Enzymatic Redox Properties and Cytotoxicity of Irreversible Nitroaromatic Thioredoxin Reductase Inhibitors in Mammalian Cells
Abstract
:1. Introduction
2. Results
2.1. Enzymatic Redox Properties of Examined Nitroaromatic Compounds
2.2. Reactions of Nitroaromatic Compounds with Reduced Glutathione
2.3. Cytotoxicity of Nitroaromatic Compounds
2.4. Inhibition of Thioredoxin Reductase and Glutathione Reductase by Nitroaromatic Compounds
3. Discussion
4. Materials and Methods
4.1. Enzymes and Chemicals
4.2. Enzymatic Assays and Chemical Reactions
4.3. Cytotoxicity Assays
4.4. Studies of Thioredoxin Reductase and Glutathione Reductase Activity in Cell Lysates
4.5. Statistical Analysis and Calculations
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Appendix A
No. | Compound | E17 (V) a | kcat/Km (M−1s−1) | log kcat/Km (avge) | |
---|---|---|---|---|---|
P-450R | ADR/ADX | ||||
1 | Nitrobenzene | −0.485 | 6.8 ± 0.8 × 102 | 3.4 ± 0.2 × 103 | 3.18 |
2 | 4-Nitrobenzoic acid | −0.425 | 2.3 ± 0.2 × 103 (2.9 ± 0.2 × 103) | 2.0 ± 0.2 × 103 (2.3 ± 0.2 × 103) | 3.33 (3.41) |
3 | 2-Nitrothiophene | −0.390 | 1.4 ± 0.1 × 104 | 4.2 ± 0.3 × 104 | 4.38 |
4 | 4-Nitroacetophenone | −0.355 | 1.7 ± 0.2 × 104 | 3.2 ± 0.3 × 104 | 4.37 |
5 | 1,3-Dinitrobenzene | −0.345 | 4.9 ± 0.2 × 104 | 5.2 ± 0.4 × 104 | 4.70 |
6 | 4-Nitrobenzaldehyde | −0.325 | 3.3 ± 0.2 × 104 | 1.7 ± 0.3 × 105 | 4.87 |
7 | 2-Nitrothiophene-5-carboxymorpholide | −0.305 | 1.4 ± 0.2 × 105 | 4.1 ± 0.6 × 105 | 5.38 |
8 | 1,2-Dinitrobenzene | −0.287 | 1.6 ± 0.1 × 105 | 1.8 ± 0.2 × 105 | 5.23 |
9 | 2-Nitrothiophene-5-aldoxime | −0.280 | 2.2 ± 0.2 × 106 (1.8 ± 0.2 × 106) | 5.4 ± 0.5 × 105 (4.2 ± 0.4 × 105) | 6.04 (5.94) |
10 | 2-Nitrothiophene-5-aldehyde | −0.260 | 2.8 ± 0.1 × 105 | 4.0 ± 0.5 × 105 | 5.52 |
11 | Nitrofurantoin | −0.255 | 9.1 ± 1.4 × 104 | 1.0 ± 0.2 × 106 | 5.48 |
12 | Nifuroxime | −0.255 | 1.1 ± 0.1 × 105 | 1.0 ± 0.1 × 106 | 5.52 |
13 | 1,4-Dinitrobenzene | −0.255 | 1.2 ± 0.1 × 106 | 2.0 ± 0.2 × 106 | 6.19 |
14 | 2,4,6-Trinitrotoluene | −0.253 | 1.0 ± 0.1 × 105 (1.4 ± 0.1 × 105) | 7.3 ± 0.2 × 105 (7.0 ± 0.2 × 105) | 5.43 (5.50) |
15 | Tetryl | −0.191 | 5.9 ± 0.2 × 106 | 8.9 ± 1.0 × 105 | 6.36 |
r2 = 0.9209, F(1,13) = 150.11.
Appendix B
No. | Compound | E17 (V) | log D | MH22a cL50 (μM) | HCT-116 GI50 (μM) |
---|---|---|---|---|---|
1 | Nitrobenzene | −0.485 | 1.91 | 1800 ± 200 | ≥5000 |
2 | 4-Nitrobenzoic acid | −0.425 | −1.66 | ≥6000 | ≥6000 |
3 | 2-Nitrothiophene | −0.390 | 1.86 | 341 ± 42 | 600 ± 70 a |
4 | 4-Nitroacetophenone | −0.355 | 1.47 | 239 ± 19 | 400 ± 80 |
5 | 1,3-Dinitrobenzene | −0.345 | 1.85 | 130 ± 14 | 350 ± 20 |
6 | 4-Nitrobenzaldehyde | −0.325 | 1.63 | 200 ± 15 | 50.0 ± 6.0 |
7 | 2-Nitrothiophene-5-carboxymorpholide | −0.305 | 1.07 | 82.0 ± 12 | n.d. |
8 | 1,2-Dinitrobenzene | −0.287 | 1.85 | 25.4 ± 3.0 | 60.0 ± 10.0 |
9 | 2-Nitrothiophene-5-aldoxime | −0.280 | 1.7 | 145 ± 30 118 ± 20 a | 20.0 ± 5.0 |
10 | 2-Nitrothiophene-5-aldehyde | −0.260 | 1.26 | 42.0 ± 5.0 | 20.0 ± 3.0 a |
11 | Nitrofurantoin | −0.255 | −0.25 | 387 ± 25 320 ± 25 a | 60.0 ± 10.0 |
12 | Nifuroxime | −0.255 | −0.34 | 40.5 ± 5.0 | 70.0 ± 10.0 |
13 | 1,4-Dinitrobenzene | −0.255 | 1.85 | 12.0 ± 1.5 | 40.0 ± 7.0 |
14 | 2,4,6-Trinitrotoluene | −0.253 | 2.31 | 17.4 ± 2.0 | 40.0 ± 8.0 |
15 | Tetryl | −0.191 | 1.38 | 7.0 ± 1.0 | 8.0 ± 1.5 |
r2 = 0.9011, F(2,12) = 54.67.
r2 = 0.9372, F(2,11) = 82.10.
References
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Compound | kcat/Km (M−1s−1) | E17(calc.) (V) | ||
---|---|---|---|---|
P-450R | ADR/ADX | NQO1 | ||
1-Chloro-2,4-dinitro- benzene | 1.7 ± 0.1 × 105 | 3.6 ± 0.2 × 105 | ≤102 (≤0.1) a | −0.285 |
NSC697923 | 5.0 ± 0.4 × 104 | 1.1 ± 0.1 × 106 | 2.5 ± 0.4 × 103 (≤0.25) a | −0.287 |
Stattic | 1.3 ± 0.1 × 105 | 1.6 ± 0.1 × 105 | 6.8 ± 0.8 × 102 (0.15 ± 0.02) a | −0.304 |
BTB06584 | 1.6 ± 0.1 × 104 | 3.7 ± 0.2 × 105 | (≤0.07) b | −0.326 |
Tri-1 | 1.7 ± 0.1 × 104 | 3.7 ± 0.2 × 104 | 2.1 ± 0.2 × 103 (0.10 ± 0.02) a | −0.365 |
Compound | E17(calc.) (V) | log D | cL50 MH22a (µM) | GI50 HCT-116 (µM) |
---|---|---|---|---|
CDNB | −0.285 | 2.46 | 1.9 ± 0.1 (28.3) | 5.0 ± 0.3 (40.2) |
NSC697923 | −0.287 | 2.70 | 1.0 ± 0.1 (25.1) | 1.0 ± 0.1 (38.3) |
Stattic | −0.304 | 1.12 | 1.8 ± 0.2 (100) | 2.0 ± 0.2 (106) |
BTB06584 | −0.326 | 5.13 | 28.0 ± 4.0 (10.5) | 80.0 ± 9.0 (36) |
Tri-1 | −0.365 | 3.29 | 6.6 ± 0.5 (77.6) | 7.0 ± 0.7 (184) |
Compound | Cell Viability (%) | ||||||
---|---|---|---|---|---|---|---|
Additions: | |||||||
None | DPPD (2.0 µM) | Desferri- Oxamine (1.0 mM) | Dicoumarol (20 µM) | α-Naphtho- Flavone (5.0 µM) | Isoniazid (1.0 mM) | Miconazole (5.0 µM) | |
1-Chloro-2,4-dinitrobenzene (2.0 µM) | 49.3 ± 4.2 | 77.3 ± 5.6 *** | 67.0 ± 4.6 ** | 69.5 ± 4.8 ** | 63.3 ± 3.8 * | 64.9 ± 4.8 * | 74.9 ± 7.7 ** |
NSC697923 (1.0 µM) | 45.1 ± 3.5 | 55.7 ± 5.2 | 61.7 ± 4.4 ** | 63.1 ± 5.0 ** | 69.0 ± 6.4 ** | 71.0 ± 5.5 *** | 71.0 ± 5.3 *** |
Stattic (2.0 µM) | 50.3 ± 5.5 | 79.1 ± 6.6 ** | 75.0 ± 3.1 ** | 76.7 ± 10 * | 38.6 ± 4.8 * | 32.6 ± 2.8 *** | 74.5 ± 10.7 * |
Tri-1 (6.0 µM) | 49.9 ± 4.5 | 67.7 ± 4.6 ** | 64.7 ± 4.3 * | 78.3 ± 4.8 *** | 70.5 ± 4.6 ** | 77.0 ± 4.8 *** | 68.6 ± 3.3 ** |
Tetryl (10.0 µM) | 45.5 ± 4.2 | 78.4 ± 5.8 *** | 79.9 ± 5.0 *** | 28.2 ± 2.8 *** | 55.4 ± 5.0 | 54.4 ± 3.6 | 24.2 ± 1.1 *** |
Compound | Enzyme | |||
---|---|---|---|---|
Rat TrxR1 | HGR | |||
IC50 (µM) | ki (min−1) | IC50 (µM) | ki (min−1) | |
1-Chloro-2,4-dinitrobenzene | 25 ± 5.0 | 0.10 ± 0.01 (10 µM) | 40 ± 6.0 | ≤0.005 (10 µM) |
NSC697923 | 1.1 ± 0.1 | 0.12 ± 0.02 (1.0 µM) | 0.5 ± 0.1 | 0.02 ± 0.003 (1.0 µM) |
Stattic | 15 ± 2.0 | 0.14 ± 0.02 (10 µM) | 14.0 ± 1.0 | 0.02 ± 0.003 (10 µM) |
Tri-1 | 2.0 ± 0.3 | 0.12 ± 0.01 (1.0 µM) | 32.0 ± 5.0 | ≤0.005 (10 µM) |
Tetryl | 10 ± 1.3 | 0.04 ± 0.01 (1.0 µM) 0.30 ± 0.04 (10 µM) | 14 ± 2.0 | ≤0.002 (10 µM) |
Compound | Cells | Compound Concentration, Incubation Time | Residual Activity (%) | |
---|---|---|---|---|
TrxR | GR | |||
Tri-1 | HCT-116 [23] | 3.3 µM, 3 h | 20 | n.d. |
BF16-F10 [44] | 2.0 µM, 3 h | 50 | n.d. | |
20 µM, 3 h | 10 | n.d. | ||
Stattic | A549 [22] | 10 µM, 4 h | 25 | n.d. |
Various human cervical carcinoma cells [24] | 20 µM, 0.5 h | n.d. | 30–50 | |
1-Chloro-2,4-dinitrobenzene | A549 [21] | 50 µM, 4 h | 30 | 80 |
HeLa [21] | 50 µM, 4 h | 30 | 100 | |
Tetryl | A549 [21] | 50 µM, 4 h | 80 | 100 |
HeLa [21] | 50 µM, 4 h | 75 | 100 |
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Nemeikaitė-Čėnienė, A.; Misevičienė, L.; Marozienė, A.; Jonušienė, V.; Čėnas, N. Enzymatic Redox Properties and Cytotoxicity of Irreversible Nitroaromatic Thioredoxin Reductase Inhibitors in Mammalian Cells. Int. J. Mol. Sci. 2023, 24, 12460. https://doi.org/10.3390/ijms241512460
Nemeikaitė-Čėnienė A, Misevičienė L, Marozienė A, Jonušienė V, Čėnas N. Enzymatic Redox Properties and Cytotoxicity of Irreversible Nitroaromatic Thioredoxin Reductase Inhibitors in Mammalian Cells. International Journal of Molecular Sciences. 2023; 24(15):12460. https://doi.org/10.3390/ijms241512460
Chicago/Turabian StyleNemeikaitė-Čėnienė, Aušra, Lina Misevičienė, Audronė Marozienė, Violeta Jonušienė, and Narimantas Čėnas. 2023. "Enzymatic Redox Properties and Cytotoxicity of Irreversible Nitroaromatic Thioredoxin Reductase Inhibitors in Mammalian Cells" International Journal of Molecular Sciences 24, no. 15: 12460. https://doi.org/10.3390/ijms241512460