nsequently SOD inhibition would promote cancer cell deathcancer cells death. Mitochondria are the major source of superoxide production and inhibition of SOD causes mitochondrial membrane damage through free-radical

Figure 1. The concentration-activity curves of the tested thiazole compound expressed as fraction of surviving cells against compound concentration for different cell lines; liver carcinoma (HEPG2) (a), colon carcinoma (HCT116) (b) and breast adenocarcinoma (c).

Figure 2. The structures of the tested thiazole compound (1), SOD quinazoline inhibitor (Hasnain et al. 2010) (2) and CYP2A6 inhibitor (Cashman et al. 2006).

attack on membrane phospholipids and loss of the ability to retain a fluorescent dye (rhodamine-123) used to indicate the loss of mitochondrial transmembrane potential [36] [37] . Damage to phospholipids results in the release of mitochondrial cytochrome c to the cytosol triggering apoptosis [38] . SOD inhibition might have different mechanisms such as inhibition of tubulin polymerization and angiogenesis [39] [40] .

SOD enzyme crystal structure reveals a catalytic site which can bind metal chelators principally due to the presence of metal cations [14] . Hasnain et al. [41] , however, discovered an allosteric site that binds 2-trifluormethyl-4-quinazoline derivatives as inhibitors of Cu-Zn loaded SOD. Molecular docking studies revealed several similarities between the binding modes of the tested thiazole and Hasnain’s 2-trifluormethyl-4-quinazoline derivative. Hasnain et al. [41] demonstrated that the trifluromethyl group is an important moiety for SOD inhibition as it invades a small hydrophobic cleft formed by the side chains of Lys-30 and Glu-100 disrupting the proper conformation of the allosteric site. The 2-pyridiyl group of our tested thiazole ligand was found to similarly invade this pocket and rests comfortably within appropriate distance with their hydrophobic side chains (Figure 3).

To assess the importance of different structural moieties of the thiazole derivative, a small set of virtual analogues of the tested pyridylthiazole with either oversized groups or truncated (smaller) functional groups were created. The bulkier virtual compound created by replacement of the 5-acetyl with pivaloyl

Figure 3. (a) Interactions of the thiazole compound with the hydrophobic cleft of Lys30 and Glu100. (b) Hydrogen bonding with the sidechain of Ser98. (c) π-π interactions between the thiazole compound and Trp32. (d) Overlay of the thiazole compound and the co-crystallized quinazoline inhibitor of SOD.

(trimethylacetyl) homologue, it showed lower scoring than our tested thiazole as the t-butyl group forced the thiazole ring away from making any significant aromatic interaction with Trp-32 and prevented the thiazole ring from making any hydrogen bonding with Ser-98. The only remaining binding interactions of the pyridine ring were with the small pocket between Lys-30 and Glu-100.

A smaller analogue without the pyridine ring was virtually constructed and included in the docking experiment to investigate the impact of the pyridyl group on binding. This compound hadrotated to place the acetyl group in the Lys30-Glu100 pocket but hydrophobic contacts appear to be less significant than that of the parent pyridyl analogue. Interestingly, the thiazole ring preferred to tighten its π-π stacking attraction with Trp-32 making a hydrogen bond with Ser-89 (Figure 4). We conclude from this docking experiment that the tested thiazole analogue has optimum structural architecture to bind and inhibit superoxide dismutase enzyme.

Cytochrome P-450 2A6 (CYP2A6) gene is one of three members of CYP2A gene subfamily in human, CYP2A7 and CYP2A13. Their transcripts (CYP2A6, CYP2A7 and CYP2A13) have been found in liver although CYP2A6 is the most abundant form [31] . CYP2A6 utilizes a heme cofactor to oxidize its substrates and the active site of this enzyme is compact containing a hydrophobic Phe-cluster formed by the residues Phe107, Phe111, Phe108, Phe209 and Phe480. In this region, coumarin substrate is directed towards a regioselective oxidation site through the only hydrogen bond donor Asn297 [42] .

Molecular docking against CYP2A6 revealed that tested thiazole analogue could bind to the (CYP2A6) in a similar fashion to the one observed in the 3-pyridyl-furan inhibitor co-crystallized with enzyme [43] .

As shown in (Figure 5) the carbonyl oxygen of tested thiazole analogue occupies virtually the same position as the methanamino nitrogen of the bound inhibitor, effectively completing the coordination shell of the heme iron. The central thiazole moiety parallels the furan moiety of the inhibitor, which allows for the optimal positioning of the pyridyl moiety, thereby establishing a key hydrogen bond to the side chain of Asn297 and two edge-to-face interactions with the aromatic side chains of Phe107 and Phe111.

Figure 4. Binding mode of a bulkier analogue of the thiazole compound (a) and a truncated analogue lacking the pyridyl moiety (b) with SOD.

To assess the importance of structural characteristics of the tested thiazole for the inhibition of CYP2A6, the same subset tested against SOD was once more tested against CYP2A6. The bulkier analogue possessing a tert-butyl group instead of methyl scored much lower. This is due to loss of essential interactions between the carbonyl oxygen and the metal ion and the hydrogen bond with Asn297. Hydrophobic interactions were rather maintained with the Phe cluster of CYP2A6. The smaller analogue lacking the pyridyl moiety also scored less due to loss of hydrophobic interactions with the Phe cluster and hydrogen bonding with Asn297. Interaction of the carbonyl oxygen with the metal ion was conserved. These findings indicate that the tested thiazole compound exhibits structural features necessary for the inhibition of CYP2A6 (Figure 6).

Figure 5. (a) Interactions of the thiazole compound with the hydrophobic Phe cluster of CYP2A6. (b) Hydrogen bonding with the side chain of Asn297. (c) Ineraction with the metal cation inside the heme cofactor of CYP2A6. (d) Overlay of the thiazole compound and the co-crystallized ligand of CYP2A6.

Figure 6. Binding mode of a bulkier analogue of the thiazole compound (a) and a truncated analogue lacking the pyridyl moiety (b) with CYP2A6.

Overall, tested thiazole analogue can be accommodated in the CYP2A6 binding site and maintain key residue interactions consistent with the reported inhibitors, this suggests that tested thiazole analogue could exert its action via inhibition of CYP2A6. CYP2A6 inhibition will contributeto programmed cancer cell death and increased sensitivity to chemotherapy in hepatocellular carcinoma [31] .

4. Conclusion

In conclusion, this study is reporting the selective antiproliferative activity of 5-acetyl-4-methyl-2-(3-pyridyl) thiazole in vitro and its possible mechanism of activity was demonstrated using docking studies. We also explained the structural determinants required for the simultaneous inhibition of SOD and CYP2A6 by conducting molecular comparisons in silico. The ability of the tested thiazole analogue to inhibit both SOD and CYP2A6 (mainly found in liver) might explain its pronounced activity as an anti-proliferative agent in liver carcinoma and the importance of inhibiting both enzymes simultaneously. Our findings may be useful in the future development of more potent cytotoxic agents directed towards liver cancer that act as adual inhibitors of SOD and CYP2A6.


We would like to acknowledge Dr. Andrew Flaus and Professor Heinz Peter Nasheuer from Center for Chromosome Biology, National University of Ireland for their help in editing this article.

Conflict of Interest

No conflict of interest associated with this work.

Cite this paper

Amin, H.K., El-Araby, A.M., Eid, S., Nasr, T., Bondock, S., Leheta, O. and Dawoud, M.E. (2017) A Thiazole Analogue Exhibits an Anti-Proli- ferative Effect in Different Human Carci- noma Cell Lines and Its Mechanism Based on Molecular Modeling. Advances in Bio- logical Chemistry, 7, 76-87. https://doi.org/10.4236/abc.2017.71005


  1. 1. Gibbs, J.B. (2000) Mechanism-Based Target Identification and Drug Discovery in Cancer Research. Science, 287, 1969-1973.

  2. 2. Cragg, G.M., Grothaus, P.G. and Newman, D.J. (2009) Impact of Natural Products on Developing New Anti-Cancer Agents. Chemical Reviews, 109, 3012-3043.

  3. 3. McFadyen, M.C., Melvin, W.T. and Murray, G.I. (2004) Cytochrome P450 Enzymes: Novel Options for Cancer Therapeutics. Molecular Cancer Therapeutics, 3, 363-371.

  4. 4. Otyepka, M., Skopalik, J., Anzenbacherova, E. and Anzenbacher, P. (2007) What Common Structural Features and Variations of Mammalian P450s Are Known to Date? Biochimica et Biophysica Acta, 1770, 376-389.

  5. 5. Yano, J.K., Denton, T.T., Cerny, M.A., Zhang, X., Johnson, E.F. and Cashman, J.R. (2006) Synthetic Inhibitors of Cytochrome P-450 2A6: Inhibitory Activity, Difference Spectra, Mechanism of Inhibition, and Protein Cocrystallization. Journal of Medicinal Chemistry, 49, 6987-7001.

  6. 6. Yano, J.K., Hsu, M.H., Griffin, K.J., Stout, C.D. and Johnson, E.F. (2005) Structures of Human Microsomal Cytochrome P450 2A6 Complexed with Coumarin and Methoxsalen. Nature structural & Molecular Biology, 12, 822-823.

  7. 7. Hoffman, S.M., Nelson, D.R. and Keeney, D.S. (2001) Organization, Structure and Evolution of the CYP2 Gene Cluster on Human Chromosome 19. Pharmacogenetics, 11, 687-698.

  8. 8. Ioannides, C. (2008) Cytochromes P450: Role in the Metabolism and Toxicity of Drugs and Other Xenobiotics. Royal Society of Chemistry, London.

  9. 9. Abu-Bakar, A., Arthur, D.M., Wikman, A.S., Rahnasto, M., Juvonen, R.O., Vepsalainen, J., Raunio, H., Ng, J.C. and Lang, M.A. (2012) Metabolism of Bilirubin by Human Cytochrome P450 2A6. Toxicology and Applied Pharmacology, 261, 50-58.

  10. 10. Raunio, H., Juvonen, R., Pasanen, M., Pelkonen, O., Paakko, P. and Soini, Y. (1998) Cytochrome P4502A6 (CYP2A6) Expression in Human Hepatocellular Carcinoma. Hepatology, 27, 427-432.

  11. 11. Nakajima, M., Itoh, M., Sakai, H., Fukami, T., Katoh, M., Yamazaki, H., Kadlubar, F.F., Imaoka, S., Funae, Y. and Yokoi, T. (2006) CYP2A13 Expressed in Human Bladder Metabolically Activates 4-Aminobiphenyl. International Journal of Cancer, 119, 2520-2526.

  12. 12. McCord, J.M. and Fridovich, I. (1969) Superoxide Dismutase. An Enzymic Function for Erythrocuprein (Hemocuprein). The Journal of Biological Chemistry, 244, 6049-6055.

  13. 13. Hough, M.A. and Hasnain, S.S. (1999) Crystallographic Structures of Bovine Copper-Zinc Superoxide Dismutase Reveal Asymmetry in Two Subunits: Functionally Important Three and Five Coordinate Copper Sites Captured in the Same Crystal. Journal of Molecular Biology, 287, 579-592.

  14. 14. Strange, R.W., Hough, M.A., Antonyuk, S.V. and Hasnain, S.S. (2012) Structural Evidence for a Copper-Bound Carbonate Intermediate in the Peroxidase and Dismutase Activities of Superoxide Dismutase. PLoS ONE, 7, e44811.

  15. 15. Bordo, D., Pesce, A., Bolognesi, M., Elena Stroppolo, M., Falconi, M. and Desideri, A. (2001) Copper-Zinc Superoxide Dismutase in Prokaryotes and Eukaryotes. In: Messerschmidt, A., Huber, R., Wieghardt, K. and Poulos, T., Eds., Handbook of Metalloproteins, John Wiley & Sons, Hoboken, 6.

  16. 16. Banci, L., Bertini, I., Cramaro, F., Del Conte, R. and Viezzoli, M.S. (2002) The Solution Structure of Reduced Dimeric Copper Zinc Superoxide Dismutase. The Structural Effects of Dimerization. European Journal of Biochemistry/FEBS, 269, 1905-1915.

  17. 17. Sharma, R.N., Xavier, F.P., Vasu, K.K., Chaturvedi, S.C. and Pancholi, S.S. (2009) Synthesis of 4-Benzyl-1,3-Thiazole Derivatives as Potential Anti-Inflammatory Agents: An Analogue-Based Drug Design Approach. Journal of Enzyme Inhibition and Medicinal Chemistry, 24, 890-897.

  18. 18. Medan, D., Wang, L., Toledo, D., Lu, B., Stehlik, C., Jiang, B.H., Shi, X. and Rojanasakul, Y. (2005) Regulation of Fas (CD95)-Induced Apoptotic and Necrotic Cell Death by Reactive Oxygen Species in Macrophages. Journal of Cellular Physiology, 203, 78-84.

  19. 19. Krchnak, V. and Holladay, M.W. (2002) Solid Phase Heterocyclic Chemistry. Chemical Reviews, 102, 61-91.

  20. 20. Carter, J.S., Kramer, S., Talley, J.J., Penning, T., Collins, P., Graneto, M.J., Seibert, K., Koboldt, C.M., Masferrer, J. and Zweifel, B. (1999) Synthesis and Activity of Sulfonamide-Substituted 4,5-Diaryl Thiazoles as Selective Cyclooxygenase-2 Inhibitors. Bioorganic & Medicinal Chemistry Letters, 9, 1171-1174.

  21. 21. Bell, F.W., Cantrell, A.S., Hogberg, M., Jaskunas, S.R., Johansson, N.G., Jordan, C.L., Kinnick, M.D., Lind, P., Morin, J.M., Jr., Noreen, R., et al. (1995) Phenethylthiazolethiourea (PETT) Compounds, a New Class of HIV-1 Reverse Transcriptase Inhibitors. 1. Synthesis and Basic Structure-Activity Relationship Studies of PETT Analogs. Journal of Medicinal Chemistry, 38, 4929-4936.

  22. 22. Desai, N.C., Rajpara, K.M., Joshi, V.V., Vaghani, H.V. and Satodiya, H.M. (2012) Synthesis and Characterization of Some New Thiazole Based Thiazolidinone Derivatives as Potent Antimicrobial and Antimycobacterial Agents. Anti-Infective Agents, 10, 75-86.

  23. 23. Jisha, M., Kamalabhai, A., Babu, G. and Biju, C. (2013) Synthesis, Characterization and in Vitro Anticancer Screening of Novel Thiazole-1, 3, 4-Oxadiazole Hybrid Analogues. Journal of Chemical and Pharmaceutical Research, 5, 64-70.

  24. 24. Rudolph, J., Theis, H., Hanke, R., Endermann, R., Johannsen, L. and Geschke, F. (2001) Seco-Cyclothialidines: New Concise Synthesis, Inhibitory Activity toward Bacterial and Human DNA Topoisomerases, and Antibacterial Properties. Journal of Medicinal Chemistry, 44, 619-626.

  25. 25. Radhakrishnan, S.K., Bhat, U.G., Hughes, D.E., Wang, I.C., Costa, R.H. and Gartel, A.L. (2006) Identification of a Chemical Inhibitor of the Oncogenic Transcription Factor Forkhead Box M1. Cancer Research, 66, 9731-9735.

  26. 26. Bhat, U.G., Zipfel, P.A., Tyler, D.S. and Gartel, A.L. (2008) Novel Anticancer Compounds Induce Apoptosis in Melanoma Cells. Cell Cycle, 7, 1851-1855.

  27. 27. Chang, S., Zhang, Z., Zhuang, X., Luo, J., Cao, X., Li, H., Tu, Z., Lu, X., Ren, X. and Ding, K. (2012) New Thiazolecarboxamides as Potent Inhibitors of Akt Kinases. Bioorganic & Medicinal Chemistry Letters, 22, 1208-1212.

  28. 28. Bondock, S., Naser, T. and Ammar, Y.A. (2013) Synthesis of Some New 2-(3-pyridyl)-4,5-disubstituted Thiazoles as Potent Antimicrobial Agents. European Journal of Medicinal Chemistry, 62, 270-279.

  29. 29. Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahon, J., Vistica, D., Warren, J.T., Bokesch, H., Kenney, S. and Boyd, M.R. (1990) New Colorimetric Cytotoxicity Assay for Anticancer-Drug Screening. Journal of the National Cancer Institute, 82, 1107-1112.

  30. 30. Kershaw, N.M., Wright, G.S., Sharma, R., Antonyuk, S.V., Strange, R.W., Berry, N.G., O’Neill, P.M. and Hasnain, S.S. (2013) X-Ray Crystallography and Computational Docking for the Detection and Development of Protein-Ligand Interactions. Current Medicinal Chemistry, 20, 569-575.

  31. 31. Pelkonen, O., Rautio, A., Raunio, H. and Pasanen, M. (2000) CYP2A6: A Human Coumarin 7-Hydroxylase. Toxicology, 144, 139-147.

  32. 32. Fridovich, I. (1995) Superoxide Radical and Superoxide Dismutases. Annual Review of Biochemistry, 64, 97-112.

  33. 33. Marklund, S.L., Westman, N.G., Lundgren, E. and Roos, G. (1982) Copper- and Zinc-Containing Superoxide Dismutase, Manganese-Containing Superoxide Dismutase, Catalase, and Glutathione Peroxidase in Normal and Neoplastic Human Cell Lines and Normal Human Tissues. Cancer Research, 42, 1955-1961.

  34. 34. Shulyakovskaya, T., Sumegi, L. and Gal, D. (1993) In Vivo Experimental Studies on the Role of Free Radicals in Photodynamic Therapy. I. Measurement of the Steady State Concentration of Free Radicals in Tumor Tissues of Mice. Biochemical and Biophysical Research Communications, 195, 581-587.

  35. 35. Van Driel, B.E. and Van Noorden, C.J. (1999) Oxygen Insensitivity of the Histochemical Assay of Glucose-6-Phosphate Dehydrogenase Activity for the Discrimination between Nonmalignant and Malignant Cells. The Journal of Histochemistry and Cytochemistry, 47, 575-582.

  36. 36. Marzo, I., Brenner, C., Zamzami, N., Jürgensmeier, J.M., Susin, S.A., Vieira, H.L., Prévost, M.-C., Xie, Z., Matsuyama, S. and Reed, J.C. (1998) Bax and Adenine Nucleotide Translocator Cooperate in the Mitochondrial Control of Apoptosis. Science, 281, 2027-2031.

  37. 37. Vander Heiden, M.G., Chandel, N.S., Williamson, E.K., Schumacker, P.T. and Thompson, C.B. (1997) Bcl-xL Regulates the Membrane Potential and Volume Homeostasis of Mitochondria. Cell, 91, 627-637.

  38. 38. Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S.M., Ahmad, M., Alnemri, E.S. and Wang, X. (1997) Cytochrome c and dATP-Dependent Formation of Apaf-1/Cas-pase-9 Complex Initiates an Apoptotic Protease Cascade. Cell, 91, 479-489.

  39. 39. D’Amato, R.J., Lin, C.M., Flynn, E., Folkman, J. and Hamel, E. (1994) 2-Methoxye-stradiol, an Endogenous Mammalian Metabolite, Inhibits Tubulin Polymerization by Interacting at the Colchicine Site. Proceedings of the National Academy of Sciences of the United States of America, 91, 3964-3968.

  40. 40. Fotsis, T., Zhang, Y., Pepper, M.S., Adlercreutz, H., Montesano, R., Nawroth, P.P. and Schweigerer, L. (1994) The Endogenous Oestrogen Metabolite 2-Methoxyoes-tradiol Inhibits Angiogenesis and Suppresses Tumour Growth. Nature, 368, 237-239.

  41. 41. Antonyuk, S., Strange, R.W. and Hasnain, S.S. (2010) Structural Discovery of Small Molecule Binding Sites in Cu-Zn Human Superoxide Dismutase Familial Amyotrophic Lateral Sclerosis Mutants Provides Insights for Lead Optimization. Journal of Medicinal Chemistry, 53, 1402-1406.

  42. 42. Kim, D., Wu, Z.L. and Guengerich, F.P. (2005) Analysis of Coumarin 7-Hydroxy-lation Activity of Cytochrome P450 2A6 Using Random Mutagenesis. The Journal of Biological Chemistry, 280, 40319-40327.

  43. 43. Khojasteh-Bakht, S.C., Koenigs, L.L., Peter, R.M., Trager, W.F. and Nelson, S.D. (1998) (R)-(+)-Menthofuran Is a Potent, Mechanism-Based Inactivator of Human Liver Cytochrome P450 2A6. Drug Metabolism and Disposition: The Biological Fate of Chemicals, 26, 701-704.


*H.K.A. and M.E.D. contributed equally to the article.

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