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INNOSC Theranostics and
Pharmacological Sciences Repurposed Drugs as inhibitors of Pfmrk

1.2 nm for both long-range and short-range interactions. within the grid box. The top five drugs – Lurasidone,
In addition, the equilibration procedure for number of Vorapaxar, Donovex, Alvesco, and Orap – exhibited the
particles, volume, and temperature (NVT) and number of lowest binding energy and demonstrated the maximum
particles, pressure, and temperature (NPT) was performed possible interaction with the amino acids situated within
for 1 ns. During this equilibration, the particle number, the enzyme’s active site (Figure 3).
volume, and temperature were held constant during Among these top five drugs, Lurasidone was found
the post-energy minimization. NVT equilibration was to exhibit the lowest binding energy of −12.03 kcal/mol
performed using the Berendsen thermostat [15,18] operating with an inhibitor constant (Ki) of 1.52 nM. Lurasidone
with a coupling constant of 0.002 fs at a temperature forms a non-covalent interaction (alkyl and pi-alkyl)
of 300 K. To identify long-range interactions, the PME with two critical residues, Ala140 and Phe143, located
method was employed with a cutoff value of 1.2 nm within the enzyme’s Protein Active Site (PAS). Vorapaxar
[19]
and a Fourier spacing of 0.16 nm. For NPT equilibration, exhibited a binding energy of −10.06 kcal/mol with a Ki
various parameters, including particle count, pressure, of 39.14 nM. Its interaction with the enzyme involves
and temperature, were fixed. Successful NPT equilibration a non-covalent interaction (pi-alkyl) with the essential
required the use of the Berendsen isotropic pressure, a residue Ile93. Besides these two drugs, Donovex, Alvesco,
time constant of 2 fs, a pressure bar of 1, and isothermal and Orap exhibited higher binding energy with higher
compaction of 4.5 × 10 bar. Following the completion Ki value. These drugs show non-covalent interactions
-5
of both equilibration steps, simulations for each system (alkyl and pi-alkyl) with Phe143 and Ala140 key residues
were executed for 100 ns with a time step (dt) of 2 fs and within the PAS of the enzyme. Furthermore, a significant
a leap-frog integrator . Finally, the simulation results of non-covalent interaction was observed between Pfmrk
[15]
all the systems were analyzed using standard commands and the drug in the ATP-binding region and around our
within the GROMACS platform. The LINear Constraint ligand-binding site. A comprehensive overview of these
Solver (LINCS) algorithm was used to restrain all bond interactions, binding free energy, and Ki, as determined
lengths [14,20] . through molecular docking, are shown in Table 2.
2.7. Molecular mechanics Poisson-Boltzmann 3.2. Molecular simulation
surface area analysis The Pfmrk and its associated ligand complexes were
The molecular mechanics Poisson-Boltzmann surface simulated for 100 ns, during which five different types of
area (MM-PBSA) method was performed using the analysis were performed to validate the docking results. To
gmx_MMPBSA tool and the pbsa.mdp script to evaluate assess the overall stability of the systems, the root-mean-
the free binding energy of the top five drugs binding to square deviation (RMSD) curve was obtained for both
Pfmrk, along with their respective binding modes. In Pfmrk and the holoprotein clusters using the gmx_rms
an effort to optimize computational efficiency, the free tool. Throughout the simulation, the gmx_gyrate tool was
energy evaluation was carried out for the final 40 ns of the used to calculate the radius of gyration (Rg) of the system.
simulation run. Dielectric constants for both solute and In addition, the root-mean-square fluctuation (RMSF)
solvent were set at 2 and 80, respectively [14,21,22] . for the total protein and complex was assessed using the
gmx_rmsf tool. To gain insights into the stability of the
2.8. Visual analysis Pfmrk and complex, the mean number of hydrogen bonds
The Discovery Studio Visualizer software was used to between them was calculated using the gmx_hbond tool.
generate a two-dimensional diagram representing potential An atomic distance cutoff of 0.35 nm was set between the
interactions within the protein-ligand complex, which donors and acceptors.
serves as a valuable resource for further study (Dassault 3.3. RMSD, Rg, and RMSF
Systemes BIOVIA 2020). Potential hydrophobic interactions
were analyzed using Protein-ligand Interaction Profiler . The comparison of the RMSD curves between Pfmrk and
[14]
the other five complexes indicates a close resemblance
3. Results between the Vorapaxar curve and that of Pfmrk. All five
drug complexes exhibited minimal fluctuations, with the
3.1. Docking results
curve of all the drugs converging within the range of 0.3 to
As mentioned earlier, the active site of Pfmrk includes 0.5 nm. Alvesco and Donovex demonstrated exceptional
non-conserved amino acids, namely, Phe15, Leu16, Lys23, stability throughout the entire simulation run of 100 ns.
Met75, Met91, ILe93, Tyr96, Ser138, Ala140, and Phe143. The Orap complex exhibited sudden fluctuations up to 40
The maximum number of amino acids was enclosed ns, after which the RMSD remained stable (Figure 4A). In

Volume 7 Issue 1 (2024) 4 https://doi.org/10.36922/itps.1313