Antioxidants, Vol. 12, Pages 63: Monocarbonyl Curcumin Analogues as Potent Inhibitors against Human Glutathione Transferase P1-1

Conceptualization, N.E.L.; methodology, P.P., V.F., D.M., B.M., U.B. and F.P.; software, V.F., A.C.P. and U.B.; validation, A.C.P., M.S., M.P. and N.E.L.; formal analysis, A.C.P., M.S., M.P. and N.E.L.; investigation, P.P. and N.E.L.; data curation, V.F., A.C.P. and U.B.; writing—original draft preparation, P.P., V.F., D.M. and B.M.; writing—A.C.P., M.S., U.B., M.P. and N.E.L.; supervision, N.E.L. All authors have read and agreed to the published version of the manuscript.

Figure 1. Structures, names, and codes of curcuminoids and of the monocarbonyl curcumin derivatives used in the present study.

Figure 1. Structures, names, and codes of curcuminoids and of the monocarbonyl curcumin derivatives used in the present study.

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Figure 2. Concentration–response curves for the determination of the IC50 values of the most potent inhibitors DM96, DM109, DM151, and DMC against hGSTP1-1.

Figure 2. Concentration–response curves for the determination of the IC50 values of the most potent inhibitors DM96, DM109, DM151, and DMC against hGSTP1-1.

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Figure 3. Kinetic inhibition studies. (a1) Lineweaver–Burk plot of the inhibition of hGSTP1-1 isoenzyme using CDNB as a variable substrate (18–980 μΜ) at different constant concentrations of DM96 (0, 2.5, 5, and 6 μM). (a2) Secondary plot of the slopes of each Lineweaver–Burk line as a function of DM96 concentration. (b1) Lineweaver–Burk plot of the inhibition of hGSTP1-1 isoenzyme using GSH as a variable substrate (37.5–3750 μΜ) at different constant concentrations of DM96 (0, 3, 5, 6, and 7 μM). (b2) Secondary plot of the slopes of each Lineweaver–Burk line as a function of DM96 concentration. (b3) Tertiary plot depicting the 1/ΔIntercept -Y and 1/Δslope as a function of the 1/[inhibitor].

Figure 3. Kinetic inhibition studies. (a1) Lineweaver–Burk plot of the inhibition of hGSTP1-1 isoenzyme using CDNB as a variable substrate (18–980 μΜ) at different constant concentrations of DM96 (0, 2.5, 5, and 6 μM). (a2) Secondary plot of the slopes of each Lineweaver–Burk line as a function of DM96 concentration. (b1) Lineweaver–Burk plot of the inhibition of hGSTP1-1 isoenzyme using GSH as a variable substrate (37.5–3750 μΜ) at different constant concentrations of DM96 (0, 3, 5, 6, and 7 μM). (b2) Secondary plot of the slopes of each Lineweaver–Burk line as a function of DM96 concentration. (b3) Tertiary plot depicting the 1/ΔIntercept -Y and 1/Δslope as a function of the 1/[inhibitor].

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Figure 4. Concentration–response curves of the cytotoxicity of the most potent inhibitors DM96, DM109, DM151, and DMC against DU-145. The data were analyzed using the GraphPad Prism version 8.

Figure 4. Concentration–response curves of the cytotoxicity of the most potent inhibitors DM96, DM109, DM151, and DMC against DU-145. The data were analyzed using the GraphPad Prism version 8.

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Figure 5. CD spectra of (A) hGSTP1-1 (0.1 mg/mL) isoenzyme in the absence (black line) or in the presence of substrate CDNB (1 mM; red line), GSH (2.5 mM; blue line), and mixture GSH + CDNB (2.5 mM + 1 mM, respectively; green line) and (B) hGSTP1-1 (0.1 mg/mL) isoenzyme in the absence (black line) or in the presence of inhibitor DM96 (5 μΜ) with substrate CDNB (1 mM; red line), GSH (2.5 mM; blue line) and mixture GSH + CDNB (2.5 mM + 1 mM, respectively; green line). Representative spectra from n = 3 independent experiments are presented.

Figure 5. CD spectra of (A) hGSTP1-1 (0.1 mg/mL) isoenzyme in the absence (black line) or in the presence of substrate CDNB (1 mM; red line), GSH (2.5 mM; blue line), and mixture GSH + CDNB (2.5 mM + 1 mM, respectively; green line) and (B) hGSTP1-1 (0.1 mg/mL) isoenzyme in the absence (black line) or in the presence of inhibitor DM96 (5 μΜ) with substrate CDNB (1 mM; red line), GSH (2.5 mM; blue line) and mixture GSH + CDNB (2.5 mM + 1 mM, respectively; green line). Representative spectra from n = 3 independent experiments are presented.

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Figure 6. RMSD curves of: (a) curcumin derivative atomic positions, and (b) backbone atomic positions throughout 20 ns molecular dynamics simulations of hGSTP1-1/DM96 (red), hGSTP1-1/DM151 (orange), hGSTP1-1/DM109 (green), and hGSTP1-1/DMC (blue) complexes.

Figure 6. RMSD curves of: (a) curcumin derivative atomic positions, and (b) backbone atomic positions throughout 20 ns molecular dynamics simulations of hGSTP1-1/DM96 (red), hGSTP1-1/DM151 (orange), hGSTP1-1/DM109 (green), and hGSTP1-1/DMC (blue) complexes.

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Figure 7. RMSF values of ligand atomic positions throughout molecular dynamics simulation production runs of hGSTP1-1 /DM96 (red), hGSTP1-1/DM151 (orange), hGSTP1-1/DM109 (green), and hGSTP1-1/DMC (blue) complexes.

Figure 7. RMSF values of ligand atomic positions throughout molecular dynamics simulation production runs of hGSTP1-1 /DM96 (red), hGSTP1-1/DM151 (orange), hGSTP1-1/DM109 (green), and hGSTP1-1/DMC (blue) complexes.

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Figure 8. Binding modes of DM96, DM151, DM109, and DMC at the active site of hGSTP1-1. Carbon atoms of the studied curcumin derivatives are presented in orange, while carbon atoms of hGSTP1-1 amino-acid residues are depicted in light blue color. Oxygen atoms are red, nitrogen atoms dark blue, and fluorine atoms green. Hydrophobic interactions are presented with gray dashed lines and hydrogen bonds with dark blue lines. All distances represent averages over all MD production run snapshots for each complex and are provided in Å. Hydrogen atoms are omitted for reasons of clarity.

Figure 8. Binding modes of DM96, DM151, DM109, and DMC at the active site of hGSTP1-1. Carbon atoms of the studied curcumin derivatives are presented in orange, while carbon atoms of hGSTP1-1 amino-acid residues are depicted in light blue color. Oxygen atoms are red, nitrogen atoms dark blue, and fluorine atoms green. Hydrophobic interactions are presented with gray dashed lines and hydrogen bonds with dark blue lines. All distances represent averages over all MD production run snapshots for each complex and are provided in Å. Hydrogen atoms are omitted for reasons of clarity.

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Table 1. Screening of the inhibition potency of the curcuminoids (curcumin and DMC) and the curcumin analogues against hGSTP1-1 isoenzyme.

Table 1. Screening of the inhibition potency of the curcuminoids (curcumin and DMC) and the curcumin analogues against hGSTP1-1 isoenzyme.

Compound CodeMolecular WeightEnzyme Inhibition (%)Curcumin368.3853.00 ± 1.98DMC338.3594.56 ± 0.25DM15274.3666.23 ± 2.57DM46334.408.50 ± 2.13DM57394.4616.75 ± 3.61DM62310.348.40 ± 0.36DM95354.3944.18 ± 3.06DM96266.2986.99 ± 1.88DM100366.4140.00 ± 1.84DM101326.3472.28 ± 1.91DM109270.2779.06 ± 2.54DM148298.2954.80 ± 0.52DM151338.3588.18 ± 3.65

Table 2. Inhibition constants and IC50 values obtained by kinetic inhibition and cytotoxicity studies of the most potent inhibitors DM96, DM151, DM109, and Curcumin II. The in-silico-determined binding free energies (kcal/mol) are also included.

Table 2. Inhibition constants and IC50 values obtained by kinetic inhibition and cytotoxicity studies of the most potent inhibitors DM96, DM151, DM109, and Curcumin II. The in-silico-determined binding free energies (kcal/mol) are also included.

InhibitorIC50 against DU-145 (μΜ)IC50 against hGSTP1-1 (μΜ)Variable SubstrateType of InhibitionInhibition Constants (μΜ)Experimental Binding Free Energies (kcal/mol)DM968.60 ± 1.075.45 ± 1.08CDNBPurely mixedKi = 3.67 ± 0.35
Ki΄ = 4.97 ± 2.86ΔGexp = −7.71
ΔG′exp = −7.52GSHPartial mixedKi = 3.69 ± 1.00
Ki΄ = 1.45 ± 0.43ΔGexp = −7.71
ΔG′exp = −8.29DM15144.59 ± 1.0811.17 ± 1.03CDNBPurely non-competitiveKi = 9.55 ± 2.36ΔGexp = −7.12GSHPurely non-competitiveKi = 5.79 ± 1.21ΔGexp = −7.43DM10946.15 ± 3.6819.53 ± 1.04CDNBPurely non-competitiveKi = 20.12 ± 5.27ΔGexp = −6.66GSHPurely non-competitiveKi = 35.12 ± 5.69ΔGexp = −5.96Curcumin II48.52 ± 1.0937.72 ± 1.02CDNBPartially mixedΚi = 3.99 ± 1.80
Ki΄ = 2.36 ± 1.37ΔGexp = −7.66
ΔG′exp = −7.98GSHPartially mixedΚi = 68.02 ± 14.60
Ki΄ = 52.83 ± 12.25ΔGexp = −5.91
ΔG′exp = −6.07

Table 3. Experimental data related to the potential of the most potent inhibitors (DM96, DM109, DM151, and DMC) to inhibit GST activity in DU-145 cell lysate.

Table 3. Experimental data related to the potential of the most potent inhibitors (DM96, DM109, DM151, and DMC) to inhibit GST activity in DU-145 cell lysate.

Compound CodeEnzyme Inhibition (%)DM9641.10 ± 1.99DM10950.16 ± 0.76DM151No inhibition was observedDMC48.30 ± 2.51

Table 4. Docking score values of the best-scored hGSTP1-1-curcumin analogur complexes obtained with the CANDOCK algorithm in conjunction with scoring function RMR6.

Table 4. Docking score values of the best-scored hGSTP1-1-curcumin analogur complexes obtained with the CANDOCK algorithm in conjunction with scoring function RMR6.

Curcumin DerivativeDocking Score Values (Arbitrary Units)DM96−40.63DM151−36.99DM109−31.30DMC−25.39

Table 5. Average RMSD values of ligand and backbone atomic positions together with average RMSF values of ligand and backbone atomic positions throughout four independent 20 ns molecular dynamics simulation production runs of hGSTP1-1/DM96, hGSTP1-1/DM151, hGSTP1-1/DM109, and hGSTP1-1/DMC complexes. The average values of all four production runs are presented as mean ± standard deviation.

Table 5. Average RMSD values of ligand and backbone atomic positions together with average RMSF values of ligand and backbone atomic positions throughout four independent 20 ns molecular dynamics simulation production runs of hGSTP1-1/DM96, hGSTP1-1/DM151, hGSTP1-1/DM109, and hGSTP1-1/DMC complexes. The average values of all four production runs are presented as mean ± standard deviation.

Curcumin DerivativeAverage Ligand RMSD (Å)Average Backbone RMSD (Å)Average Ligand RMSF (Å)Average Backbone RMSF (Å)DM960.35 ± 0.02 0.76 ± 0.03 1.25 ± 0.04 0.77 ± 0.02 DM1510.44 ± 0.030.85 ± 0.041.55 ± 0.030.79 ± 0.01DM1090.63 ± 0.031.01 ± 0.071.81 ± 0.040.86 ± 0.06DMC2.10 ± 0.081.14 ± 0.061.83 ± 0.050.90 ± 0.07

Table 6. The average electrostatic (ele) and van der Waals (vdW) non-bonded interactions of DM96, DM151, DM109, and DMC in water (the free state W) as well as in complex with hGSTP1-1 (the bound state P) along with the corresponding binding free energies.

Table 6. The average electrostatic (ele) and van der Waals (vdW) non-bonded interactions of DM96, DM151, DM109, and DMC in water (the free state W) as well as in complex with hGSTP1-1 (the bound state P) along with the corresponding binding free energies.

Energies〈VvdWL−P〉
(kcal/mol) 〈VvdWL−W〉
(kcal/mol) 〈VeleL−P〉
(kcal/mol) 〈VeleL−W〉
(kcal/mol) 〈VeleL−P〉0
(kcal/mol) ΔGbindingL−P **
(kcal/mol)DM96LIE average *−40.52 ± 0.44−23.49 ± 0.03−36.05 ± 0.1−36.7 ± 0.04/−7.83 ± 0.27LRA average *−40.52 ± 0.44−23.49 ± 0.03−36.05 ± 0.1−36.7 ± 0.04−0.28 ± 0.02−8.11 ± 0.28DM151LIE average *−39.88 ± 0.54−28.23 ± 0.04−47.52 ± 0.43−45.91 ± 0.19/−7.49 ± 0.50LRA average *−39.88 ± 0.54−28.23 ± 0.04−47.52 ± 0.43−45.91 ± 0.19−0.23 ± 0.01−7.73 ± 0.51DM109LIE average *−34.49 ± 0.07−24.83 ± 0.01−21.41 ± 0.05−20.89 ± 0.01/−5.38 ± 0.07LRA average *−34.49 ± 0.07−24.83 ± 0.01−21.41 ± 0.05−20.89 ± 0.01−0.22 ± 0.01−5.60 ± 0.08DMCLIE average *−37.08 ± 0.06−28.80 ± 0.02−55.11 ± 0.29−54.4 ± 0.12/−4.88 ± 0.20LRA average *−37.08 ± 0.06−28.80 ± 0.02−55.11 ± 0.29−54.4 ± 0.12−0.21 ± 0,01−5.09 ± 0.21

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