Aerobic Cytotoxicity of Aromatic N-Oxides: The Role of NAD(P)H:Quinone Oxidoreductase (NQO1)
Abstract
:1. Introduction
2. Results
2.1. Reactions of Aromatic N-Oxides with Flavoenzymes Dehydrogenases-Electrontransferases
No. | Compound | E17 (V) a | kcat/Km (M−1·s−1) | log kcat/Km (avge) | |
---|---|---|---|---|---|
P-450R | PfFNR | ||||
1 | 7-CF3O-tirapazamine | 4.6 ± 0.4 × 104 | 3.8 ± 0.4 × 104 | 4.62 | |
2 | 3-CH3CONH-1,2,4-benzotriazine-1,4-dioxide | 7.0 ± 0.5 × 104 | 6.2 ± 0.5 × 104 | 4.82 | |
3 | 3-CH3OCONH-1,2,4-benzotriazine-1,4-dioxide | 8.0 ± 0.9 × 104 | 4.8 ± 0.4 × 104 | 4.78 | |
4 | 3-CF3SO2NH-1,2,4-benzotriazine-1,4-dioxide | 2.5 ± 0.3 × 104 | 2.6 ± 0.3 × 104 | 4.41 | |
5 | 3-CH3SO2NH-1,2,4-benzotriazine-1,4-dioxide | 2.7 ± 0.3 × 103 | 7.9 ± 0.5 × 103 | 3.67 | |
6 | 2-CF3-quinoxaline-1,4-dioxide | (−0.465) b | 2.7 ± 0.3 × 104 | 8.9 ± 0.7 × 103 | 4.19 |
7 | 2-NH2-3-CN-quinoxaline-1,4-dioxide | 4.7 ± 0.4 × 103 | 1.8 ± 0.2 × 104 | 3.96 | |
8 | 1,2,4-Benzotriazine-1-oxide | (−0.431) b | 1.7 ± 0.2 × 104 | 4.3 ± 0.3 × 103 | 3.94 |
9 | 3-CH3CONH-1,2,4-benzotriazine-1-oxide | 8.7 ± 0.9 × 103 | 1.6 ± 0.1 × 103 | 3.58 | |
ArN→O with available E17 values | |||||
10 | 1,2,4-Benzotriazine-1,4-dioxide | −0.318 | 4.3 ± 0.4 × 105 c | 2.5 ± 0.3 × 104 | 5.00 |
11 | 7-CF3-tirapazamine | −0.345 | 8.7 ± 0.7 × 104 c | 5.2 ± 0.4 × 104 d | 4.83 |
12 | 7-Cl-tirapazamine | −0.400 | 6.9 ± 0.7 × 104 c | 3.7 ± 0.4 × 104 d | 4.71 |
13 | 7-F-tirapazamine | −0.400 | 3.4 ± 0.3 × 104 c | 2.7 ± 0.2 × 104 d | 4.48 |
14 | Tirapazamine | −0.455 | 1.1 ± 0.1 × 104 c | 4.4 ± 0.5 × 103 d | 3.84 |
15 | 7-CH3-tirapazamine | −0.474 | 8.6 ± 0.7 × 103 c | 5.0 ± 0.6 × 103 d | 3.82 |
16 | 7-C2H5O-tirapazamine | −0.494 | 4.5 ± 0.5 × 103 c | 4.5 ± 0.5 × 103 d | 3.65 |
17 | 3-Amino-1,2,4-benzotriazine-1-oxide | −0.568 | 2.8 ± 0.2 × 103 c | 3.2 ± 0.2 × 103 d | 3.48 |
18 | Quinoxaline-1,4-dioxide | −0.575 | 3.3 ± 0.2 × 103 c | 8.2 ± 0.9 × 102 d | 3.22 |
2.2. NQO1-Catalyzed Reduction of Aromatic N-Oxides
2.3. Cytotoxicity of Aromatic N-Oxides
3. Discussion
4. Materials and Methods
4.1. Enzymes and Chemicals
4.2. Enzymatic Assays
4.3. Cytotoxicity Assays
4.4. Statistical Analysis and Calculations
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
ArN→O | Heteroaromatic N-oxide |
BCNU | 1,3-bis(2-chloroethyl)-1-nitrosourea |
cL50 | Concentration for 50% cell survival |
DESF | Desferrioxamine |
DIC | Dicoumarol |
DPPD | N,N′-diphenyl-p-phenylene diamine |
E17 | Single-electron reduction midpoint potential at pH 7.0 |
GI50 | Concentration for 50% inhibition of maximal cell proliferation |
kcat | Catalytic constant |
kcat/Km | Bimolecular rate constant |
kcat/Km (avge) | Geometric average of kcat/Km in P-450R- and PfFNR-catalyzed reactions |
kcat/Km (NQO1) | kcat/Km in NQO1-catalyzed reaction |
log D | Octanol/water distribution coefficient at pH 7.0 |
NQO1 | NAD(P)H:quinone oxidoreductase |
P-450R | NADPH:cytochrome P-450 reductase |
PfFNR | Plasmodium falciparum ferredoxin:NADP+ oxidoreductase |
SOD | Superoxide dismutase |
TPZ | Tirapazamine |
References
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No. | Compound | kcat (s−1) | kcat/Km (M−1.s−1) | VdWvol (Å3) a |
---|---|---|---|---|
1 | 7-CF3O-tirapazamine | 0.11 ± 0.02 | 1.6 ± 0.1 × 103 | 175.8 |
2 | 3-CH3CONH-1,2,4-benzotriazine-1,4-dioxide | 3.40 ± 0.20 | 1.3 ± 0.1 × 104 | 172.3 |
3.00 ± 0.20 b | 1.0 ± 0.1 × 104 b | |||
3 | 3-CH3OCONH-1,2,4-benzotriazine-1,4-dioxide | 2.40 ± 0.22 | 3.0 ± 0.2 × 103 | 180.7 |
4 | 3-CF3SO2NH-1,2,4-benzotriazine-1,4-dioxide | 0.34 ± 0.06 | 1.0 ± 0.2 × 103 | 203.5 |
5 | 3-CH3SO2NH-1,2,4-benzotriazine-1,4-dioxide | 0.40 ± 0.05 | 1.2 ± 0.2 × 103 | 184.9 |
6 | 2-CF3-quinoxaline-1,4-dioxide | 13.5 ± 1.72 | 2.4 ± 0.2 × 104 | 168.2 |
7 | 2-NH2-3-CN-quinoxaline-1,4-dioxide | 0.11 ± 0.02 | 1.4 ± 0.1 × 103 | 146.2 |
8 | 1,2,4-Benzotriazine-1-oxide | 1.10 ± 0.12 | 2.1 ± 0.2 × 103 | 111.7 |
9 | 3-CH3CONH-1,2,4-benzotriazine-1-oxide | 0.06 ± 0.02 | 5.0 ± 1.0 × 102 | 163.5 |
10 | 1,2,4-Benzotriazine-1,4-dioxide c | 1.00 ± 0.10 | 5.1 ± 0.7 × 103 | 120.5 |
11 | 7-CF3-tirapazamine c | 1.30 ± 0.10 | 3.7 ± 0.4 × 103 | 167.0 |
12 | 7-Cl-tirapazamine c | 1.00 ± 0.10 | 4.3 ± 0.4 × 103 | 152.6 |
13 | 7-F-tirapazamine c | 0.70 ± 0.10 | 4.9 ± 0.6 × 103 | 143.5 |
14 | Tirapazamine c | 0.20 ± 0.10 | 2.4 ± 0.2 × 103 | 131.5 |
15 | 7-CH3-tirapazamine c | 0.30 ± 0.05 | 1.7 ± 0.1 × 103 | 148.8 |
16 | 7-C2H5O-tirapazamine c | 0.30 ± 0.04 | 1.0 ± 0.1 × 103 | 174.9 |
17 | 3-Amino-1,2,4-benzotriazine-1-oxide c | 0.10 ± 0.02 | 1.6 ± 0.2 × 103 | 122,7 |
18 | Quinoxaline-1,4-dioxide c | 0.60 ± 0.10 | 7.9 ± 0.9 × 103 | 126.8 |
No. | Compound | log kcat/Km (avge) | log D | cL50 (µM) MH22a | GI50 (µM) HCT-116 |
---|---|---|---|---|---|
1 | 7-CF3O-tirapazamine | 4.62 | 1.32 | 3.6 ± 0.7 | 17 ± 3.0 |
2 | 3-CH3CONH-1,2,4-benzotriazine-1,4-dioxide | 4.82 | −0.82 | 1.5 ± 0.3 | 2.5 ± 0.5 |
3 | 3-CH3OCONH-1,2,4-benzotriazine-1,4-dioxide | 4.78 | −0.08 | 9.4 ± 1.4 | 6.3 ± 1.0 |
4 | 3-CF3SO2NH-1,2,4-benzotriazine-1,4-dioxide | 4.41 | −0.40 | 44 ± 6.5 | 125 ± 19 |
5 | 3-CH3SO2NH-1,2,4-benzotriazine-1,4-dioxide | 3.67 | −2.42 | 184 ± 25 | 185 ± 23 |
6 | 2-CF3-quinoxaline-1,4-dioxide | 4.19 | 0.42 | 10 ± 2.0 | 12.5 ± 2.0 |
7 | 2-NH2-3-CN-quinoxaline-1,4-dioxide | 3.96 | −0.18 | 358 ± 52 | 125 ± 17 |
8 | 1,2,4-Benzotriazine-1-oxide | 3.94 | 0.45 | 168 ± 21 | 225 ± 27 |
9 | 3-CH3CONH-1,2,4-benzotriazine-1-oxide | 3.58 | 0.37 | ≥600 | ≥1000 |
10 | 1,2,4-Benzotriazine-1,4-dioxide | 5.00 | −0.70 | 11 ± 1.5 a | 18 ± 2.0 |
11 | 7-CF3-tirapazamine | 4.83 | 0.76 | 3.4 ± 0.4 a | 6.0 ± 1.0 a |
12 | 7-Cl-tirapazamine | 4.71 | 0.49 | 3.1 ± 0.5 a | 13 ± 1.5 a |
13 | 7-F-tirapazamine | 4.48 | 0.03 | 7.2 ± 1.0 a | 25 ± 4.0 |
14 | Tirapazamine | 3.84 | 0.11 | 31 ± 5.5 a 28 ± 4.0 | 75 ± 7.0 a 64 ± 7.0 |
15 | 7-CH3-tirapazamine | 3.82 | 0.40 | 83 ± 10 a | 50 ± 6.0 a |
16 | 7-C2H5O-tirapazamine | 3.65 | 0.08 | 64 ± 10 a | 60 ± 7.0 a |
17 | 3-Amino-1,2,4-benzotriazine-1-oxide | 3.48 | 0.30 | ≥600 a | ≥600 a |
18 | Quinoxaline-1,4-dioxide | 3.22 | −0.90 | 325 ± 40 a | ≥800 |
No. | Compound | Cell Viability (%) | ||||
---|---|---|---|---|---|---|
No Additions | Additions: | |||||
DESF (1.0 mM) | DPPD (2.5 µM) | BCNU (20 µM) | DIC (20 µM) | |||
1 | 3-CH3CONH-1,2,4-benzotriazine-1,4-dioxide, 1.5 µM | 47.2 ± 4.0 | 71.8 ± 4.0 *** | 64.9 ± 3.8 *** | 34.0 ± 3.0 ** | 83.7 ± 6.1 *** |
2 | 2-NH2-3-CN-quinoxaline-1,4-dioxide, 350 µM | 47.3 ± 4.2 | 83.5 ± 7.0 ** | 72.1 ± 5.7 ** | 35.3 ± 3.6 * | 71.6 ± 5.1 ** |
3 | 1,2,4-Benzotriazine-1,4-dioxide, 10 µM | 55.6 ± 4.5 | n.d. | n.d. | n.d. | 81.2 ± 7.4 ** |
4 | 3-CF3SO2NH-1,2,4-benzotriazine-1,4-dioxide, 40 µM | 55.8 ± 4.9 | n.d. | n.d. | n.d. | 79.0 ± 3.1 ** |
5 | 2-CF3-quinoxaline-1,4-dioxide, 10 µM | 57.6 ± 4.5 | n.d. | n.d. | n.d. | 83.1 ± 5.6 ** |
Cell Line | a | b | c | d | r2 |
---|---|---|---|---|---|
7.22 ± 0.80 | −1.36 ± 0.19 | - | - | 0.7638 | |
MH22a | 7.07 ± 0.84 | −1.33 ± 0.20 | −0.09 ± 0.13 | - | 0.7704 |
8.44 ± 0.88 | −1.20 ± 0.18 | - | −0.55 ± 0.24 | 0.8266 | |
8.30 ± 0.88 | −1.14 ± 0.19 | −0.13 ± 0.118 | −0.59 ± 0.24 | 0.8405 | |
6.68 ± 0.73 | −1.19 ± 0.17 | - | - | 0.7440 | |
HCT-116 | 6.63 ± 0.79 | −1.18 ± 0.19 | −0.03 ± 0.13 | - | 0.7448 |
7.74 ± 0.82 | −1.05 ± 0.17 | - | −0.48 ± 0.22 | 0.8052 | |
7.67 ± 0.85 | −1.02 ± 0.18 | −0.06 ± 0.11 | −0.50 ± 0.23 | 0.8093 |
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Nemeikaitė-Čėnienė, A.; Šarlauskas, J.; Misevičienė, L.; Marozienė, A.; Jonušienė, V.; Lesanavičius, M.; Čėnas, N. Aerobic Cytotoxicity of Aromatic N-Oxides: The Role of NAD(P)H:Quinone Oxidoreductase (NQO1). Int. J. Mol. Sci. 2020, 21, 8754. https://doi.org/10.3390/ijms21228754
Nemeikaitė-Čėnienė A, Šarlauskas J, Misevičienė L, Marozienė A, Jonušienė V, Lesanavičius M, Čėnas N. Aerobic Cytotoxicity of Aromatic N-Oxides: The Role of NAD(P)H:Quinone Oxidoreductase (NQO1). International Journal of Molecular Sciences. 2020; 21(22):8754. https://doi.org/10.3390/ijms21228754
Chicago/Turabian StyleNemeikaitė-Čėnienė, Aušra, Jonas Šarlauskas, Lina Misevičienė, Audronė Marozienė, Violeta Jonušienė, Mindaugas Lesanavičius, and Narimantas Čėnas. 2020. "Aerobic Cytotoxicity of Aromatic N-Oxides: The Role of NAD(P)H:Quinone Oxidoreductase (NQO1)" International Journal of Molecular Sciences 21, no. 22: 8754. https://doi.org/10.3390/ijms21228754