2.1 In Vitro ACE Inhibition Assay
Since there was no suitable ACE2 kits compatible with the Glomax Discover Microplate Reader used in this project, in vitro inhibition assays were only performed against ACE1.
The inhibition of ACE1 was tested using the absorbance-based calometric ACE1 Kit-WST (Dojindo Laboratories, Mashiki-machi, Japan) following the manufacturer’s instructions. Custom synthesized hemorphin peptides listed in Table 1 were ordered from Watson Biosciences (Houston, TX, USA). Sample serial dilutions were prepared with concentrations of 0.1, 1, 2, 5, 15, 25, 75 µM for camel LVVHem6 and LVVHem6; and 5, 10, 25, 35, 50, 75, 100 µM for camel LVVHem5, camel LVVHem7, VVHem6, LVVHem7, LVVHem5, and camel7. Briefly, 10 µL of each concentration of hemorphin was added to the wells of a 96-well microplate. 20 µL and 40 µL of deionized water were added in blank 1 and blank 2 wells, respectively.
20 µL of the substrate buffer was added to each well, followed by 20 µL of enzyme working solution in all of the sample wells and blank 1.
The plate was incubated for 1 h at 37℃. fter the incubation period, 200 µL of indicator working solution was added to each well and then incubated at room temperature for 5 min. Following incubation, plate absorbance was measured at 450 nm using Glomax Discover Microplate Reader (Promega, Madison, WI, USA). The inhibition of ACE1 was measured using the below equation:
𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 𝑟𝑎𝑡𝑒 (%) =(Blank1 − Sample)
(Blank1 − Blank2)× 100
where blank1 and blank2 are 20 µL and 40 µL deionized water, respectively.
The half-maximal inhibitory concentration (IC50) was calculated using GraphPad Prism version 9 (GraphPad, San Diego, CA) from a non- linear regression plot of inhibition against peptide concentration.
Initially, all eighteen peptides were screened in a single run. The top eight peptides in terms of ACE1 inhibition were selected for triplicate runs followed by further computational analysis.
2.2 Protein Structure Pre-Processing
The three-dimensional structure of ACE1 (PDB ID: 2XY9) (Akif et al., 2011) and ACE2 (PDB ID: 1R4L) (Towler et al., 2004) were retrieved from RCSB Protein Data Bank (PDB). The retrieved protein structures were pre-processed using the Protein Preparation Wizard of Schrödinger Suite 2021-1 (Schrödinger Release, 2021a). The protein preparation involved proper bond orders assignment, ionization states adjustment, orientation of disorientated groups, disulphide bond creation, unwanted water molecule metal and co-factors removal, terminus amide capping, partial charge assignment, and the addition of side chains and missing atoms. Additionally, hydrogen atoms were added and pH 7 was set as the standard protonation state. Finally, the protein structures were optimized and minimized to ensure geometrically structural stability (Sastry et al., 2013).
2.3 Active Site Identification and Grid Generation
Using default parameters and the OPLS 2001 force field, a receptor grid was setup in the vicinity of the active site of the pre-processed protein by selecting the active site residues. For this step, a van der waal’s scaling factor of 1 and a charge cutoff of 0. 5 was used. cubic search space centered around the centroid of the active site residues of ACE1 and ACE2 was then generated.
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2.4 Peptide Docking
Peptide docking was performed to determine the most probable binding mode of each peptide with ACE1 and ACE2, and to analyze the molecular interactions in the complex, as well as to estimate the binding free energy. Standard precision (SP) flexible docking was performed using Schrödinger Glide version 2021-1 for the docking (Friesner et al., 2004) using default parameters. The peptides were generated using the 3D builder tool to create extended conformations of the peptides to ensure flexible docking to ACE1 and ACE2. The poses were ranked on the basis of the GlideScore (GScore) scoring function (Friesner et al., 2006). Finally, the three lowest GScore values of the best docked poses were selected for further analysis.
2.5 Analysis of Docking Results and Binding Free Energy Calculation
Schrödinger Maestro was employed for post docking visualization and data analysis of different types of interactions, including hydrogen bonds, hydrophobic interactions, salt bridges, - and -cation contacts (Schrödinger Release, 2021b). Using molecular mechanics-generalized Born surface area (MM-GBSA), binding free energy of the best docked poses was computed. The MM-GBSA was calculated using Schrödinger Prime using the VSGB 2.0 implicit solvent model and the OPLS 2005 force field (Schrödinger Release, 2021c; Li et al., 2011).
2.6 Molecular Dynamics (MD) Simulations
The function of most proteins is contingent upon its dynamics. Thus, it is essential to assess the dynamic motions of the docked complexes to calculate the strength of inter- and intra- molecular contacts and the stability of the ACE1-hemorphin and ACE2-hemorphin complexes. For this, MD
simulations of the docked complexes were performed to evaluate the dynamics and stability of the best binding pose of the top-eight peptides identified in the in vitro inhibition assays. MD simulations of the protein- peptide complexes were performed using Desmond employing the OPLS 2005 force field (Schrödinger Release, 2021d).
Sixteen simulation systems – ACE1 and ACE2 with each of the top- eight peptides against ACE1 – were prepared. One hundred nanosecond (ns) MD simulation was performed for each protein-hemorphin complex on workstations equipped with graphics processing units (GPUs). Single point charge (SPC) water model was employed for system solvation in an orthorhombic box of water molecules, with a 10 Å buffer distance (Mark and Nilsson, 2001). The simulation system was then neutralized with an appropriate number of counterions and a salt concentration of 0.15 M NaCl was established. Prior to carrying out the MD simulations, the systems were subjected to the steepest descent minimization with Desmond’s default protocol.
ll the systems were also subjected to Desmond’s default eight stage relaxation protocol prior to the start of the 100 ns run (Zhang et al., 2012).
For the simulations, the Nose-Hoover thermostat and the isotropic Martyna- Tobias-Klein barostat were used to maintain a temperature at 300 K, pressure at 1 atm (Martyna et al., 1992; Martyna et al., 1994).
Long-range and short-range and coulombic interactions were evaluated with a cutoff of 9.0 Å using the the smooth particle mesh Ewald method (PME) and short-range approach, respectively (Essmann, 1995). A time-reversible reference system propagator algorithm (RESPA) integrator was used with an an outer time step 6.0 fs and inner time step of 2.0 fs (Humphreys et al., 1994). Data was retrieved to simulation trajectories every 100 ps. Finally, protein-ligand contacts, root mean square fluctuation
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(RMSF) and root mean square deviation (RMSD) of the complexes were calculated from the trajectories.