiting PHD2, our goal is to increase HIF stabilization, activating genes that promote cell survival, angiogenesis, and metabolism under low oxygen conditions. While in silico screening can narrow down potential candidates, further experimental validation is necessary. This involves in vitro assays and cellular studies to confirm the inhibitory activity and specificity of identified compounds against PHD2. The study's findings show significant promise for developing therapeutic agents targeting the PHD2-HIF-1α axis, enhancing adaptive responses during ischemic events. Understanding HIF-1 and PHD2 interactions in various ischemic diseases provides valuable insights for designing pharmacological interventions. This could mitigate detrimental effects of oxygen deprivation and improve patient outcomes, addressing unmet clinical needs in managing ischemic diseases. This approach, integrating computational screening with experimental validation, underscores the potential translational impact in developing effective therapies for ischemic conditions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJIP: Methodology, formal validation, initial manuscript draft, NK: Visualization, AP: Visualization, English editing, PKJ: manuscript corrections, ASK: support in draft preparation, CGJ: supervision, review and editing, RKM: review and editing, KM: software, writing, JP: Conceptualization, Methodology, writing-review and editing, project administration, supervision, and editing manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgment\u003c/h2\u003e \u003cp\u003eThe author expresses gratitude to Google Inc. for generously providing Google Research Cloud Credit (EDU Credit - wilsonjessica \u0026minus;\u0026thinsp;209474439) and to the SDM Research Institute for Biomedical Sciences and Shri Dharmasthala Manjunatheshwara University, and Department of Biochemistry and Industrial Chemistry, Mangalore University for their facility.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e \u003cp\u003eAll data generated or analysed during this study are included in this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMajmundar, A. J., Wong, W. J. \u0026amp; Simon, M. C. Hypoxia-Inducible Factors and the Response to Hypoxic Stress. Molecular Cell 40, 294\u0026ndash;309 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeidemann, A. \u0026amp; Johnson, R. S. Biology of HIF-1a. Cell Death and Differentiation 15, 621\u0026ndash;627 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuo, Z. \u003cem\u003eet al.\u003c/em\u003e Hypoxia signaling in human health and diseases: implications and prospects for therapeutics. Sig Transduct Target Ther 7, 218 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcDonough, M. A. \u003cem\u003eet al.\u003c/em\u003e Cellular oxygen sensing: Crystal structure of hypoxia-inducible factor prolyl hydroxylase (PHD2). Proc Natl Acad Sci U S A 103, 9814\u0026ndash;9819 (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSemenza, G. L. Oxygen sensing, hypoxia-inducible factors, and disease pathophysiology. Annu Rev Pathol 9, 47\u0026ndash;71 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGroulx, I. \u0026amp; Lee, S. Oxygen-dependent ubiquitination and degradation of hypoxia-inducible factor requires nuclear-cytoplasmic trafficking of the von Hippel-Lindau tumor suppressor protein. Mol Cell Biol 22, 5319\u0026ndash;5336 (2002).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao, Y. \u003cem\u003eet al.\u003c/em\u003e Hypoxia-induced signaling in the cardiovascular system: pathogenesis and therapeutic targets. Sig Transduct Target Ther 8, 431 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBerra, E., Roux, D., Richard, D. E. \u0026amp; Pouyss\u0026eacute;gur, J. Hypoxia-inducible factor-1α (HIF-1α) escapes O(2)-driven proteasomal degradation irrespective of its subcellular localization: nucleus or cytoplasm. EMBO Reports 2, 615\u0026ndash;620 (2001).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePoon, E., Harris, A. L. \u0026amp; Ashcroft, M. Targeting the hypoxia-inducible factor (HIF) pathway in cancer. \u003cem\u003eExpert Reviews in Molecular Medicine\u003c/em\u003e 11, null-null (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBereczki, D., Balla, J. \u0026amp; Bereczki, D. Heme Oxygenase-1: Clinical Relevance in Ischemic Stroke. Curr Pharm Des 24, 2229\u0026ndash;2235 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eP\u0026aring;lsson-McDermott, E. M. \u0026amp; O\u0026rsquo;Neill, L. A. J. Targeting immunometabolism as an anti-inflammatory strategy. Cell Res 30, 300\u0026ndash;314 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOgle, M. E., Gu, X., Espinera, A. R. \u0026amp; Wei, L. Inhibition of prolyl hydroxylases by dimethyloxaloylglycine after stroke reduces ischemic brain injury and requires hypoxia inducible factor-1α. Neurobiology of Disease 45, 733\u0026ndash;742 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSuhara, T. \u003cem\u003eet al.\u003c/em\u003e Inhibition of the oxygen sensor PHD2 in the liver improves survival in lactic acidosis by activating the Cori cycle. \u003cem\u003eProc. Natl. Acad. Sci. U.S.A.\u003c/em\u003e 112, 11642\u0026ndash;11647 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFlagg, S. C., Martin, C. B., Taabazuing, C. Y., Holmes, B. E. \u0026amp; Knapp, M. J. Screening chelating inhibitors of HIF-prolyl hydroxylase domain 2 (PHD2) and factor inhibiting HIF (FIH). Journal of Inorganic Biochemistry 113, 25\u0026ndash;30 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbboud, M. I. \u003cem\u003eet al.\u003c/em\u003e 2-Oxoglutarate regulates binding of hydroxylated hypoxia-inducible factor to prolyl hydroxylase domain 2. Chem Commun (Camb) 54, 3130\u0026ndash;3133 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFoulks, J. M. \u003cem\u003eet al.\u003c/em\u003e PAF-acetylhydrolase expressed during megakaryocyte differentiation inactivates PAF-like lipids. Blood 113, 6699\u0026ndash;6706 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeSai, C. \u0026amp; Hays Shapshak, A. Cerebral Ischemia. in \u003cem\u003eStatPearls\u003c/em\u003e (StatPearls Publishing, 2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSpeer, R. E. \u003cem\u003eet al.\u003c/em\u003e Hypoxia-inducible factor prolyl hydroxylases as targets for neuroprotection by \u0026ldquo;antioxidant\u0026rdquo; metal chelators: From ferroptosis to stroke. Free Radical Biology and Medicine 62, 26\u0026ndash;36 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBento, C. F. \u0026amp; Pereira, P. Regulation of hypoxia-inducible factor 1 and the loss of the cellular response to hypoxia in diabetes. Diabetologia 54, 1946\u0026ndash;1956 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaase, V. H. The VHL tumor suppressor: master regulator of HIF. Curr Pharm Des 15, 3895\u0026ndash;3903 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEllermann, M. \u0026amp; Arthur, J. C. Siderophore-mediated iron acquisition and modulation of host-bacterial interactions. Free Radic Biol Med 105, 68\u0026ndash;78 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhasheii, B., Mahmoodi, P. \u0026amp; Mohammadzadeh, A. Siderophores: Importance in bacterial pathogenesis and applications in medicine and industry. Microbiological Research 250, 126790 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSiegert, I. \u003cem\u003eet al.\u003c/em\u003e Ferritin-Mediated Iron Sequestration Stabilizes Hypoxia-Inducible Factor-1α upon LPS Activation in the Presence of Ample Oxygen. Cell Reports 13, 2048\u0026ndash;2055 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDev, S. \u003cem\u003eet al.\u003c/em\u003e Role of extracellular Hydrogen peroxide in regulation of iron homeostasis genes in neuronal cells: Implication in iron accumulation. Free Radical Biology and Medicine 86, 78\u0026ndash;89 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMinervini, G., Quaglia, F. \u0026amp; Tosatto, S. C. E. Insights into the proline hydroxylase (PHD) family, molecular evolution and its impact on human health. Biochimie 116, 114\u0026ndash;124 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, Z., Han, F., Du, Y., Shi, H. \u0026amp; Zhou, W. Hypoxic microenvironment in cancer: molecular mechanisms and therapeutic interventions. Sig Transduct Target Ther 8, 70 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarthels, D. \u0026amp; Das, H. Current advances in ischemic stroke research and therapies. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease 1866, 165260 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSemenza, G. L. HIF-1: mediator of physiological and pathophysiological responses to hypoxia. Journal of Applied Physiology 88, 1474 (2000).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRajendran, G. \u003cem\u003eet al.\u003c/em\u003e Inhibition of Endothelial PHD2 Suppresses Post-Ischemic Kidney Inflammation through Hypoxia-Inducible Factor-1. \u003cem\u003eJASN\u003c/em\u003e 31, 501\u0026ndash;516 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSemenza, G. L. Hypoxia-Inducible Factors in Physiology and Medicine. Cell 148, 399\u0026ndash;408 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAvivi, A., Resnick, M. B., Nevo, E., Joel, A. \u0026amp; Levy, A. P. Adaptive hypoxic tolerance in the subterranean mole rat Spalax ehrenbergi: the role of vascular endothelial growth factor. FEBS Letters 452, 133\u0026ndash;140.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePoyya, J., Joshi, C. G., Kumar, D. J. \u0026amp; Nagendra, H. Sequence Analysis and Phylogenetic Studies of Hypoxia-Inducible Factor-1α. Cancer Informatics 16, 117693511771224 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeite de Oliveira, R. \u003cem\u003eet al.\u003c/em\u003e Gene-Targeting of Phd2 Improves Tumor Response to Chemotherapy and Prevents Side-Toxicity. Cancer Cell 22, 263\u0026ndash;277 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZiello, J. E., Jovin, I. S. \u0026amp; Huang, Y. Hypoxia-Inducible Factor (HIF)-1 regulatory pathway and its potential for therapeutic intervention in malignancy and ischemia. Yale J Biol Med 80, 51\u0026ndash;60 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWatts, E. R. \u0026amp; Walmsley, S. R. Inflammation and Hypoxia: HIF and PHD Isoform Selectivity. Trends in Molecular Medicine 25, 33\u0026ndash;46 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBerman, H. M. The Protein Data Bank. Nucleic Acids Research 28, 235\u0026ndash;242 (2000).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarder, E. \u003cem\u003eet al.\u003c/em\u003e OPLS3: A Force Field Providing Broad Coverage of Drug-like Small Molecules and Proteins. Journal of Chemical Theory and Computation 12, 281\u0026ndash;296 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFriesner, R. A. \u003cem\u003eet al.\u003c/em\u003e Extra Precision Glide: Docking and Scoring Incorporating a Model of Hydrophobic Enclosure for Protein\u0026thinsp;\u0026ndash;\u0026thinsp;Ligand Complexes. Journal of Medicinal Chemistry 49, 6177\u0026ndash;6196 (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHalgren, T. A. \u003cem\u003eet al.\u003c/em\u003e Glide: A New Approach for Rapid, Accurate Docking and Scoring. 2. Enrichment Factors in Database Screening. Journal of Medicinal Chemistry 47, 1750\u0026ndash;1759 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGodschalk, F., Genheden, S., S\u0026ouml;derhjelm, P. \u0026amp; Ryde, U. Comparison of MM/GBSA calculations based on explicit and implicit solvent simulations. Phys. Chem. Chem. Phys. 15, 7731 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHou, T., Wang, J., Li, Y. \u0026amp; Wang, W. Assessing the performance of the molecular mechanics/Poisson Boltzmann surface area and molecular mechanics/generalized Born surface area methods. II. The accuracy of ranking poses generated from docking. J Comput Chem 32, 866\u0026ndash;877 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePoyya, J. \u003cem\u003eet al.\u003c/em\u003e Receptor based virtual screening of potential novel inhibitors of tigar [TP53 (tumour protein 53)-induced glycolysis and apoptosis regulator. Medical Hypotheses 110683 (2021) doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.mehy.2021.110683\u003c/span\u003e\u003cspan address=\"10.1016/j.mehy.2021.110683\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShaw, D. E. Desmond Maestro 2020 by DE Shaw Research. (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMark, P. \u0026amp; Nilsson, L. Structure and Dynamics of the TIP3P, SPC, and SPC/E Water Models at 298 K. J. Phys. Chem. A 105, 9954\u0026ndash;9960 (2001).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFong, G.-H. \u0026amp; Takeda, K. Role and regulation of prolyl hydroxylase domain proteins. Cell Death Differ 15, 635\u0026ndash;641 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiang, Z. \u0026amp; Li, Q. X. π-Cation Interactions in Molecular Recognition: Perspectives on Pharmaceuticals and Pesticides. J Agric Food Chem 66, 3315\u0026ndash;3323 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBissantz, C., Kuhn, B. \u0026amp; Stahl, M. A medicinal chemist\u0026rsquo;s guide to molecular interactions. J Med Chem 53, 5061\u0026ndash;5084 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChowdhury, R. \u003cem\u003eet al.\u003c/em\u003e Selective Small Molecule Probes for the Hypoxia Inducible Factor (HIF) Prolyl Hydroxylases. ACS Chem. Biol. 8, 1488\u0026ndash;1496 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFlanigan, W. R. \u0026amp; Jain, I. H. The Goldilocks Oxygen Principle: not too little and not too much. Nat Cardiovasc Res 1, 1101\u0026ndash;1103 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKalogeris, T., Baines, C. P., Krenz, M. \u0026amp; Korthuis, R. J. Cell Biology of Ischemia/Reperfusion Injury. in \u003cem\u003eInternational Review of Cell and Molecular Biology\u003c/em\u003e vol.\u0026nbsp;298 229\u0026ndash;317 (Elsevier, 2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLoor, G. \u0026amp; Schumacker, P. T. Role of hypoxia-inducible factor in cell survival during myocardial ischemia\u0026ndash;reperfusion. Cell Death Differ 15, 686\u0026ndash;690 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eConde, E. \u003cem\u003eet al.\u003c/em\u003e HIF-1α induction during reperfusion avoids maladaptive repair after renal ischemia/reperfusion involving miR127-3p. Sci Rep 7, 41099 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMohsen, Y. \u003cem\u003eet al.\u003c/em\u003e The Different Types of Metallophores Produced by Salmonella enterica: A Review. Microbiology Research 14, 1457\u0026ndash;1469 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWinterbourn, C. C., Kettle, A. J. \u0026amp; Hampton, M. B. Reactive Oxygen Species and Neutrophil Function. Annu. Rev. Biochem. 85, 765\u0026ndash;792 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHou, K. \u003cem\u003eet al.\u003c/em\u003e Microbiota in health and diseases. Sig Transduct Target Ther 7, 135 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRolhion, N. \u0026amp; Chassaing, B. When pathogenic bacteria meet the intestinal microbiota. Philos Trans R Soc Lond B Biol Sci 371, 20150504 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaha, P. \u003cem\u003eet al.\u003c/em\u003e Enterobactin, an iron chelating bacterial siderophore, arrests cancer cell proliferation. Biochemical Pharmacology 168, 71\u0026ndash;81 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGokarn, K., Sarangdhar, V. \u0026amp; Pal, R. B. Effect of microbial siderophores on mammalian non-malignant and malignant cell lines. BMC Complement Altern Med 17, 145 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoivunen, P., Hirsil\u0026auml;, M., G\u0026uuml;nzler, V., Kivirikko, K. I. \u0026amp; Myllyharju, J. Catalytic properties of the asparaginyl hydroxylase (FIH) in the oxygen sensing pathway are distinct from those of its prolyl 4-hydroxylases. J Biol Chem 279, 9899\u0026ndash;9904 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTian, Y.-M. \u003cem\u003eet al.\u003c/em\u003e Differential sensitivity of hypoxia inducible factor hydroxylation sites to hypoxia and hydroxylase inhibitors. J Biol Chem 286, 13041\u0026ndash;13051 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJaakkola, P. \u003cem\u003eet al.\u003c/em\u003e Targeting of HIF-α to the von Hippel-Lindau Ubiquitylation Complex by O \u003csub\u003e2\u003c/sub\u003e -Regulated Prolyl Hydroxylation. Science 292, 468\u0026ndash;472 (2001).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChowdhury, R. \u003cem\u003eet al.\u003c/em\u003e Structural basis for oxygen degradation domain selectivity of the HIF prolyl hydroxylases. Nat Commun 7, 12673 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchofield, C. J. \u0026amp; Ratcliffe, P. J. Oxygen sensing by HIF hydroxylases. Nat Rev Mol Cell Biol 5, 343\u0026ndash;354 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEltzschig, H. K., Bratton, D. L. \u0026amp; Colgan, S. P. Targeting hypoxia signalling for the treatment of ischaemic and inflammatory diseases. Nat Rev Drug Discov 13, 852\u0026ndash;869 (2014).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Ischemia, Hypoxia, HIF-1α signaling, Prolyl Hydroxylase Domain 2, Gut siderophores","lastPublishedDoi":"10.21203/rs.3.rs-3714812/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3714812/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn oxygen-deprived conditions cells respond by activating adaptive mechanisms to bolster their survival and protect tissue integrity. A key player in this process is the HIF-1α signaling cascade, meticulously regulated by Prolyl Hydroxylase Domain 2 (PHD2), which plays a central role in orchestrating cellular responses to fluctuating oxygen levels.\u003c/p\u003e \u003cp\u003eThe primary aim of this investigation is to explore potential PHD2 inhibitors using in-silico methods by employing molecular docking and dynamic simulation techniques. Low molecular-weight secondary metabolites known as Siderophores secreted from gut microbiota were selected and subjected for stringent assessment against PHD2. Molecular docking analysis revealed that Salmochelin SX (-9.527 Kcal/mol), Mycobactin (-9.166 Kcal/mol), Staphyloferrin A (-7.819 Kcal/mol), and Enterobactin (-7.302 Kcal/mol) displayed substantial affinities for the protein, suggesting them as potential inhibitors.\u003c/p\u003e \u003cp\u003eFurthermore, Molecular Dynamic Simulation studies unveiled a noteworthy interaction between the metal ion, Fe\u003csup\u003e2+\u003c/sup\u003e, and the ligand molecules, indicating enhanced stability in these interactions. The iron-chelating property is a salient characteristic of many siderophores, and in the same lines our in-silico investigations have yielded promising results.\u003c/p\u003e \u003cp\u003eHowever, it is essential to underscore that the validation of this study remains a critical step to substantiate our hypothesis. Further research and experimental investigations are necessary to confirm the practical implications of these findings and to assess the potential of the identified compounds as effective PHD2 inhibitors.\u003c/p\u003e","manuscriptTitle":"Molecular Docking and Dynamic Simulation Studies of PHD2 Interactions with Gut Siderophores: Implications for HIF-1α Stabilization","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-09 20:56:46","doi":"10.21203/rs.3.rs-3714812/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"260bedff-2681-4a1b-a6c4-68e5fdc9f716","date":"2023-12-21T09:36:19+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-12-21T07:45:08+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-12-21T07:05:45+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2023-12-11T18:47:02+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-12-11T17:31:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2023-12-06T11:58:52+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"660de60c-61c9-4cc6-80a2-db3c02bbb4c3","owner":[],"postedDate":"August 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":27741703,"name":"Biological sciences/Computational biology and bioinformatics"},{"id":27741704,"name":"Biological sciences/Drug discovery"}],"tags":[],"updatedAt":"2024-08-09T20:56:46+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-09 20:56:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3714812","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3714812","identity":"rs-3714812","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}