Top ranking ligands (Natural compounds from Zinc database) after virtual screening against EhOASS using GLIDE and GOLD docking programs.

<p>Top ranking ligands (Natural compounds from Zinc database) after virtual screening against EhOASS using GLIDE and GOLD docking programs.</p

Virtual Screening, Identification and In Vitro Testing of Novel Inhibitors of O-Acetyl-L-Serine Sulfhydrylase of <em>Entamoeba histolytica</em>

<div><p>The explosive epidemicity of amoebiasis caused by the facultative gastrointestinal protozoan parasite <em>Entamoeba histolytica</em> is a major public health problem in developing countries. Multidrug resistance and side effects of various available antiamoebic drugs necessitate the design of novel antiamobeic agents. The cysteine biosynthetic pathway is the critical target for drug design due to its significance in the growth, survival and other cellular activities of <em>E. histolytica</em>. Here, we have screened 0.15 million natural compounds from the ZINC database against the active site of the EhOASS enzyme (PDB ID. 3BM5, 2PQM), whose structure we previously determined to 2.4 Å and 1.86 Å resolution. For this purpose, the incremental construction algorithm of GLIDE and the genetic algorithm of GOLD were used. We analyzed docking results for top ranking compounds using a consensus scoring function of X-Score to calculate the binding affinity and using ligplot to measure protein-ligand interactions. Fifteen compounds that possess good inhibitory activity against EhOASS active site were identified that may act as potential high affinity inhibitors. <em>In vitro</em> screening of a few commercially available compounds established their biological activity. The first ranked compound ZINC08931589 had a binding affinity of ∼8.05 µM and inhibited about 73% activity at 0.1 mM concentration, indicating good correlation between <em>in silico</em> prediction and <em>in vitro</em> inhibition studies. This compound is thus a good starting point for further development of strong inhibitors.</p> </div

The plot between ΔF/F vs. inhibitor concentration follows a typical hyperbolic similar to Michaelis-Menton curve, the plot between 1/ΔF/F vs. inhibitor concentration (the reciprocal plot) follows that of a Lineweaver-Burk plot, the Kd values of inhibitor A) ZINC08931589 and B) ZINC03984585 were determined from these plots.

<p>The plot between ΔF/F vs. inhibitor concentration follows a typical hyperbolic similar to Michaelis-Menton curve, the plot between 1/ΔF/F vs. inhibitor concentration (the reciprocal plot) follows that of a Lineweaver-Burk plot, the Kd values of inhibitor A) ZINC08931589 and B) ZINC03984585 were determined from these plots.</p

Inhibition studies of EhOASS.

<p>The concentration of the inhibitor was kept at 100 µM and the readings were taken under two substrate concentrations; 0.5 mM (Km of the substrate) and 100 µM (for competitive inhibition, inhibitor and substrate were used same concentration).</p

Post-docking interactions between active site residues of protein with ligand (ZINC03984585).

<p>(A) The protein is depicted in surface view and ligand ZINC03984585 as stick in the binding pocket. (B) Schematic drawing of types of interactions of the ligands generated using Ligplot.</p

Calculated Hydrogen and Hydrophobic interactions for the top ten best ranking ligands based on Glide score and ligplot.

<p>Calculated Hydrogen and Hydrophobic interactions for the top ten best ranking ligands based on Glide score and ligplot.</p

Active site of EhOASS with reaction centre PLP (shown in blue) located at the middle of N and C terminal domain.

<p>A) EhOASS (in ribbon) and reaction centre PLP (in sticks). B) EhOASS with electrostatic surface view and reaction centre PLP deep-seated (in sticks) is in binding pocket.</p

Regulation of cysteine biosynthetic pathway through feedback inhibition of SAT by cysteine.

<p>Regulation of cysteine biosynthetic pathway through feedback inhibition of SAT by cysteine.</p