Identification of Small Molecule Inhibitors Targeting Phosphoserine Phosphatase: A Novel Target for the Development of Antiamoebic Drugs

Abstract

Amoebiasis, a widespread disease caused by the protozoan parasite Entamoeba histolytica, poses challenges due to the adverse effects of existing antiamoebic drugs and rising drug resistance. Novel targeted drugs are in need of the hour to combat the prevalence of this disease. Given the significance of cysteine for Entamoeba survival, the rate-determining step in the serine (the sole substrate of cysteine synthesis) biosynthetic pathway, i.e., the conversion of 3-phosphoserine to l-serine catalyzed by phosphoserine phosphatase (PSP), emerges as a promising drug target. Our previous study unveils the essential role of EhPSP in amoebas' survival, particularly under oxidative stress, by increasing cysteine production. The study also revealed that EhPSP differs significantly from its human counterpart, both structurally and biochemically, highlighting its potential as a viable target for developing new antiamoebic drugs. In the present study, employing in silico screening of vast natural and synthetic small chemical compound libraries, we identified 21 potential EhPSP inhibitor molecules. Out of the 21 compounds examined, only five could inhibit the catalytic activity of EhPSP. The inhibition capability of these five compounds was subsequently validated by in silico binding free energy calculations, SPR-based real-time binding studies, and molecular simulations to assess the stability of the EhPSP-inhibitor complexes. By identifying the five potential inhibitors that can target cysteine synthesis via EhPSP, our findings establish EhPSP as a drug candidate that can serve as a foundation for antiamoebic drug research.

Figure 1

Enzyme inhibition activity with different compounds. Plot for % inhibition versus the log of inhibitor concentrations for EhPSP. Inhibition curves for (A) AN-967/15490027, (B) A0-476/43407254, (C) AE-562/12222184, (D) STK184776, and (E) raltitrexed at various concentrations. (F) Tabulated IC₅₀ values, docking scores, and MM-GBSA values (ΔG of binding).

Figure 2

2D configuration of the lead molecules and substrate of EhPSP. The identified potential molecules belonged to diverse classes, such as AO-476/43407254, a phenylpyridazine; AE-562/12222184, a naphthalenesulfonate; STK184776, an azole; raltitrexed, a quinoline folate analogue; and AN-967/15490027, an alkaloid.

Figure 3

3D representation displaying the binding poses of the potential inhibitors within the active site of EhPSP. EhPSP complexed with OPLS (substrate) and inhibitors AE-562/12222184, AN-967/15490027, raltitrexed, AO-476/43407254, and STK184776. The compounds are displayed as balls and sticks, while the protein is represented in the surface view (A) and cartoon representation (B), with inhibitor molecules in the active site. The figure was generated using Maestro (version 2019-1).

Figure 4

Illustration of the interactions between potential inhibitors and the active site residues of EhPSP. Interactions are plotted for substrate (A) OPLS and compounds (B) AE-562/12222184, (C) AN-967/15490027, (D) raltitrexed, (E) STK184776, and (F) AO-476/43407254. The figure was generated by using LigPlot. Hydrogen bonds are represented by dashed lines between the atoms involved, while hydrophobic contacts are depicted as an arc with spokes radiating toward the ligand atoms they interact.

Figure 5

Binding studies of EhPSP with the substrate OPLS and inhibitors using SPR. The sensorgram depicting binding responses for the (A) substrate OPLS and small molecules (B) AO-476/43407254 and (C) AN-967/15490027 at different concentrations. (D) Tabular representation of the dissociation constant (K_D) values of the substrate and two inhibitors.

Figure 6

MD simulations of EhPSP–potential inhibitor complexes. Panel (A) illustrates the RMSF trajectory analysis of the backbone atom within the EhPSP–inhibitor complexes, while panel (B) presents the RMSD plotted against time for the EhPSP–inhibitor complexes. RMSD and RMSF were computed based on the initial and final conformation differences. The graphs encompass various runs, with the unbound EhPSP shown in blue, OPLS in green, AO-476/43407254 in a distinctive yellow shade, STK184776 in black, raltitrexed in red, AE-562/12222184 in purple, and AN-967/15490027 in algal green blue.

Figure 7

Histogram depicting various interactions and their respective levels between the potential inhibitors and the binding pocket of EhPSP, as monitored throughout the simulations. The panel represents the interactions of the binding pocket of EhPSP with the compounds: (A) OPLS, (B) AE-562/12222184, (C) AN-967/15490027, (D) AO-476/43407254, (E) raltitrexed, and (F) STK184776.