SUMMARY. One of the way to enhance the efficiency of alkylating chemotherapy is reducing the level of the repair enzyme MGMT (O6-methylguanine-DNA methyltransferase) in cancer cells. The standard MGMT inhibitor, O6-benzylguanine (BG), has exhibited cytotoxicity towards hematopoietic cells in the third stage of clinical trials, making the search for new alternative inhibitors relevant. In this study, we have conducted research to determine the cytotoxicity and efficacy of new potential MGMT inhibitors, which were modeled using molecular flexible docking. At the first stage of the study, MTT and clonogenic assays were performed to assess cytotoxicity, in which HEp-2 cells were cultured with the tested compounds at a concentration of 10 µM. At the second stage, the efficacy of the compounds was evaluated. One of the methods used was a clonogenic assay, in which the cell treatment consisted of combinations of the tested compounds (10 µM) and the alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) at different con-
centrations. Another method was Western blot analysis, for which proteins were extracted from HEp-2 cells treated with potential inhibitors in combination with MNNG. The obtained results were analyzed in Microsoft Excel 2016, Origin8.1 and ImageLab. As a result, 4 of the 5 examined compounds demonstrated low cytotoxicity at a concentration of 10 µM in HEp-2 cells compared to the standard inhibitor BG. According to the clonogenic assay, compound 41B (5-Benzo[1,3]dioxol-5-ylmethylene-thiazolidin-2,4-dione) was the most effective, and compounds 41 (5-(5-Chloro-2-hydroxy-benzylidene)-4-thioxo-thiazolidin-2-one) and 89 (2-[5-(4-Bromo-phenyl)-pyrimidin-4-yl]-5-ethoxy-phenol) also showed high efficiency. The results of Western blot analysis showed a significant decrease in MGMT protein after treatment with compounds 41, 41B and 89, thereby confirming the inhibitory properties of these compounds.
Keywords: Repair enzyme MGMT, new potential MGMT inhibitors, alkylating agent, cell line HEp-2
Full text and supplemented materials
References
Arslan, F.T., Yurdakok-Dikmen, B., Akgedik, R., and Topcu, G., Nitrosoguanidine-induced genotoxicity and oxidative stress in human gastric adenocarcinoma cells, Mutat. Res., 2016, vol. 797, pp. 28–34.
Chae, M.-Y., Swenn, K., Kanugula, S., Dolan, M.E., Pegg, A.E., and Moschel, R.C., 8-substituted O6-benzylguanine, substituted 6(4)-(benzyloxy)pyrimidine, and related derivatives as inactivators of human O6-alkylguanine-DNA alkyltransferase, J. Med. Chem., 1995, vol. 38, no. 2, pp. 359–365. https://doi.org/10.1021/jm00002a018
Christmann, M., Verbeek, B., Roos, W.P., and Kaina, B., O6-Methylguanine-DNA methyltransferase (MGMT) in normal tissues and tumors: Enzyme activity, promoter methylation and immunohistochemistry, Biochim. Biophys. Acta, Rev. Cancer, 2011, vol. 1816, no. 2, pp. 179–190. https://doi.org/10.1016/j.bbcan.2011.06.002
Dolan, M., Chae, M., Pegg, A., et al., Metabolism of O6-benzylguanine, an inactivator of O6-alkylguanine-DNA alkyltransferase, Cancer Res., 1994, vol. 54, no. 19, pp. 5123–5130. https://doi.org/10.1158/0008-5472.CAN-14-2047
Green, S.J. and Michael, R., Molecular Cloning, New York: Cold Spring Harbor Lab., 2012.
Griffin, R.J., Arris, C.E., Bleasdale, C., Boyle, F.T., Calvert, A.H., Curtin, N.J., Dalby, C., Kanugula, S., Lembicz, N.K., Newell, D.R., Pegg, A.E., and Golding, B.T., Resistance-Modifying Agents. 8. Inhibition of O 6-Alkylguanine-DNA Alkyltransferase by O 6-Alkenyl-, O 6-Cycloalkenyl-, and O 6-(2-Oxoalkyl)guanines and Potentiation of Temozolomide Cytotoxicity in Vitro by O6-(1-Cyclopentenylmethyl)guanine, J. Med. Chem., 2000, vol. 43, no. 22, pp. 4071–4083. https://doi.org/10.1021/jm000961o
Kaina, B., Margison, G.P., and Christmann, M., Targeting O 6-methylguanine-DNA methyltransferase with specific inhibitors as a strategy in cancer therapy, Cell. Mol. Life Sci., 2010, vol. 67, no. 21, pp. 3663–3681. https://doi.org/10.1007/s00018-010-0491-7
Kotsarenko, K.V., Lylo, V.V., Macewicz, L.L., Dasyukevich, O.I., Poltoratskaya, L.V., and Burkovskaya, T.E., Changes in the MGMT gene expression under the influence of exogenous cytokines in human cells in vitro, Cytol. Genet., 2013, vol. 47, no. 4, pp. 202–209. https://doi.org/10.3103/S0095452713040087
Kotsarenko, K., Lylo, V., Ruban, T., Macewicz, L., and Lukash, L., Effects of some growth factors and cytokines on the expression of the repair enzyme MGMT and protein MARP in human cells in vitro, Biochem. Genet., 2018, vol. 56, pp. 459–477. https://doi.org/10.1007/s10528-018-9854-9
Lipinski, C.A., Lead- and drug-like compounds: the rule-of-five revolution, Drug Discovery Today: Technol., 2004, vol. 1, no. 4, pp. 337–341. https://doi.org/10.1016/j.ddtec.2004.11.007
Lopez, S., Margison, G.P., McElhinney, R.S., Cordeiro, A., McMurry, T.B H., and Rozas, I., Towards more specific O 6-methylguanine-DNA methyltransferase (MGMT) inactivators, Bioorg. Med. Chem., 2011, vol. 19, no. 5, pp. 1658–1665. https://doi.org/10.1016/j.bmc.2011.01.038
Lukash, L.L., Bodt, J., Pegg, A.E., Dolan, M.E., Maher, V.M., and McCormick, J., Effect of O 6-alkylguanine-DNA alkyltransferase on the frequency and spectrum of mutations induced by N-methyl-N’-nitro-N-nitrosoguanidine in the HPRT gene of diploid human fibroblasts, Mutat. Res., 1991, vol. 250, no. 12, pp. 397–409. https://doi.org/10.1016/0027-5107(91)90196-U
McElhinney, R., Donnelly, D., McCormick, A., et al., Inactivation of O 6-alkylguanine-DNA alkyltransferase. 1. Novel O 6-(hetarylmethyl)guanines having basic rings in the side chain, J. Med. Chem., 1998, vol. 41, no. 26, pp. 5265–5271. https://doi.org/10.1021/jm9804388
Mitra, S., MGMT: A personal perspective, DNA Repair, 2007, vol. 6, no. 8, pp. 1064–1070. https://doi.org/10.1016/j.dnarep.2007.03.007
Mohanty, S., Sharma, P., Gupta, P.K., and Chatterjee, S., Nitrosoguanidine-induced DNA damage response in Escherichia coli cells, Mutat. Res., 2019, vol. 842, pp. 18–27. https://doi.org/10.1016/j.mrgentox.2019.04.006
Morris, G.M., Huey, R., Lindstrom, W., Sanner, M.F., Belew, R.K., Goodsell, D.S., and Olson, A.J., AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexiblity, J. Comput. Chem., 2009, vol. 30, pp. 2785–2791.
Moschel, R.C., McDougall, M., Dolan, M.E., Pegg, A.E., and Phillips, D.R., Structural features of substituted purine derivatives compatible with depletion of human O 6-alkylguanine-DNA alkyltransferase, J. Med. Chem., 1992, vol. 35, no. 23, pp. 4486–4491. https://doi.org/10.1021/jm00103a019
Oshiro, S., Tsugu, H., Komatsu, F., Ohmura, T., Ohta, M., Sakamoto, S., Fukushima, T., and Inoue, T., Efficacy of Temozolomide Treatment in Patients with High-grade Glioma, Anticancer Res., 2009, vol. 29, pp. 911–918. https://ar.iiarjournals.org/content/29/3/911.full.
Pauly, G., Loktionova, N., Fang, Q., et al., Substitution of aminomethyl at the meta-position enhances the inactivation of O 6-alkylguanine-DNA alkyltransferase by O 6-benzylguanine, J. Med. Chem., 2008, vol. 51, no. 22, pp. 7144–7153. https://doi.org/10.1021/jm800758y
Pedretti, A., Mazzolari, A., Gervasoni, S., Fumagalli, L., and Vistoli, G., The VEGA suite of programs: an versatile platform for cheminformatics and drug design projects, Bioinformatics, 2021, vol. 37, no. 8, pp. 1174–1175. https://doi.org/10.1093/bioinformatics/btaa774
Pegg, A.E., Repair of O 6-alkylguanine by alkyltransferases, Mutat. Res., 2000, vol. 462, nos. 2–3, pp. 83–100. https://doi.org/10.1016/s1383-5742(00)00017-x
Pegg, A.E., Multifaceted roles of alkyltransferase and related proteins in DNA Repair, DNA damage, resistance to chemotherapy, and research tools, Chem. Res. Toxicol., 2011, vol. 24, no. 5, pp. 618–639. https://doi.org/10.1021/tx200031q
Quinn, J.A., Jiang, S.X., Carter, J., Reardon, D.A., Desjardins, A., Vredenburgh, J.J., Friedman, H.S., Phase II trial of gliadel plus O 6-benzylguanine in Adults with recurrent glioblastoma multiforme, Clin. Cancer Res., 2009, vol. 15, no. 3, pp. 1064–1068. https://doi.org/10.1158/1078-0432.ccr-08-2130
Ranson, M., Lomeguatrib, a potent inhibitor of O 6-alkylguanine-DNA-alkyltransferase: Phase I safety, pharmacodynamic, and pharmacokinetic trial and evaluation in combination with temozolomide in patients with advanced solid tumors, Clin. Cancer Res., 2006, vol. 12, no. 5, pp. 1577–1584. https://doi.org/10.1158/1078-0432.ccr-05-2198
Ruiz, F., Gil-Redondo, R., Morreale, A., et al., Structure-based discovery of novel non-nucleosidic DNA alkyltransferase inhibitors: virtual screening and in vitro and in vivo activities, J. Chem. Inf. Modell., 2008, vol. 48, no. 10, pp. 1972–1982. https://doi.org/10.1021/ci800202t
Sharma, S., Salehi, S., Yang, Y., and Vessella, R.L., Role of MGMT in tumor development, progression, diagnosis, treatment and prognosis, Anticancer Res., 2009, vol. 29, no. 10, pp. 3759–3768. https://doi.org/10.1016/j.etap.2019.03.012
Terashima, I. and Kohda, K., Inhibition of human O 6-alkylguanine-DNA alkyltransferase and potentiation of the cytotoxicity of chloroethylnitrosourea by 4(6)-(benzyloxy)-2,6(4)-diamino-5-(nitro or nitroso)pyrimidine derivatives and analogues, J. Med. Chem., 1998, vol. 41, no. 4, pp. 503–508. https://doi.org/10.1021/jm970712f
Verbeek, B., Southgate, T.D., Gilham, D.E., and Margison, G.P., O 6-methylguanine-DNA methyltransferase inactivation and chemotherapy, Br. Med. Bull., 2008, vol. 85, no. 1, pp. 17–33. https://doi.org/10.1093/bmb/ldm036
Volynets, G.P., Ruban, T.P., Yatsyshina, A.P., Matsevich, L.L., Bdzhola, V.G., Yarmolyuk, S.M., and Lukash, L.L., RF Patent 127059, 2018. https:// base.uipv.org/searchINV/search.php?action=viewdetails&IdClaim=2.492.48.
Volynets, G.P., Yatsyshina, A.P., Ruban, T.P., Matse-vich, L.L., Nidoyeva, Z.M., Balanda, A.O., Bdzhola, V.G., Yarmolyuk, S.M., and Lukash, L.L., Ukraine Patent 122373, 2020. https://base.uipv.org/ searchINV/search.php?action=viewdetails&IdClaim =271905.
Wang, C., Abegg, D., Hoch, D., and Adibekian, A., Chemoproteomics-enabled discovery of a potent and selective inhibitor of the DNA repair protein MGMT, Angew. Chem., Int. Ed., 2016, vol. 55, no. 10, pp. 2911–2915. https://doi.org/10.1002/anie.201510203
Yu, W., Zhang, L., Wei, Q., and Shao, A., O6-methylguanine-DNA methyltransferase (MGMT): challenges and new opportunities in glioma chemotherapy, Front. Oncol., 2020, vol. 9, p. 1547. https://doi.org/10.3389/fonc.2019.01547
Zhang, X., Zhou, Y., Hu, Y., and Huang, P., Nitrosoguanidine-induced DNA damage and cell cycle arrest in human liver cells, Environ. Toxicol. Pharmacol., 2019, vol. 68, pp. 66–71. https://doi.org/10.1016/j.etap.2019.03.012