Page 90 - IMO-2-2

Innovative Medicines & Omics Femtomolar inhibition of pseudoeriocitrin

protein preparation. For this purpose, target proteins targets. The homology models and Ramachandran plots
and enzymes found in nematodes, as well as their human are shown in the Appendices. Two web servers, the SWISS-
homologs with crystallized structures, were obtained from MODEL and Zhang lab I-TASSER, 22-24 were utilized
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the Brookhaven Protein Databank (http://www.rcsb.org/ to develop a sequence overlap-based model. The most
pdb). These target proteins and enzymes are known targets appropriate 3D structure was selected based on Global
of anthelmintic drugs, such as albendazole (β-tubulin Model Quality Estimating (GMQE) and Qualitative
inhibition), thiabendazole (fumarate reductase inhibition), Model Energy Analysis (QMEAN) values. For QMEAN,
and certain inhibitors used in Onchocerca lienalis infection values below 4.0 indicate reliability, while for GMQE, the
(carnitine o-palmitoyltransferase inhibition). Human highest value between 0 and 1 represents the most reliable
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homolog proteins were used to evaluate the selectivity predicted structure.
of the inhibitor. The selected proteins were as follows:
Ascaris suum fumarate reductase (AsFR) enzyme (PDB ID: 2.4. Docking process
4YSX, mitochondrial rhodoquinol-fumarate reductase, Proteins retrieved from the Protein Data Bank or those
resolution: 2.25 Å, bound with NN23 inhibitor); human with 3D structures predicted by homology modeling were
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fumarate reductase (hFR) enzyme (PDB ID: 6VAX, docked with ligands using AutoDock4.2. All energies
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resolution: 2.59 Å); human β-tubulin protein (PDB used in the calculation of molecular free binding energy
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ID: 6E7C, resolution: 3.65 Å); Haemonchus contortus (ΔG) in the AutoDock program were described by Morris
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β-tubulin protein (PDB ID: 1OJ0, in complex with ABZ, et al. The formula to calculate free binding energy is as
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theoretical structure); rat CPT 2, a target of anthelmintic follows:
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drug (PDB ID: 2H4T, resolution: 1.90 Å, bound with
dodecane [C H ]; and PDB ID: 2FW3, resolution: 2.50 ΔG=RTlnK i (I)
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Å, in complex with antidiabetic drug ST1326 ). The work R is the gas constant (R = 8.314 J/K/mol). T represents
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of Taylor et al. was used as a reference for identifying some the temperature of the environment measured in Kelvin.
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target proteins. Since the crystallized form of β-tubulin
from H. contortus was unavailable in protein databases, The Ki represents half of the substrate concentration
required for maximum inhibition of protein-ligand
its theoretical structure was used. Ions and ligands, except binding.
for cofactors and water molecules, were removed. Missing
hydrogen atoms were added, and the residues of the The grid box size was determined based on the ligand
proteins were checked for missing atoms and bonds. After size or the number of torsions. The cofactor of the protein
adding all hydrogen atoms, the proteins were optimized was chosen as the center, or in the absence of a cofactor,
using the “Clean Geometry” tool, followed by the Charm the native ligand bound to the protein was placed at the
forcefield. The optimized proteins were saved in PDB center of the grid box. For the β-tubulin protein docking
format using Discovery Studio 2020 Client, then opened simulation, the ligand bound to the protein was selected
using ADT to add Gasteiger charges, and finally saved in as the center. For the CPT 2 enzyme, the coordinates
PDBQT format. provided by Taylor et al. were used as a reference. Atoms
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at the active site of the protein were allowed to move freely,
2.3. Homology modeling while the rest of the protein remained rigid. To dock
The known protein sequences for S. obvelata were searched proteins with their natural ligands in their co-crystallized
using the UniProt Knowledgebase. Although the COX1 form, the natural ligands were used as a reference. The
and COX2 proteins from the mitochondrial genome are dielectric constant was set to 10, the ionic strength to
not known targets of anthelmintics, they were selected 0.145, the dimensions of the grid box to 60 × 60 × 60, and
because they are vital to oxyurid nematodes. Since the the grid point to 0.375 Å. Since the number of rotational
genome sequence of S. obvelata and the experimental bonds was <10, the maximum number of generations was
crystal structure of the S. obvelata COX1 (SoCOX1) set to 27,000 and the maximum number of extensions was
and COX2 (SoCOX2) proteins are unknown, homology set to 2,500,000. The docking procedure was performed
modeling was employed to predict their 3D structures. using the Lamarckian Genetic Algorithm 4.2. AutoDock
Proteins from other oxyurid nematodes, E. vermicularis 4.2 scoring functions were used to generate ten different
β-tubulin (EvTub) protein and Caenorhabditis elegans conformations for each ligand. After sorting the ligands by
glucose transporter 1 (CeGLUT1) receptor were also their free binding energy, the interactions at the binding
in silico modeled using homology modeling, as they are site of the ligands were analyzed using ADT and Biovia
essential for nematodes and represent potential drug Discovery Studio 2020 Client.

Volume 2 Issue 2 (2025) 84 doi: 10.36922/imo.6026