XANTHINE OXIDASE INHIBITORY PROPERTIES OF 1,2,3,4-TETRAHYDROISOQUINOLINE DERIVATIVES

Xanthine oxidase (XO) is a versatile metalloflavoprotein enzyme that is best known for its rate-limiting role in the purine degradation pathway. Therapeutic inhibition of XO is based on its role in a variety of diseases that is attributed either to the hyperproduction of uric acid, or the hyperproduction of reactive oxygen species. Herein, we report the assessment of XO inhibitory properties of 24 1,2,3,4-tetrahydroisoquinoline derivatives, among which compound 16 exhibited IC 50 value of 135.72 ± 2.71 µM. The interaction of compound 16 with XO enzyme was simulated using the Site Finder module, molecular docking and molecular dynamics. Molecular modeling suggests that interactions with Met 1038, Gln 1040, Thr 1077, Gln 1194 and Val 1259 are an important factor for inhibitor affinity toward the XO enzyme. Our proposed binding model might be beneficial for the discovery of new active 1,2,3,4-tetrahydroisoquino-line-based inhibitors of XO enzyme. Acta Medica Medianae 2021;60(1):48-55.


Introduction
Xanthine oxidoreductase (XOR) is a versatile molybdopterin-containing flavoprotein enzyme that exists in two interconvertible forms, xanthine oxidase (XO) and xanthine dehydrogenase (XDH) (1). The enzyme is best known for its rate-limiting role in the purine degradation pathway, where it catalyzes oxidative hydroxylation of hypoxanthine and xanthine, subsequently producing uric acid (2, 3). Dehydrogenase form is predominant in healthy tissues, while conversion to XO occurs in pathological conditions, after XDH proteolysis or oxidation of some of its sulfhydryl residues (1). Superoxide anion radical and hydrogen peroxide that are generated as the byproducts of enzyme activity are responsible for oxidative stress which usually accompanies elevated XO activity. The role of XO in diseases is attributed either to the hyperproduction of uric acid, or the hyperproduction of reactive oxygen species. Therefore, pharmacological inhibition of xanthine oxidase is proven to be invaluable for the treatment of hyperuricemia and gout in the first place, but might also be beneficial for plethora of conditions, such as cholecystitis, hemorrhagic shock, ischemia-reperfusion injuries, hypercholesterolemia and carcinogenesis (4).
Given the broad therapeutic potential of XO inhibitors, the aim of the current study was to assess a group of 24 1,2,3,4-tetrahydroisoquinoline derivatives for potential inhibitory properties against XO and to perform molecular docking and molecular dynamics simulation on active compounds, in order to elucidate key structural features responsible for XO inhibitory activity.

Evaluation of xanthine oxidase inhibition
Compounds were studied for inhibitory properties against bovine milk xanthine oxidase. Spectrophotometric measurement of uric acid formation at 293 nm was used for in vitro evaluation of enzyme inhibition, as described in our previous studies (6,7). Initially, all compounds were assayed at a concentration of 150 µM, while those inhibiting more than 50% of enzyme activity were subsequently tested in a broader concentration range to allow for IC50 determination. Allopurinol was used as a positive control. All experiments were performed in triplicate and averaged.

Ligand preparation
Examined inhibitor was generated using the builder panel in the Molecular Operating Environment (MOE) 2019.0101 software (8). Using the MOE LigX module, partial atomic charges were ascribed and possible ionization states were generated at a pH of 7.0. The MMFF94x force field was used for optimization and the resulting structure was used for modeling studies. Conformational search was carried out by MOE LowModelMD method which performs molecular dynamic perturbations along with low frequency vibrational modes with energy window of 7 kcal/mol, and conformational limits of 1000.

Receptor preparation
The X-ray crystallographic structure of XO enzyme (PDB code: 1N5X), retrieved from the Protein Data Bank, was prepared using the Structure Preparation process in MOE. After the correction, hydrogens were added and partial charges (Gasteiger methodology) were calculated. Energy minimization (AMBER14:EHT, RMS gradient: 0.100) was performed.

Binding site selection
The Site Finder module of the MOE was used to identify possible ligand-binding sites within the optimized structure of XO enzyme. Hydrophobic or hydrophilic alpha spheres served as probes denoting zones of tight atom packing. These alpha spheres were utilized to define and rank potential ligandbinding sites according to their propensity for ligand binding (PLB) score, which was based on the amino acid composition of the pocket (9).

Docking protocol
The molecular docking study was performed using the MOE to understand the ligand/protein interactions in detail. The default Triangle Matcher placement method was used for the induced fit docking. GBVI/WSA dG scoring function which estimates the free energy of binding of the ligand from a given pose was used to rank the final poses. The ligand/protein complex with lowest relative binding free energy (ΔG) score was selected for further study.

Molecular dynamics simulation
The molecular dynamics simulation of compound 16 on XO enzyme, was carried out using the Desmond Molecular Dynamics System (Desmond) 2018.4 software (10). The structure of the added water was based on the simple point charge (SPC) solvent model. The system was neutralized with Na + ions to balance the net charge of the whole simulation box to neutral. The final system contained approximately 123,000 atoms. The system was passed through a 6-step relaxation protocol before molecular dynamics simulations. The relaxed system was simulated for 10 ns, using a normal pressure temperature (NPT) ensemble with a Nosé-Hoover thermostat at 300 K and Martyna-Tobias-Klein barostat at 1.01325 bar pressure. Atomic coordinate data and system energies were recorded every 1 ps. The root mean square deviation (RMSD) and root mean square fluctuation (RMSF) of the inhibitor/XO enzyme complex were analyzed with respect to the simulation time.
By combining a novel and highly effective algorithm for rapid binding-site evaluation with easy-to-use property visualization tools, Site Finder provides researchers with efficient means to identify and characterize binding sites (9). The results from the Site Finder analysis highlighted that catalytic residues like Gln 767, Glu 802, Arg 880, Phe 914, Phe 1009 and Glu 1261 (11,12) constituted the topranked binding pocket of XO enzyme (Table 2, Figure 1).
The intermolecular contacts between examined inhibitor and XO enzyme were analyzed using the ligand interaction diagram of MOE suite (Table 3, Figure 2). It illustrates the existence of hydrogen bond and pi-H interactions. Additionally, the bond distances, bond energy and binding free energy between the inhibitor and receptor atoms were also examined ( Table 3). The molecular docking highlighted the importance of Met 1038, Gln 1040, Thr 1077, Gln 1194 and Val 1259 in the formation of inhibitor/XO enzyme complex (Table 3, Figure 2). Observed interactions between 16 and non-catalytic residues (Table 3, Figure 2), are similar to molecular interplay involving non-purine XO inhibitors (13)(14)(15)(16). Some of recently synthesized pyrazole derivatives were found to inhibit XO enzyme via Met 1038, Gln 1040 and Gln 1194 residues (13). Furthermore, inhibitory effect of verbascoside on XO activity, can be attributed to the formation of hydrogen bond with Gln 1194 residue (14). Moreover, molecular simulation revealed that newly synthesized hesperidin derivatives interacted with XO residues Met 1038, Gln 1040, Thr 1077, Gln 1194 and Val 1259 (15). Additionally, molecular docking revealed that 1,4-dicaffeoylquinic acid interacted with XO enzyme via Gln 1040 and Thr 1077 residues (16).    while the solvent exposed part is shown as a red surface.
The study was further extended to assess the stability of 16/XO enzyme complex through the molecular dynamics simulation. The RMSD and RMSF plots for XO enzyme and examined inhibitor showed that docking complex was stable during entire simulation period (Figures 3-4). The RMSD for Cα, side chains and heavy atoms remained within the limit of 2 Å. The similar situation was noted for RMSF values. The obtained results indicated small structural rearrangements, less conformational changes and confirmed stability of 16/XO enzyme complex (17).

Conclusion
A series of 24 1,2,3,4-tetrahydroisoquinoline derivatives were screened for potential XO inhibitory properties. Among them, only compound 16 (IC50 = 135.72 ± 2.71 µM) exhibited IC50 value below 150 µM, and was further subjected to molecular docking and molecular dynamic simulation. Molecular modeling suggests that interactions with Met 1038, Gln 1040, Thr 1077, Gln 1194 and Val 1259 are an important factor for inhibitor affinity toward the XO enzyme. Observed interactions with non-catalytic residues, might be beneficial for the discovery of new active 1,2,3,4-tetrahydroisoquinoline-based inhibitors of XO enzyme.