Genotoxicity of Novel Pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine Sulfonamides in Normal and Cancer Cells In Vitro
Abstract
:1. Introduction
2. Results
2.1. Trypan Blue Staining
2.2. Alamar Blue
2.3. Alkaline Comet Assay
2.3.1. Cancer Cells
2.3.2. Normal Human Fibroblasts (WI-38 Cell Line)
2.4. Neutral Comet Assay
2.5. γH2AX Staining
2.6. Computational Studies
2.6.1. Molecular Docking
2.6.2. Molecular Dynamics
3. Discussion
4. Materials and Methods
4.1. Chemicals
4.2. Cell Culture
4.3. Cell Treatment
4.4. Trypan Blue Staining
4.5. Alamar Blue
4.6. Comet Assays
4.6.1. Alkaline Comet Assay
4.6.2. Neutral Comet Assay
4.7. γH2AX Staining
4.8. Data Analysis
4.8.1. Alkaline and Neutral Comet Assays
4.8.2. γH2AX Staining
4.9. Computational Studies
4.9.1. Molecular Docking
4.9.2. Molecular Dynamics
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
ABL | ABL protein kinase |
AKT | RAC-alpha serine/threonine-protein kinase |
ATM | serine/threonine protein kinase ATM |
ATR | serine/threonine protein kinase ATR |
BAX/BAK | pro-apoptotic protein BAX/BAK |
BSA | bovine serum albumin |
BTK | Bruton’s tyrosine kinase |
CA | carbonic anhydrase |
CDK | cyclin-dependent kinase |
CHK1 | serine/threonine protein kinase CHK1 |
DAPI | 4′,6-diamidino-2-phenylindole |
DDR | DNA damage response |
DSB | double-strand breaks |
FBS | fetal bovine serum |
IRF1 | Interferon regulatory factor 1 |
JAK | tyrosine-protein kinase JAK |
MMP | mitochondria membrane potential |
mTOR | mammalian target of rapamycin |
NMP | normal melting point agarose |
PARP1 | poly[ADP-ribose] polymerase 1 |
PBS | buffered saline |
PD-1 | programmed death 1 |
PD-L1 | programmed death ligand 1 |
PHA | paraformaldehyde |
PI3K | phosphatidylinositol-4,5-bisphosphate 3-kinase |
s-ICAM-1 | soluble intercellular adhesion molecule-1 |
SSBs | single-strand breaks |
STAT1/3 | signal transducer and activator of transcription 1/3 |
TP53 | cellular tumor antigen p53 |
TRIS | tris(hydroxymethyl)aminomethane |
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MM Compound | Binding Energy (Kcal/mol) | Binding Residues | Reference Ligand | Binding Energy (Kcal/mol) | Binding Residues |
---|---|---|---|---|---|
APE-1 DNA-(apurinic or apyrimidinic site) endonuclease 6MKO | |||||
MM134 | −9.18 | Asp308, His309, Gly176, Gly178, Arg177, Ala230, Trp280, Asn174 | Methoxyamine (No native ligand was complexed) | −5.74 | Asn212, Asp210, His309, Asn68, Asp308, Glu96 |
MM136 | −8.07 | Gly178, Arg177, Gly176, Asp308, Trp280, His309, Leu282, Phe266 | |||
MM137 | −9.64 | Asp308, His309, Gly176, Ala230, Trp280, Phe266, Asn212, Tyr171 | |||
MM139 | −7.63 | Tyr269, Phe266, Met270, Thr268, Ala230, Asn174, Gly231 | |||
ATR Serine/threonine-protein kinase ATR 4WAF | |||||
MM134 | −10.06 | Asp933, Ile848, Tyr836, Ile932, Val850, Met922, Trp780, Thr856, His855 | N,N-dimethyl-4-[(6R)-6-methyl-5-(1H-pyrrolo[2,3-b]pyridin-4-yl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-3-yl]benzenesulfonamide | −10.62 | Ile848, Ile932, Val850, Val851, Met922, Glu849, Thr856, His855, Gln859, Lys802, Met800 |
MM136 | −10.32 | Tyr836, Ile932, Val850, Arg852, Val851, Met922, Asn853, Ser854, Trp780 | |||
MM137 | −10.95 | Tyr836, Val851, Met922, Val850, Ile932, Trp780, Ser854, Arg852 | |||
MM139 | −10.34 | Ile848, Ile932, Val850, Trp780, Thr856, His855 | |||
ATM Serine/threonine-protein kinase ATR 7NI5 | |||||
MM134 | −9.02 | Pro2775, Gln2874, Trp2769, Leu2767, Leu2715, Ile2888, Tyr2755 | KU-55933 | −10.58 | Tyr2755, Cys2770, Glu2768, Leu2767, Lys2717, Trp2769, Leu2715, Pro2699, Ala2693, Pro2775, Leu2877 |
MM136 | −9.17 | Asp2720, Lys2717, Leu2877, Trp2769, Thr2773, Cys2770, Tyr2755, Leu2767, Ile2888, Asn2697 | |||
MM137 | −9.16 | Ala2693, Glu2778, Ile2888, Lys2717, Tyr2755, Leu2767, Cys2770, Trp2769, Leu2877, Pro2775, Val2774 | |||
MM139 | −10.05 | Trp2769, Leu2877, Tyr2755, Ile2888, Gly2694, Pro2699, Leu2715, Cys2770 | |||
CHK1 Serine/threonine-protein kinase Chk1 2YM8 | |||||
MM134 | −9.75 | Cys87, Ala36, Leu137, Leu15, Val23, Leu84, Lys38, Phe149 | (R)-5-(8-chloroisoquinolin-3-ylamino)-3-(1-(dimethylamino)propan-2-yloxy)pyrazine-2-carbonitrile | −9.14 | Ala36, Leu84, Glu85, Val68, Lys38, Glu134, Asn135, Leu137, Val23, Cys87, Tyr86, Leu15 |
MM136 | −10.44 | Cys87, Tyr86, Leu137, Leu84, Ala36, Val68, Phe149, Lys38, Val23, Gly90 | |||
MM137 | −10–82 | Cys87, Ala36, Leu137, Val23, Val68, Lys38, Leu84, Phe149, Asn59, Tyr86 | |||
MM139 | −10.61 | Cys87, Phe149, Lys38, Val23, Ala36, Leu137, Leu15 | |||
CHK2 Serine/threonine-protein kinase Chk2 2W0J | |||||
MM134 | −9.72 | Leu301, Lys249, Asn352, Ile299, Glu308, Val234, Leu354, Leu226 | 4,4′-diacetyldiphenylurea-bis(guanyl-hydrazone) | −9.76 | Glu273, Thr367, Ile299, Lys249, Leu301, Val234, Leu303, Leu226, Glu305 |
MM136 | −9.64 | Leu226, Met304, Ala247, Leu301, Ile299, Lys249, Thr367, Leu354, Val234 | |||
MM137 | −9.72 | Leu226, Met304, Ala247, Val234, Leu354, Lys249, Leu301, Ile299 | |||
MM139 | −10.23 | Phe369, Lys249, Ala247, Leu354, Leu226, Met304, Leu303, Val234, Thr367 | |||
PARP-1 Poly [ADP-ribose] polymerase 1 7ONS | |||||
MM134 | −10.51 | Glu763, Tyr889, Asp766, Ala880, Leu877, Arg878, Leu769, Pro881, Tyr896 | 7-[[4-(1,5-dimethylimidazol-2-yl)piperazin-1-yl]methyl]-3-ethyl-1~{H}-quinolin-2-one | −10.36 | Gly863, Arg878, Leu769, Pro881, Asp770, Asn767, Tyr896, Tyr907, Lys903, His862, Ala898 |
MM136 | −9.88 | Tyr907, His862, Asp766, Asp770, Leu877, Ile872, Arg878, Ala880 | |||
MM137 | −10.29 | Arg878, Ala898, Tyr907, Tyr896, His862, Ile879 | |||
MM139 | −10.36 | Ala880, Leu769, Pro881, Arg878, Asn767, Arg865, His909, Tyr907, His862, Asp770 | |||
RPA70 Replication protein A 70 kDa DNA-binding subunit 4IJL | |||||
MM134 | −5.81 | Arg41, Ile95, Ile83 | {[5-(3-chloro-1-benzothiophen-2-yl)-4-phenyl-4H-1,2,4-triazol-3-yl]sulfanyl}acetic acid | −5.97 | Leu87, Met57, Arg41, Ala59, Thr60, Ile95, Val93, Asn85 |
MM136 | −6.16 | Met97, Ala59, Gln61, Ile83, Ile95 | |||
MM137 | −5.71 | Arg41, Met92, Ala59, Ile95, Met57, Ile83 | |||
MM139 | −7.08 | Met57, Ile95, Ile83 | |||
TOP1 Topoisomerase I 1TL8 | |||||
MM134 | −9.81 | Met428, Asn352, Lys436, Arg364, Lys425, Thrr426, DA13, DA14 | 2,3-dimethoxy-12H-[1,3]dioxolo[5,6]indeno[1,2]isoquinolin-6-ium | −9.53 | Asn722, Thr718, Arg364, DT10, DG12, DA13 |
MM136 | −10.44 | Lys425, Arg364, Glu356, Asn722, DT10, DG12, DA13 | |||
MM137 | −10.69 | Lys425, Lys374, Arg364, DT10, DG12, DA13 | |||
MM139 | −10.34 | Lys532, DT10 | |||
TOP2B Topoisomerase II 3QX3 | |||||
MM134 | −8.54 | Glu477, Gly478, DC8, DA12, DG13 | Etoposide | −9.98 | Asp479, Arg503, Gly478, Met782, DC8, DA12, DG13 |
MM136 | −8.24 | Arg503, Ala779, Met782, DA12, DG13 | |||
MM137 | −8.32 | Ser480, Leu502, Asp559, Asp557 | |||
MM139 | −7.76 | Gly478, Arg503, DC8, DA12, DG13 | |||
WEE1 Wee1-like protein kinase 2IN6 | |||||
MM134 | −9.14 | Asp386, Ile305, Cys379, Phe433, Val313, Ala326, Ser307 | PD311839 | −9.45 | Gly382, Cys379, Phe433, Ala326, Glu377, Val313, Val360, Asn376, Lys328, Ile305, Gly306, Ser307 |
MM136 | −9.99 | Cys379, Asn376, Ile374, His350, Gly382, Val313, Ala326, Phe433, Val360, Lys328, Asp463 | |||
MM137 | −10.07 | Ile305, Val313, Lys328, Asp463, Ile374, Asn376, Phe433, Val360, Ala326, Cys379 | |||
MM139 | −9.92 | Lys328, Val313, Ala326, Phe433, Ile305, Tyr378, Gly382, Ser383 |
Compound | Cell Line | ||
---|---|---|---|
IC50 Value ± SD | |||
BxPC-3 | PC-3 | WI-28 | |
MM134 | 0.32 ± 0.1 | 0.16 ± 0.02 | 0.65 ± 0.07 |
MM136 | 0.25 ± 0.08 | 0.13 ± 0.01 | 0.48 ± 0.09 |
MM137 | 0.16 ± 0.04 | 0.11 ± 0.007 | 0.27 ± 0.04 |
MM139 | 0.33 ± 0.14 | 0.17 ± 0.003 | 0.64 ± 0.06 |
Target | Full Name | PDB Code | x-D | y-D | z-D | Spacing (Ả) | X-Center | Y-Center | Z-Center |
---|---|---|---|---|---|---|---|---|---|
APE-1 | DNA-(apurinic or apyrimidinic site) endonuclease | 6MKO | 50 | 50 | 50 | 0.381 | 20.283 | 22.147 | 20.508 |
ATR | Serine/threonine-protein kinase ATR | 4WAF | 40 | 40 | 46 | 0.397 | −1.215 | 8.294 | −17.439 |
ATM | Serine/threonine-protein kinase ATR | 7NI5 | 40 | 40 | 40 | 0.397 | 111.559 | 150.337 | 210.394 |
CHK1 | Serine/threonine-protein kinase Chk1 | 2YM8 | 40 | 40 | 40 | 0.414 | 15.394 | −1.219 | 11.745 |
CHK2 | Serine/threonine-protein kinase Chk2 | 2W0J | 40 | 40 | 46 | 0.408 | 37.373 | −31.962 | 9.087 |
PARP-1 | Poly [ADP-ribose] polymerase 1 | 7ONS | 40 | 40 | 46 | 0.397 | 10.962 | 42.743 | 7.819 |
RPA70 | Replication protein A 70 kDa DNA-binding subunit | 4IJL | 40 | 40 | 46 | 0.369 | −5.944 | −9.334 | 1.963 |
TOP1 | Topoisomerase I | 1TL8 | 40 | 40 | 40 | 0.392 | 22.245 | −4.32 | 27.329 |
TOP2B | Topoisomerase II | 3QX3 | 40 | 40 | 40 | 0.553 | 32.884 | 95.413 | 50.785 |
WEE1 | Wee1-like protein kinase | 2IN6 | 40 | 40 | 40 | 0.408 | 3.987 | 52.579 | 26.054 |
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Kciuk, M.; Mujwar, S.; Marciniak, B.; Gielecińska, A.; Bukowski, K.; Mojzych, M.; Kontek, R. Genotoxicity of Novel Pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine Sulfonamides in Normal and Cancer Cells In Vitro. Int. J. Mol. Sci. 2023, 24, 4053. https://doi.org/10.3390/ijms24044053
Kciuk M, Mujwar S, Marciniak B, Gielecińska A, Bukowski K, Mojzych M, Kontek R. Genotoxicity of Novel Pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine Sulfonamides in Normal and Cancer Cells In Vitro. International Journal of Molecular Sciences. 2023; 24(4):4053. https://doi.org/10.3390/ijms24044053
Chicago/Turabian StyleKciuk, Mateusz, Somdutt Mujwar, Beata Marciniak, Adrianna Gielecińska, Karol Bukowski, Mariusz Mojzych, and Renata Kontek. 2023. "Genotoxicity of Novel Pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine Sulfonamides in Normal and Cancer Cells In Vitro" International Journal of Molecular Sciences 24, no. 4: 4053. https://doi.org/10.3390/ijms24044053