Structure–Activity Relationships and Docking Studies of Hydroxychavicol and Its Analogs as Xanthine Oxidase Inhibitors

Abstract

Hydroxychavicol (HC), which is obtained from the leaves of Piper betle LINN. (Piperaceae), inhibits xanthine oxidase (XO) with an IC₅₀ value of 16.7 µM, making it more potent than the clinically used allopurinol (IC₅₀=30.7 µM). Herein, a structure–activity relationship analysis of the polar part analogs of HC was conducted and an inhibitor was discovered with a potency 13 times that of HC. Kinetic studies have revealed that HC and its active analog inhibit XO in an uncompetitive manner. The binding structure prediction of these inhibitor molecules to the XO complex with xanthine suggested that both compounds (HC and its analog) could simultaneously form hydrogen bonds with xanthine and XO.

Xanthine oxidase (XO) is a key enzyme in the purine metabolic pathway, catalyzing the oxidation of hypoxanthine to uric acid. In addition, it is known as a target protein for the treatment of hyperuricemia and gout.^1,2) It also produces reactive oxygen species (ROS) in parallel with the oxidation process. ROS have been associated with some pathological conditions, such as the post-ischemic reperfusion injury, diabetes, and chronic heart failure.³⁾ Therefore, the selective inhibitor of XO may result in a broad-spectrum chemotherapeutic agent applicable to gout, cancer, chronic inflammation, and oxidative damage.⁴⁾

Allopurinol (Fig. 1), a well-known XO inhibitor, has been widely prescribed for the treatment of gout for over 40 years.⁵⁾ Since allopurinol is a purine analog, it could affect the activities of an enzyme associated with purine and pyrimidine metabolism, thereby causing life-threatening side effects, such as the hypersensitivity, i.e., the Stevens–Johnsons syndrome.⁶⁾ For this reason, there is a critical need for the discovery of novel XO inhibitors independent of purine or pyrimidine scaffolds.⁴⁾ In this decade, highly potent XO inhibitors, such as febuxostat and topiroxostat (Fig. 1) with IC₅₀ values of 4.4 and 69 nM, respectively, have been developed.^7,8) Although these compounds are free from purine or pyrimidine scaffolds, liver dysfunction has been reported as a serious side effect from their use.⁹⁾ Therefore, novel XO inhibitors with fewer and milder side effects are required.^10,11)

Fig. 1. Structure of Alloprinol, Febxostat, Topiroxostat and HC

IC₅₀ values (µM) are shown in parenthesis.

From the extract of Piper betle LINN. (Piperaceae) leaves originating from South Asia and Southeast Asia, in 2009, Matsuda et al. discovered hydroxychavicol (HC), a molecule that possesses an XO inhibitory activity that is twice that of allopurinol¹²⁾ (Fig. 1). Although the XO inhibitory activity of HC is relatively low compared with febxostat and topiroxostat, its ligand efficiency index¹³⁾ value is as high as 0.60, indicating that HC is a good candidate as a seed compound for a modification study. Recently, Honda and Masuda reported that pyrogallol (Fig. 1) in coffee also possessed XO inhibitory activity.¹⁴⁾ It is interesting that such polyphonols contained in natural plants indicated XO inhibiting potency. In this research, therefore, the structure–activity relationship (SAR) of HC was investigated with a focus on the polar component of the molecule. The number and position of the hydroxy groups on the benzene ring were modified. In addition, the contribution of the allyl group was briefly examined. On the other hand, the inhibition pattern of HC and its active analog were studied, and the binding structures of these compounds were predicted by a docking study.

Results and Discussion

Chemistry

The designed HC analogs, including several known compounds, are listed in Table 1 in addition to allopurinol and HC. These compounds were synthesized according to previously published methods.^15–23) Briefly, HC was obtained by removing a methylene group of safrole. Chavibetol was synthesized via successive iodination and subsequent allylation by Stille coupling, followed by the deacetylation of methoxyphenyl acetate. 3-Allylphenol (1) was obtained from 1-bromo-3-methoxybenzene with an allyl Grignard reagent. 4-Allylphenol (2) was obtained by the demethylation of 4-allylanisole. 2-Allylhydroquinone (3) and 2-allylresorcinol (4) were obtained from 2-bromo-1,4-dimethoxybenzene and 1-bromo-3,5-dimethoxybenzene via the Grignard reaction and demethylation, respectively. 3-Allylcatechol (5) was synthesized from the corresponding methyl ether via demethylation. The monoallylation of resorcinol and subsequent Claisen rearrangement leads to 4-allylresorcinol (6) and 2-allylresorcinol (7) at once. Finally, using the same procedure, 5-allylpyrogallol (8) was synthesized from pyrogallol. It should be noted that all reactions were not optimized.

Table 1. In Vitro Xanthine Oxidase Inhibitory Activities of HC and Its Analogues

XO Inhibition

XO inhibition was measured according to the method developed by Chang et al.²⁴⁾ Allopurinol was used as a positive control, and all experiments were performed in triplicate. The experimental data were tested for statistical significance using the Bonferroni–Dunn multiple range test method. The IC₅₀ values of HC and its analogs in vitro are listed in Table 1.

SAR

First, we briefly examined the contribution of the alkyl group. Catechol, formed by dropping the allyl group from HC, and 4-methylcatechol were inactive. These results indicate that the allyl group may significantly contribute to the inhibitory activity of HC. However, while phenol and catechol lost their inhibitory activity, pyrogallol was as potent as allopurinol, with an IC₅₀ value of 36.5 µM. The third hydroxy group plays an effective role in enhancing any interaction. These results indicate that the allyl group is not essential in XO inhibition; however, it is an important pharmacophoric moiety for the HC analogs. Therefore, we examined the SAR of the polar site retaining the allyl group.

Because allylbenzene (Table 1) wherein two hydroxy groups on the benzene ring are removed from HC was inactive, as the next step, the hydroxy groups were capped by methyl group(s) to validate whether the hydroxy groups contribute as a hydrogen binding donor. Both monomethylated analogs, i.e., eugenol and chavibetol, and the dimethylated analog, i.e., methyleugenol, were inactive. This suggests that the hydroxy groups would be involved in hydrogen bond formation as proton donors. Subsequently, we examined whether the removal of one hydroxy group could be tolerated for the inhibitory activity; thus, three positional isomers, 2-, 3-, and 4-allylphenols, were tested. All three compounds were found to be inactive, indicating that at least two hydroxy groups were mandatory for XO inhibition. For this reason, all the dihydroxy derivatives of allylbenzene were tested. In total, there are six isomers, compounds 3–7 (Table 1) and HC. Interestingly, all isomers except HC were found to be inactive, indicating that the existence of at least two hydroxy groups at both the 3- and 4-positions is required for the inhibitory activity. Finally, 5-allylpyrogallol (8) comprising three hydroxy groups on allylbenzene at the 3-, 4-, and 5-positions was synthesized, and it indicated an IC₅₀ value of 1.27 µM. This indicates that 8 is 13 times and 29 times more potent than HC and pyrogallol, respectively.

Inhibition Patterns

To identify inhibition patterns of HC and 8, the inhibition kinetics of both compounds were analyzed. The Lineweaver–Burk plots shown in Fig. 2 revealed that both compounds inhibited XO by uncompetitive inhibition, indicating that these compounds inhibit XO by binding to the XO complex with the substrate. It should be noted that the type of inhibition of allopurinol and topiroxostat is competitive, and that of febuxostat is mixed.^25–27) Since the binding sites of HC and 8 were difficult to identify, it was assumed that each compound bound just adjacent to the substrate in the enzyme–substrate complex. From these observations, all hydroxy groups of catechol or pyrogallol units were found to form hydrogen bonds with XO and/or the substrate.

Fig. 2. Lineweaver–Burk Plot Analyses of XO by HC (a) and 8 (b) in the Absence (Control) and in the Presence of HC with Xanthine as the Substrate

(a) ◆: control, ▲: HC- 15 µM, ●: HC- 20 µM. (b) ◆: control, ▲: 8- 1 µM, *: 8- 2 µM, ×: 8- 3 µM, ●: 8- 5 µM, ■: and dashed line alloprinol 20 µM.

Docking Study

Finally, a docking study was performed to investigate the binding structures for HC and 8. The uncompetitive inhibition manner suggested docking both compounds against XO under a complex state with a substrate. Therefore, the docking study of HC was performed using the crystal structure of XO with xanthine (PDB ID: 3EUB)²⁸⁾ and the docking site was set to around the area where xanthine was bound. Using the MOE dock,²⁹⁾ three binding structures were suggested for HC: A, B, and C (Fig. 3). In candidate A, the catechol part of HC formed three hydrogen bonds, two of which were with xanthine and one was with the carboxyl group of Glu802 sidechain of XO (Fig. 3a). Conversely, the candidates B and C formed two hydrogen bonds with xanthine and XO (Figs. 3b, c). The binding free energies ΔG_bind for the three candidate structures were predicted using the molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) method³⁰⁾ and are listed in Table 2. Candidate A formed more hydrogen bonds than candidates B and C and thus had the highest affinity. It should be noted that HC in candidates B and C could not maintain the initial structures during molecular dynamics (MD) simulations (Figs. 3e, f), whereas candidate A maintained the docked structure keeping three hydrogen bonds (Fig. 3d). Consequently, candidate structure A was adopted as the most appropriate binding structure of HC. This structure can explain the active/inactive SAR of the monohydroxy and dihydroxy analogs.

Fig. 3. The Candidate Binding Structures for HC to the XO-Xanthine Complex

a), b), and c) Candidate structures A, B, and C. d), e) and f) Structures after three MD simulations for candidate A, B, and C (first: yellow, second: magenta, third: cyan). The blue dashed lines show hydrogen bonds.

Table 2. ΔG_bind (kcal/mol) by the MM-PBSA Method for the Predicted Binding Structures^a)

Structure		ΔGbind	ΔEvdW	ΔEele	ΔGPB	ΔGSA
HC	Candidate A	−18.6	−18.3	−7.5	8.7	−1.5
	Candidate B	−14.2	−14.2	−7.7	8.9	−1.3
	Candidate C	−15.3	−16.6	−4.5	7.1	−1.4
8		−21.4	−14.7	−24.7	19.6	−1.6

a) Average of n=3.

Utilizing the same protocol, the binding structure and the binding free energy of 8 were predicted. The most stable structure and its binding free energy are shown in Fig. 4 and Table 2, respectively. Compound 8 formed four hydrogen bonds through xanthine and XO, increasing preferable electrostatic interactions compared with HC. Due to this effect, 8 obtained a higher affinity than HC by 2.8 kcal/mol. The difference of the predicted ΔG_bind values between HC and 8 did not reproduce the experimentally estimated value of 1.4 kcal/mol, with a twofold overestimation. This discrepancy might be caused by the contribution of the binding entropy, which was being ignored by the prediction scheme.

Fig. 4. Predicted Binding Structure of 8 to the XO Complex with Xanthine

Hydrogen bonds were shown in blue dashed lines.

Conclusion

Overall, a SAR study of HC and its analogs was performed and a novel inhibitor (8) was discovered, with a potency 13 times that of HC (IC₅₀=1.27 µM). These compounds inhibited XO through an uncompetitive inhibitory mechanism. The docking study of HC and 8 suggested that HC and 8 formed three and four hydrogen bonds, respectively, with the XO–xanthine complex and that the predicted binding affinity qualitatively reproduced experimental values. To validate these predictions, hybrid compounds of HC (or 8) with xanthine (or allopurinol) are currently under development. If our prediction is pertinent, we can obtain more potent compounds bound in a competitive manner.

Experimental

Chemistry

General Methods

Xanthine oxidase, methyleugenol and allopurinol were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Phenol and pyrogallol were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Other chemicals and reagents were reagent grade and were purchased unless otherwise noted. Melting points were taken on Yanaco MP-S3 melting point apparatus and not corrected. NMR spectra were recorded on JEOL JNM-AI400 (¹H 400 MHz, ¹³C 100 MHz) or JEOL JNM-ECA500 (¹H 500 MHz, ¹³C 125 MHz) spectrometers in CDCl₃ with tetramethylsilane (TMS) as an internal standard. Mass spectra (electron ionization (EI)) were recorded on a JMS-HX100 spectrometer. Infrared spectra were recorded on a Jasco FT/IR-460 Plus spectrophotometer. Microanalysis was performed on Yanaco MT-3. Unless otherwise noted, all materials were obtained from commercial suppliers and used without further purification. TLC was carried out on a Merck precoated Silica gel 60 F254. Unless otherwise noted, all materials were obtained from commercial suppliers and used without further purification. Flash column chromatography was performed on Merck Silica gel 60 (230–400 mesh ASTM) according to Still’s method. All spectroscopic data for known compounds were in complete accord with literature values.

Registry Numbers of Synthesized Compounds

Hydroxychavicol: 1126-61-0, Chavibetol: 501-19-9, 3-Allylphenol (1): 1446-24-8, 4-Allylphenol (2): 501-92-8, 2-Allylbenzene-1,4-diol (3): 5721-21-1, 5-Allylbenzene-1,3-diol (4): 142039-78-9, 3-Allylcatechol (5): 1125-74-2, 4-Allylresorcinol (6): 1616-52-0, 2-Allylresorcinnol (7): 1746-89-0, 5-Allylpyrogallol (8): 850896-26-3.

Syntheses of the Test Compounds

Hydroxychavicol¹⁵⁾

To a solution of safrole 0.45 mL (3 mmol), BBr₃ 1.22 g (3.3 mmol) in CH₂Cl₂ (6.0 mL) was added a solution of 1.0 M BBr₃ in CH₂Cl₂ 7.5 mL (7.5 mmol) dropwise at −78°C. The solution was warmed up to 0°C for 1 h. The reaction mixture was added to sat. NaHCO₃ aq and extracted with CH₂Cl₂ and the subsequent purification by flash column chromatography (hexane–Et₂O=2 : 1 and then CHCl₃–EtOAc=8 : 1) afforded hydroxychavicol (0.12 g, 27%).

Chavibetol¹⁶⁾

To a solution of 2-methoxyphenyl acetate (0.75 mL, 5.1 mmol) in CH₂Cl₂ (18 mL) iodine monochloride (0.30 mL, 5.7 mmol) was added at 0°C under N₂ and stirred for 2 h. The reaction mixture was added to a cold sat. Na₂S₂O₃ aq. and extracted with CH₂Cl₂ and the subsequent purification by flash column chromatography (hexane–CHCl₃=4 : 1) afforded 5-iodo-2-methoxyphenyl acetate (1.5 g, 99%). To a solution of 5-iodo-2-methoxyphenyl acetate (0.84 g, 2.9 mmol), cesium fluoride (1.0 g, 7.2 mmol) and Pd(PPh₃)₄ (55 mg, 0.048 mmol) in tetrahydrofuran (THF) (30 mL) was added and stirred. To the mixture allyltributyltin (1.2 mL, 3.6 mmol) was added and stirred for 10 h at 45°C and then added water. The reaction mixture was shaken vigorously and filtered off. The filtrate was extracted with ethyl acetate and the subsequent purification by flash column chromatography (hexane–Et₂O=3 : 1) afforded 5-allyl-2-methoxyphenyl acetate (0.42 mg, 71%). To a solution of 5-allyl-2-methoxyphenyl acetate (0.42 mg, 2.0 mmol) in MeOH (1.7 mL), (THF 1.7 mL) and water (0.60 mL), LiOH·H₂O (0.337 mg, 8.0 mmol) was added and stirred for 3.5 h. The reaction mixture was added 1 M HCl aq. and extracted with Et₂O. The extract afforded chavibetol (0.28 g, 84%) without further purification.

3-Allylphenol (1)¹⁷⁾

To magnesium (290 g, 12 mmol), 1-bromo-3-methoxybenzene (1 mL, 8 mmol) in Et₂O (40 mL) was added dropwise under nitrogen atmosphere. The mixture was refluxed until magnesium was consumed and cooled to 0°C subsequently. To the solution allylbromide (2.4 mL, 28 mmol) was added dropwise. After stirring for 2 h at room temperature, the mixture was quenched with sat. NH₄Cl aq. and extracted with ether and the subsequent purification by flash column chromatography (hexane–ethyl acetate=19 : 1) afforded 1-allyl-3-methoxybenzene (667 mg, 56%). To a stirred solution of 1-allyl-3-methoxybenzene (667 mg, 4.5 mmol) in CH₂Cl₂ (60 mL), an 1 M solution of BBr₃ in CH₂Cl₂ 5.5 mL (5.5 mmol) was added dropwise at −78°C under nitrogen atmosphere. The solution was allowed to warm up to 0°C and stirred for 2 h at room temperature. The reaction mixture was quenched with water and extracted with CH₂Cl₂, and the subsequent purification by flash column chromatography (hexane–diethyl ether=3 : 1) afforded 3-allylphenol (302 mg, 50%).

4-Allylphenol (2)¹⁸⁾

To a stirred solution of 1-allyl-4-methoxybenzene (0.32 mL, 2 mmol) in CH₂Cl₂ (30 mL), an 1 M solution of BBr₃ in CH₂Cl₂ 7.8 mL (7.8 mmol) was added dropwise at −78°C under nitrogen atmosphere. The solution was allowed to warm up to 0°C and stirred for 2 h at room temperature. The reaction mixture was quenched with water and extracted with CH₂Cl₂, and the subsequent purification by flash column chromatography (hexane–ethyl acetate=5 : 1) afforded 4-allylphenol (15 mg, 5.2%).

2-Allylhydroquinone (3)¹⁹⁾

To magnesium (290 g, 12 mmol), 2-bromo-1,4-dimethoxybenzene (1.2 mL, 8.0 mmol) in Et₂O (40 mL) was added dropwise under nitrogen atmosphere. The mixture was refluxed until magnesium was consumed and cooled to 0°C. Subsequently to the solution allylbromide (2.1 mL, 8.4 mmol) was added dropwise. After stirring for 2 h at room temperature the mixture was quenched with sat. NH₄Cl aq. and extracted with ether and the subsequent purification by flash column chromatography (hexane–ethyl acetate=19 : 1) afforded 1-allyl-2,5- dimethoxybenzene (1.33g, 93%). The mixture of CS₂ (70 mL), Al (2.0 g 74 mmol) and I₂ (15 g 118 mmol) was refluxed for 2 h and cooled down to room temperature. To the stirred solution, a solution of 1-allyl-2,5-dimethoxybenzene (1.33 g, 7.5 mmol) in CS₂ (70 mL) was added dropwise. The reaction mixture was stirred under reflux for 12 h. The reaction mixture was quenched with water and extracted with Et₂O. The combined organic phase washed with 1 M Na₂S₂O₃ aqueous solution and dried over Na₂SO₄. The solvent was removed and the resulting residue was dissolved in THF, subsequent purification by flash column chromatography (CHCl₃–methanol=5 : 1) afforded 2-allylhydroquinone (35 mg, 3%).

3-Allylresorcinol (4)²⁰⁾

To magnesium (290 g, 12 mmol), 1-bromo-3,5-dimethoxybenzene (0.86 g 4.0 mmol) in THF (20 mL) was added dropwise under nitrogen atmosphere. The mixture was refluxed until magnesium was consumed and cooled to 0°C subsequently. To the solution allylbromide (1.2 mL, 15 mmol) was added dropwise. After stirring for 2 h at room temperature the mixture was quenched with sat. NH₄Cl aq. was added and extracted with ether and the subsequent purification by flash column chromatography (hexane–ethyl acetate=19 : 1) afforded 1-allyl-3,5-dimethoxybenzene (356 mg, 50%). The mixture of CS₂ (35 mL), Al (0.40 g 74 mmol) and I₂ (3 g 118 mmol) was refluxed for 2hs and cooled down to room temperature. To the stirred solution, a solution of 1-allyl-3,5-dimethoxybenzene (0.75 g, 4.2 mmol) in CS₂ (35 mL) was added dropwise. The reaction mixture was stirred under reflux for 12 h. The reaction mixture was quenched with water and extracted with Et₂O. The combined organic phase washed with 1 M Na₂S₂O₃ aqueous solution and dried over Na₂SO₄. The solvent was removed and the resulting residue was dissolved in THF, subsequent purification by flash column chromatography (CHCl₃–methanol=5 : 1) afforded 2-allylhydroquinone (34 mg, 6%).

3-Allylcatechol (5)²¹⁾

To a stirred solution of 2-allyl-6-methoxyphenol (0.32 mL, 2 mmol) in CH₂Cl₂ (30 mL), an 1.0 M solution of BBr₃ in CH₂Cl₂ 7.8 mL (7.8 mmol) was added dropwise at −78°C under nitrogen atmosphere. The solution was allowed to warm up to 0°C and stirred for 2 h at room temperature. The reaction mixture was quenched with water and extracted with CH₂Cl₂. And the subsequent purification by flash column chromatography (hexane–ethyl acetate=5 : 1) afforded 3-allylcatechol (15 mg, 31%).

4-Allylresorcinol (6) and 2-Allylresorcinol (7)²²⁾

To a solution of resorcinol (0.77 g, 7.0 mmol) in dry acetone (7 mL), allylbromide (0.80 mL, 9.2 mmol) and K₂CO₃ (1.1 g, 8.1 mmol) was added and stirred for 8 h. The reaction mixture was filtered off. The filtrate was concentrated and purified by flash column chromatography (hexane–Et₂O=10: 1) afforded 3-(2-propenyloxy)phenol (0.43 g, 41%). 3-(2-Propenyloxy)phenol (0.43 g 0.28 mmol) was heated for 30 min at 210°C. The reaction mixture was purified with flash column chromatography (hexane–Et₂O=10 : 1) afforded 4-allylresorcinol (6) (0.22 g, 51%) and 2-allylresorcinnol (7) (0.090 g, 21%).

5-Allylpyrogallol (8)²³⁾

Suspension of allyl bromide (0.21 mL, 2.4 mmol) and sodium iodide (0.51 g, 3.4 mmol) in acetone (5 mL) was stirred under nitrogen atmosphere at room temperature and prevented light by aluminum foil. In other flask, suspension of pyllogarol (0.32 g, 2.5 mmol) and potassium bicarbonate (0.35 g, 2.5 mol) in acetone (5 mL) was stirred under nitrogen atmosphere at room temperature and prevented light by aluminum foil. To a suspension of potassium salt of pyllogarol, allyl iodide solution was added dropwise at room temperature. The mixture was refluxed for 6 d. The solvent was evaporated and the residue was washed with sat. NH₄Cl solution. The aqueous phase was extracted with CHCl₃ and the subsequent purification by flash column chromatography (ethyl acetate) afforded 5-allylpyrogallol (18 mg, 4.4%).

XO Inhibitory Assay²⁴⁾

Xanthine (3.0 mg) was dissolved in 100 mL of 0.1 M phosphate buffer (pH 7.8) with gentle heating to make 200 µM of xanthine buffer. The test solution was prepared by dissolving each sample into dimethyl sulfoxide. The assay mixture consisting of 10 µL of test solution and 990 µL of xanthine buffer was prepared for the reaction. The reaction was initiated by addition of 2 µL enzyme solution (20 units/mL). The mixture was vortexed and incubated at room temperature for 4 min. The concentration of uric acid was measured by the optical density (OD) at 295 nm by using a spectrophotometer (UV-2450PC, Shimadzu, Kyoto, Japan).

Computational Studies

Docking studies were performed based on the crystal structure of the XO-xanthine complex (PDB ID: 3EUB).²⁸⁾ Mammalian XO is formed with α,β-subunits. The α-subunit consists of the FAD region and two 2Fe-2S clusters. The β-subunit contains a molybdopterin (Mo-pt) cofactor. In this study, we used only Mo-pt containing region (Ala590-Thr1315) of the β-subunit for the calculation to reduce computation time. Electrostatic potential (ESP) around Mo-pt was calculated using HF/6-31G*/LANL2DZ with Gaussian09³¹⁾ and restrained electrostatics potential (RESP) charges³²⁾ for Mo-pt were assigned with Antechamber.³³⁾ The hydrogen atoms were added using SYBYL³⁴⁾ and all acidic and basic amino-acid residues were set to charged state. To eliminate the distortion of hydrogen atoms, the structure optimization was done only to hydrogen atoms by the MMFF94x³⁵⁾ force field until the maximum gradient was <0.0001 kcal/(mol·Å). The structures of HC and 8 were optimized using HF/6-31G*, and RESP charges for each atom were assigned with Antechamber.

Alpha spheres were generated around xanthine by Site Finder implemented in Molecular Operating Environment (MOE).²⁹⁾ Using MOE Dock, 100000 binding poses of HC were generated by a Triangle Matcher method and were evaluated using five scoring functions of London dG, ASE, Affinity dG, Alpha HB and GBVI/WSA dG. Binding poses with the best 100 scores were subjected to structure optimization using the MMFF94x force field and then scored again by the same evaluation functions. Out of five binding structures with the best score for each scoring function, three structures which formed hydrogen bonds with both xanthine and XO were selected as binding structure candidates.

Water molecules with the TIP3P³⁶⁾ potential were spherically generated from the center of the inhibitor molecule with the radius of 30 Å. The FF99SB³⁷⁾ and gaff³⁸⁾ force field were assigned to XO and inhibitors, respectively. After structure optimization, molecular dynamics simulation was performed using Amber11³⁹⁾ fixing the position of the Mo-pt moiety and restraining the position of the protein atoms locating 20 Å far from inhibitor with 100 kcal/(mol·Å²). All MD simulations were carried out at 300 K with the SHAKE⁴⁰⁾ procedure to constrain hydrogen atoms from moving. An equilibration step of 500 ps, after a 50 ps heating step, was followed by a production run of 2000 ps, with a time step of 1.0 fs, and trajectories were collected every 5 ps. This simulation was repeated three times changing the initial velocity. Using total of 1200 snapshot structures, the binding free energy was calculated by the mmpbsa_py_energy script of Amber11.

Acknowledgment

This study was supported by the Ministry of Education, Culture, Sports, Science and Technology (MEXT)-Supported Program for the Strategic Research Foundation at Private Universities (S1411037, 2014–2018).

Conflict of Interest

The authors declare no conflict of interest.

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