Molecular docking is the computational simulation of protein-ligand interactions based on the relative orientation and binding affinity during the molecular recognition process. Molecular docking tools are extremely important for experimental design and the virtual screening of compounds for pharmacological use. Autodock Vina is a computational programme developed by the Molecular Graphics Lab that allows the prediction of small molecule binding to a macromolecule or protein with a known crystal structure (Morris). AutoDock Vina has been successfully employed for the virtual screening of SARS-CoV-2 main protease (Mpro) inhibitors or (Abdellah) and ligands of the human small transmembrane protein sigma 1 receptor .
By using AutoDock Vina, we aimed to identify a plant-derived compound that could serve as a potential urease inhibitor for our chassis to secrete. We mainly focused on quercetin structural analogues retrieved from the PubChem chemical database in order to identify a compound that could serve as a potential urease inhibitor. We screened a total of 27 compounds and performed three iterations of the cycle over three different search spaces in both the Helicobacter pylori and the Klebsiella aerogenes ureases. We compared the output binding modes produced by Vina in the molecular visualization tool PyMol
Figure 1. Crystal structure of Helicobacter pylori urease view on PyMol (right and magenta) Crystal Structure of Klebsiella aerogenes Urease (left and cyan). The active site containing the two Nickel ions is highlighted in green.
The three dimensional X-ray crystallographic structure of the urease enzymes from H. pylori and K. aerogens (PDB ID: 1E9Z and 1FWJ) were retrieved from the Protein Data Bank (PDB). The ligand structures were prepared for docking by using the Open Babel software tool and converted into pdbqt files [open babel]. The PyRx tool was connected to Autodock vina to remove solvent molecules from the protein structure. The search space used for docking with AutoDock Vina was selected with PyRx. This search space encompasses the active site of the urease enzyme (search space is specified below).
For each protein structure, we iterated around the active site to increase the accuracy of the search space where AutoDock Vina would identify possible ligand-protein binding conformations. Compared to the first the search space, the second search space was enlarged, and the third was minimized. All these search spaces included the active site of the ureases and the flap motif.
Based on the biosafety considerations for our urease inhibitor, we have searched the PubChem Database for Quercetin derivatives to evaluate for molecular docking. We evaluated a total of 27 compounds for molecular docking as summarised in Table 1 and compared their binding affinities (kcal/mol) with the binding affinities from urea and acetohydroxamic acid. We were unable to run Quercetin in (PubChem CID: 5280343) AutoDock Vina as Quercetin produced too many binding modes (up to 739) with both tested urease enzymes. Consequently, we used Quercetin depositor-supplied synonyms compounds including 3,5,7,3',4'-Pentahydroxyflavone, HMS3414J21, Quercetin_117-39-5_ID441586707, Quercetin_6151-25-3_ID438465356, and Quercetin_NCGC00015870-48_ID434294964 (table 1). All these Quercetin compounds have the same chemical formula C15H10O7 but differ in their stereo conformation and binding orientation .
|Compound||Formula||PubChem CID||Compound||Formula||PubChem CID|
|Urea||CH4N2O or NH2CONH2||1176||Acetohydroxamic Acid||C2H5NO2||1990|
|Quercetin 3,4'-diglucoside||C27H30O17||5320835||Quercetin 3-O-malonylglucoside||C24H22O15||5282159>|
|Quercetin 7-O-glucoside||C21H20O12||5381351||Quercetin D5||C15H10O7||12305312|
Table 1.-Compounds selected for molecular docking with AutoDock Vina against the Heliobacter pylori and Klebsiella aerogenes ureases.
AutoDock vina output files result in up to 9 different binding possibilities for each compound in decreasing binding affinities with the protein macromolecule. From the different search spaces we narrowed down our results based on the compounds that resulted in the highest binding affinity on the first possible binding conformation with the ureases and these are summarised in table 2.
Table 2 - Compounds that exhibited the highest binding affinity to the Helicobacter pylori and Klebsiella aerogenes ureases. The affinity values represent the first binding mode in kcal/mol.
|Helicobacter pylori||Compound||Search Space 1 (kcal/mol)||Search Space 2 (kcal/mol)||Search Space 3 (kcal/mol)|
|Klebsiella aerogenes||Compound||Search Space 1 (kcal/mol)||Search Space 2 (kcal/mol)||Search Space 3 (kcal/mol)|
Ligands that exhibit similar binding affinities might also exhibit different kinetics and binding conformations with a target molecule. Consequently, we analysed the binding profiles of the compounds in Table 2 with both bacterial ureases in PyMol to further visualise how the ligands interacted with the enzymes. We noticed that although a compound exhibited a high binding affinity such as Quercetin HMS3414J21 and Quercetin_NCGC00015870-48 (ID434294964), not all compounds would interact with the active site of the urease within their first 3 binding conformations. Using PyMol, we further identified three compounds that bound with high affinity to both urease enzymes within their first binding conformation: Quercetin (3,5,7,3',4'-Pentahydroxyflavone) and the structural derivatives Quercetin 3,4'-diglucoside and Quercetin 7-O-glucoside (Table 3 and Figures 2 and 3).
Table 3 - Compounds that exhibited the highest binding affinity to the Helicobacter pylori and Klebsiella aerogenes ureases within the first binding conformation viewed on PyMol. The affinity values represent the first binding mode in kcal/mol.
|Heliobacter pylori||Compound||Affinity (kcal/mol)|
|Klebsiella aerogenes ureases||Compound||Affinity (kcal/mol)|
Figure 2.- Crystal structure of Helicobacter pylori urease view on PyMol in complex with Quercetin 3,4'-diglucoside (a), Quercetin 7-O-glucoside (b), and 3,5,7,3',4'-Pentahydroxyflavone or Quercetin (c). The ligand visuals are in pdbqt format and correspond to the output file produced by AutoDock Vina. The di-nickel ion active site of the urease is displayed in green and the ligand in light blue. Residues in the flap motif 321Cys (blue), 322/323Hist (Yellow), and 223Asp (Orange).
Figure 3.- Crystal structure of Klebsiella aerogenes urease view on PyMol in complex with Quercetin 3,4'-diglucoside (a), Quercetin 7-O-glucoside (b), and 3,5,7,3',4'-Pentahydroxyflavone or Quercetin (c). The ligand visuals are in pdbqt format and correspond to the output file produced by AutoDock Vina. The di-nickel ion active site of the urease is displayed in green and the ligand in light blue. Residues in the flap motif 321Cys (blue), 322/323Hist (Yellow), and 223Asp (Orange).
The docking simulations showed that ligands often interacted with the Asparagine (Asp) residue (orange residue on figure 2 and 3) located at position 221 in the H. pylori and at position 223 in theK. aerogenes urease. This residue is in close proximity to the αLys220 residue, previously suggested to play a role in the urease-inhibition process . For future improvements, we would like to design the metabolic pathway to further engineer our chassis to secrete Quercetin 3,4'-diglucoside, Quercetin 7-O-glucoside, and 3,5,7,3',4'-Pentahydroxyflavone (Quercetin). Additionally, we would like to perform wet laboratory experiments to assess whether these selected compounds display potential urease inhibitory activity in vitro. Nevertheless, we hope that our work will inspire future iGEM teams to harness molecular docking tools such as AutoDock Vina for the virtual screening of compounds with potential therapeutic use.