3.1 Computational modeling
VS was used to screen potential FtsZ inhibitor molecules from the ChemDiv compound library (~ 1.6 million molecules). To make the whole VS process fast and accurate, before molecular docking, pharmacophore models and structural model were used for screening.
3.1.1 Construction and screening of pharmacophore models
A pharmacophore model based on receptor-ligand interactions was constructed and validated using the "Receptor-Ligand Pharmacophore Generation" module in Discovery Studio 2016. In this study, the best crystal structure complex of FtsZ was selected. First, 10 pharmacophores were generated using the "Receptor-Ligand Pharmacophore Generation" module of Discovery Studio 2016, and each model could contain 3–4 features. The specificity of the pharmacophore was then tested using Discovery Studio's compound library by selecting "DruglikeDiverse [5384mol]" in the "Search 3D Database" module. " in the "Search 3D Database" module, and poorly specific models were excluded by comparing the percentage of results screened for each model. Finally, the compliant pharmacophore models were selected based on the key hydrogen bonding interactions in the FtsZ eutectic complex.
A 3D conformational database of ChemDiv compounds was constructed using the "Build 3D Database" module of Discovery Studio. All conformations in the database were mapped to pharmacophore models using a rigid fitting algorithm ("FAST" search) in the "Build 3D Database" module. The FitValue score is a measure of the similarity of each conformation to the pharmacophore model. Compounds with a score greater than 2 were retained in order of FitValue score.
3.1.2 Construction and screening of shape model
Shape models were constructed using ROCS (3.3.1.2 Inc., OpenEye Scientific Software Inc.) based on the binding conformation of the homologous ligand RJ5 in the eutectic structure. The shape models were used for the next step of screening, where shape similarity was measured by the ShapeTanimoto score. ShapeTanimoto score values greater than 0.65 compounds were saved during the screening process.
3.1.3 Molecular docking
Molecular docking was performed using FRED. First, a maximum of 200 conformations per compound were generated using OMEGA, placed at the binding site to the receptor, and scored using the Chemgauss4 scoring function. Compounds with scores below − 13 were retained and visually inspected for binding modes. Compounds that formed hydrogen bonding interactions with VAL207, LEU209 or Asn263 were retained.
Structural clustering based on FCFP_6 fingerprints was performed using Discovery Studio's “Cluster Ligands” module. One or two compounds from each cluster were selected by visual inspection, prioritizing compounds with higher FitValue, ShapeTanimoto score and Chemgauss4 score and better synthetic feasibility.
3.1.4 Molecular dynamics simulation
Molecular dynamics (MD) simulation was performed according to the published protocol[14, 15]. The FtsZ protein topology file was constructed with the GROMOS96 43A1 force field [16]. The ligand topology file was constructed by the LigParGen server (http://zarbi.chem.yale.edu/ligpargen/) [17], with the PDB coordinates as the input. Then, the GROMACS software (version 2019.4) was used to perform MD simulations of the FtsZ-ligand complex [18]. The system consisted of single point charge water to solvate the entire system. The water box was then extended by 10 Å from the periphery of the system in each dimension. Than, 16 Na + were added to the system to make the total charge become zero. The MD simulation included energy minimization, equilibration, and production phases. The simulation started with 5000 steps of energy minimization based on steepest descent algorithm. In the equilibrium phase, 500 ps simulation for NVT and 500 ps simulation for NPT were included. The system was maintained at a pressure of 1 atm using Parrinello Rahman and a constant temperature of 300 K using V-rescale. Lastly, 100-ns MD simulation without restraint was performed at NPT. The coordinates of the system were saved every 100 ps during the simulation.
3.2 In vitro biological evaluation
3.2.1 FtsZ protein expression and purification
The gene for FtsZ of S. aureus origin was synthesised in vitro, and the FtsZ gene was cloned into the pET-28b (+) vector using BamHI and HindIII as cleavage sites, and a 6×histidine tag (His-Tag) was pre-inserted at the N-terminal end of the expressed one for purification by affinity chromatography. The pET-28b-FtsZ exprssion plasmid was transformed into BL21 (DE3) competent cells, and FtsZ protein expression was induced using 0.5 mmol/L of Isopropyl b-D-1-thiogalactopyranoside (IPTG) for 16 h at 16℃. The bacteria are collected after fermentation, and resuspended and lysed using sonication in lysis buffer (50 mM HEPES, 500 mM NaCl, 1 mM EDTA, Ph 7.4). The lysate was centrifuged at 4°C, 12500 rpm for 1 h, and the resulting supernatant was subjected to affinity chromatography using a Ni2+ chelated affinity chromatography column with nickel binding for 1 h. Elution was performed with elution buffer (50 mM HEPES, 500 mM NaCl, 250 mM imidazole, 1 mM EDTA, pH = 7.4). The purified proteins were subjected to concentration determination and SDS-PAGE running gel for purity determination. FtsZ protein was aliquoted, flash frozen, and stored at -80℃.
3.2.2 FtsZ activity and inhibition assay
Principle of FtsZ activity assay: GTPase can catalyse the decomposition of GTP into GDP and phosphate ions, which can form green complexes with malachite green and molybdate. The GTPase activity of FtsZ was calculated by detecting the amount of free phosphate generated from hydrolysed GTP per unit time of FtsZ protein at 620 nm[19]. To determine the activity of FtsZ, add 5 µM FtsZ, buffer (250 mM HEPES, 250 mM KCl, 5 mM EDTA), GTP, water and MgCl2 to a 96-well plate (100 µL), mix well and react for 20 min at 37 ℃, then add the acidic solution (5 µL) and react for 10 min at room temperature (25 ℃), protected from light. Add blue solution (15 µL and react for 20 min at 25°C, protected from light. The absorbance was measured at 620 nm.
Using the above established enzyme activity assay, 38 compound samples were initially screened at a final concentration of 50 µM. The compounds with enzyme inhibition greater than 50% obtained from the screening were subjected to multiplicative dilution to obtain the inhibition rate at different concentrations, the logarithm of the inhibitor concentration was taken as the horizontal coordinate and the enzyme activity as the vertical coordinate, and then the IC50 value could be obtained by fitting the curve using the software Graphpad Prism 5.
3.2.3 Antimicrobial Testing
MIC is an important index for evaluating the in vitro antimicrobial effect of compounds, and the twofold dilution method is usually used to determine the MIC values of the target compounds and control drugs[20, 21]. The concentration of the sample storage solution to be tested was dissolved in DMSO to 6.4 mg/mL. A bacterial suspension of S. aureus strain (equivalent to a bacterial suspension of 0.5 McFarland turbidity standard), was diluted with liquid LB medium to obtain a final inoculum of 105 CFU/mL. 196 µL of the inoculum was added to A1-H1 of a 96-well plate, and the rest of 100 µL of liquid LB medium was added to each well. Add 4 µL of sample storage solution to A1-H1 of the 96-well plate and mix well. Then 100 µL was pipetted from well A1-H1 and added to A2-H2 and mixed well. Another 100 µL was pipetted from A2-H2 and added to A3-H3 and mixed well, and so on until it was added to wells A10-H10, and 100 µL of liquid was pipetted and discarded from the tenth column of wells. The above 96-well plate was placed at 37℃ for 17–20 h. After incubation, bacterial growth was observed and the minimum inhibitory concentration (MIC) value was determined by visual inspection as the lowest dilution of the compound without turbidity.