EZH2 Inhibitor GSK126: Metabolism, drug transporter and rat pharmacokinetic studies

Amit Kumar, Vijay Kumar, Janet Lightner, Anaheed Little, Promsuk Jutabha, Hitoshi Endou, Peter Rice, Philip Reigan, Rajeev Vibhakar, Peter Harris, Ilango Balakrishnan Ilango, Sujatha Venkataraman, Marielle Nebout, Jean-Francois Peyron, Michael F. Wempe


Histone lysine methyl transferase 2 (EZH2) inhibitor GSK126 and a novel deuterated internal standard GSK126-d7 were chemically prepared. We performed in vitro experiments using the prepared GSK126 to: i) confirm in vitro EZH2 inhibitory activity; ii) conduct Sprague-Dawley (SD) rat liver microsomal incubations and identified Phase I metabolites; iii) determine whether or not GSK126 was an Organic Anion Transporter (OAT) substrate; and, iv) determine oral bioavailability by conducting oral and orbital sinus dosing (OSD) experiments and determining blood concentration versus time profiles. GSK126 was shown to decrease the expression of H3K27Me3 protein in medulloblastoma D283 cells and was able to decrease cell viability in KO99L cells, a novel T cell lymphoma cell line. Three in vitro hepatic Phase I mono-oxidative metabolites (L-M1, L-M2 and L-M3) were observed and also detected in rat liver and urine samples from the in vivo studies.  GSK126 was found to be an OAT1 and OAT2 substrate, but not an OAT3 or OAT4 substrate. Our Pharmacokinetic (PK) results indicate: 1) GSK126 has very poor oral bioavailability (< 2%); 2) co-administration of probenecid, a prototypical OAT inhibitor, did not significantly alter observed PK; and 3) tissue distribution studies demonstrate that GSK126 predominately distributes to the liver and kidneys after an OSD.


EZH2 Inhibitor; GSK126 and GSK126-d7; OAT’s; Drug Metabolism; Rat Pharmacokinetic Study

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Alimova, I.; Venkataraman, S.; Harris, P.; Marquez, V. E.; Northcott, P. A.; Dubuc, A.; Taylor, M. D.; Foreman, N. K.; Vibhakar, R. 2012. Targeting the enhancer of zeste homologue 2 in medulloblastoma. Int J Cancer, 131:1800-1809.

Alimova, I.; Birks, D. K.; Harris, P. S.; Knipstein, J. A.; Venkataraman, S.; Marquez, V. E.; Foreman, N. K.; Vibhakar, R. 2013. Inhibition of EZH2 suppresses self-renewal and induces radiation sensitivity in atypical rhabdoid teratoid tumor cells. Neuro Oncol, 15:149-60.

Anzai, N.; Wakui, S.; Jutabha, P.; Muto, T.; Hayashi, M.; Hayashi, K.; Domae, M.; Uchida, K.; Wempe, M. F.; Endou, H. 2011. Human Drug Transporter Gene-expressing Cells are Useful Alternatives to Predict Pharmacokinetics in Man. AATEX, 16:66-73.

Be´guelin, W.; Popovic, R.; Teater, M.; Jiang, Y.; Bunting, K. L.; Rosen, M.; Shen, H.; Yang, S. N.; Wang, L.; Ezponda, T.; Martinez-Garcia, E.; Zhang, H.; Zheng, Y.; Verma, S. K.; McCabe, M. T.; Ott, H. M.; Van Aller, G. S.; Kruger, R. G.; Liu, Y.; McHugh, C. F.; Scott, D. W.; Chung, Y. R.; Kelleher, N.; Shaknovich, R.; Creasy, C. L.; Gascoyne, R. D.; Wong, K-K.; Cerchietti, L.; Levine, R. L.; Abdel-Wahab, O.; Licht, J. D.; Elemento, O.; Melnick, A. M. 2013. EZH2 is required for germinal center formation and somatic EZH2 mutations promote lymphoid transformation. Cancer Cell, 23:677–692.

Bhasin, M.; Reinherz, E. L.; Reche, P. A. 2006. Recognition and classification of histones using support vector machine. Journal of Computational Biology, 13:102-112.

Brackley, J.; Burgess, J.L.; Grant, S.; Johnson, N.; Knight, S.D.; LaFrance, L.; Miller, W.H.; Newlander, K.; Romeril, S.; Rouse, M.B.; Tian, X.; Verma, S.K. 2011. International Application Published Under the Patent Cooperation Treaty (PCT). WO 2011/140324 A1.

Cardoso, C.; Mignon, C.; Hetet, G.; Grandchamps, B.; Fontes, M.; Colleaux, L. 2000. The human EZH2 gene: genomic organisation and revised mapping in 7q35 within the critical region for malignant myeloid disorders. European Journal of Human Genetics, 8:174–180.

Chiba, S.; Ikawa, T.; Takeshita, H.; Kanno, S.; Nagai, T.; Takada, M.; Mukai, T.; Wempe, M. F. 2013. Human organic cation transporter 2 (hOCT2): Inhibitor studies using S2-hOCT2 cells. Toxicology, 310:98-103.

Crea, F.; Hurt, E. M.; Mathews, L. A.; Cabarcas, S. M.; Sun, L.; Marquez, V. E.; Danesi, R.; Farrar, W. L. 2011. Pharmacologic disruption of Polycomb Repressive Complex 2 inhibits tumorigenicity and tumor progression in prostate cancer. Molecular Cancer, 10:40.

Enomoto, A.; Takeda, M.; Shimoda, M.; Narikawa, S.; Kobayashi, Y.; Kobayashi, Y.; Yamamoto, T.; Sekine, T.; Cha, S. H.; Niwa, T.; Endou, H. 2002. Interaction of human organic anion transporters 2 and 4 with organic anion transport inhibitors. J Pharmacol Exp Ther, 301:797-802.

Hagenbeek, T. J.; Naspetti, M.; Malergue, F.; Garcon, F.; Nunes, J. A.; Cleutjens, K. B.; Trapman, J.; Krimpenfort, P.; Spits, H. 2004. The loss of PTEN allows TCR alphabeta lineage thymocytes to bypass IL-7 and Pre-TCR-mediated signaling. J Exp Med, 200:883-894.

Harris, P. S.; Venkataraman, S.; Alimova, I.; Birks, D. K.; Donson, A. M.; Knipstein, J.; Dubuc, A.; Taylor, M. D.; Handler, M. H.; Foreman, N. K.; Vibhakar, R. 2012. Polo-like kinase 1 (PLK1) inhibition suppresses cell growth and enhances radiation sensitivity in medulloblastoma cells. BMC Cancer, 12:80, 1471-2407-12-80.

Hosoyamada, M.; Obinata, M.; Suzuki, M.; Endou, H. 1996. Cisplatin-induced toxicity in immortalized renal cell lines established from transgenic mice harboring temperature sensitive SV40 large T-antigen gene. Arch Toxicol., 70:284-292.

Kimura, H.; Takeda, M.; Narikawa, S.; Enomoto, A.; Ichida, K.; Endou, H. 2002. Human organic anion transporters and human organic cation transporters mediate renal transport of prostaglandins. J Pharmacol Exp Ther., 301:293-298.

Kleer, C. G.; Cao, Q.; Varambally, S.; Shen, R.; Ota, I.; Tomlins, S. A.; Ghosh, D.; Sewalt, R. G.; Otte, A. P.; Hayes, D. F.; Sabel, M. S.; Livant, D.; Weiss, S. J.; Rubin, M. A.; Chinnaiyan, A. M. 2003. EZH2 is a marker of aggressive breast cancer and promotes neoplastic transformation of breast epithelial cells. Proc. Natl Acad. Sci., 100:11606–11611.

Kleer, C.G. 2009. Carcinoma of the breast with medullary-like features: diagnostic challenges and relationship with BRCA1 and EZH2 functions. Arch Pathol Lab Med., 133:1822-1825.

Knutson, S. K.; Wigle, T. J.; Warholic, N. M.; Sneeringer, C. J.; Allain, C. J.; Klaus, C. R.; DSacks, J.; Raimondi, A.; Majer, C. R.; Song, J.; Scott, M. P.; Jin, L.; Smith, J. J.; Olhava, E. J.; Chesworth, R.; Moyer, M. P.; Richon, C. M.; Copeland, R. A.; Keilhack, H.; Pollock, R. M.; Kuntz, K. W. 2012. A selective inhibitor of EZH2 blocks H3K27 methylation and kills mutant lymphoma cells. Nature chemical Biology, 8:890-896.

Konze, K. D.; Ma, A.; Li, F.; Barsyte-Lovejoy, D.; Parton, T.; MacNevin, C. J.; Liu, F.; Gao, C.; Huang, X-P.; Kuznetsova, E.; Rougie, M.; Jiang, A.; Pattenden, S. G.; Norris, J. L.; James, L. I.; Roth, B. L.; Brown, P. J.; Frye, S. V.; Arrowsmith, C. H.; Hahn, K. M.; Wang, G. G.; Vedadi, M.; Jin, J. 2013. An orally bioavailable chemical probe of the Lysine Methyltransferases EZH2 and EZH1. ACS Chem. Biol., 8:1324−1334.

McCabe, M. T.; Ott, H. M.; Ganji, G.; Korenchuk, S.; Thompson, C.; Van Aller, G. S.; Liu, Y.; Graves, A. P.; Pietra III, A. D.; Diaz, E.; LaFrance, L. V.; Mellinger, M.; Duquenne, C.; Tian, X.; Kruger, R. G.; McHugh, C. F.; Brandt, M.; Miller, W. H.; Dhanak, D.; Verma, S. K.; Tummino, P. J.; Creasy, C. L. 2012. EZH2 inhibition as a therapeutic strategy for lymphoma with EZH2-activating mutations. Nature, 492:108-112.

Miranda, T. B.; Cortez, C. C.; Yoo, C. B.; Liang, G.; Abe, M.; Kelly, T. K.; Marquez, V. E.; Jones, P. A. 2009. DZNep is a global histone methylation inhibitor that reactivates developmental genes not silenced by DNA methylation. Mol Cancer Ther, 8:1579-1588.

Pritchard, J. B.; Miller, D. S. 1993. Mechanisms mediating renal secretion of organic anions and cations. Physiol Rev, 73:765-796.

Qi, W.; Chana, H.; Teng, L.; Li, L.; Chuai, S.; Zhang, R.; Zeng, J.; Li, M.; Fan, H.; Lin, Y.; Gu, J.; Ardayfio, O.; Zhang, J-H.; Yan, X.; Fang, J.; Mi, Y.; Zhang, M.; Zhou, T.; Feng, G.; Chen, Z.; Li, G.; Yang, T.; Zhao, K.; Liu, X.; Yu, Z.; Lu, C.X.; Atadja, P.; Li, E. 2012. Selective inhibition of Ezh2 by a small molecule inhibitor blocks tumor cells proliferation. Pro. Nat. Acc. Sci., 109:21360-21365.

Rosilio, C.; Nebout. M.; Imbert. V.; Griessinger, E.; Neffati, Z.; Benadiba, J.; Hagenbeek, T.; Spits, H.; Reverso, J.; Ambrosetti, D.; Michiels, J. F.; Bailly-Maitre, B.; Endou, H.; Wempe, M. F. Peyron, J. F. 2015. L-type amino-acid transporter 1 (LAT1): a therapeutic target supporting growth and survival of T-cell lymphoblastic lymphoma/T-cell acute lymphoblastic leukemia. Leukemia, in press. Dec 8. doi: 10.1038/leu.2014.338. [Epub ahead of print] PMID: 25482130.

Smits, M.; Nilsson, J.; Mir, S. E.; Van der Stoop, P. M.; Hulleman, E.; Niers, J. M.; de Witt Hamer, P. C.; Marquez, V. E.; Cloos, J.; Krichevsky, A. M.; Noske, D. P., Tannous, B. A.; Würdinger, T. 2010. miR-101 is down-regulated in glioblastoma resulting in EZH2-induced proliferation, migration, and angiogenesis. Oncotarget, 1:710 – 720.

Takawa, M.; Masuda, K.; Kunizaki, M.; Daigo, Y.; Takagi, K.; Iwai, Y.; Cho, H. S.; Toyokawa, G.; Yamane, Y.; Maejima, K.; Field, H. I.; Kobayashi, T.; Akasu, T.; Sugiyama, M.; Tsuchiya, E.; Atomi, Y.; Ponder, B. A.; Nakamura, Y.; Hamamoto, R. 2011. Validation of the histone methyltransferase EZH2 as a therapeutic target for various types of human cancer and as a prognostic marker. Cancer Sci., 102:1298-1305.

Takeda, M.; Khamdang, S.; Narikawa, S.; Kimura, H.; Kobayashi, Y.; Yamamoto, T.; Cha, S. H.; Sekine, T.; Endou, H. 2002. Human organic anion transporters and human organic cation transporters mediate renal antiviral transport. J Pharmacol Exp Ther, 300:918-924.

Tan, J.; Yang, X.; Zhuang, L.; Jiang, X.; Chen, W.; Lee, P. L.; Karuturi, R. K.; Tan, P. B.; Liu, E. T.; Yu, Q. 2007. Pharmacologic disruption of Polycomb-repressive complex 2-mediated gene repression selectively induces apoptosis in cancer cells. Genes Dev, 21:1050–1063.

Varambally, S.; Dhanasekaran, S. M.; Zhou, M.; Barrette, T. R., Kumar-Sinha, C.; Sanda, M. G.; Ghosh, D.; Pienta, K. J.; Sewalt, R. G.; Otte, A. P.; Rubin, M. A.; Chinnaiyan, A. M. 2002. The polycomb group protein EZH2 is involved in progression of prostate cancer. Nature, 419:624-629.

Varambally, S.; Cao, Q.; Mani, R. S.; Shankar, S.; Wang, X.; Ateeq, B.; Laxman, B.; Cao, X.; Jing, X.; Ramnarayanan, K.; Brenner, J. C.; Yu, J.; Kim, J. H.; Han, B.; Tan, P.; Kumar-Sinha, C.; Lonigro, R. J.; Palanisamy, N.; Maher, C. A.; Chinnaiyan, A. M. 2008. Genomic loss of microRNA-101 leads to overexpression of histone methyltransferase EZH2 in cancer. Science, 322:1695-1699.

Verma, S. K.; Tian, X.; LaFrance, L. V.; Duquenne, C.; Suarez, D. P.; Newlander, K. A.; Romeril, S. P.; Burgess, J. L.; Grant, S. W.; Brackley, J. A.; Graves, A. P.; Scherzer, D. A.; Shu, A.; Thompson, C.; Ott, H. M.; Van Aller, G. S.; Machutta, C. A.; Diaz, E.; Jiang, Y.; Johnson, N. W.; Knight, S. D.; Kruger, R. G.; McCabe, M. T.; Dhanak, D.; Tummino, P. J.; Creasy, C. L.; Miller, W. H. 2012.

Identification of Potent, Selective, Cell-Active Inhibitors of the Histone Lysine Methyltransferase EZH2. Med. Chem. Lett., 3:1091−1096.

Wagener, N.; Holland, D.; Bulkescher, J.; Crnković-Mertens, I.; Hoppe-Seyler, K.; Zentgraf, H.; Pritsch, M.; Buse, S.; Pfitzenmaier, J.; Haferkamp, A.; Hohenfellner, M.; Hoppe-Seyler, F. 2008. The enhancer of zeste homolog 2 gene contributes to cell proliferation and apoptosis resistance in renal cell carcinoma cells. Int. J. Cancer, 123:1545-1550.

Wagener, N.; Macher-Goeppinger, S.; Pritsch, M.; Hüsing, J.; Hoppe-Seyler, K.; Schirmacher, P.; Pfitzenmaier, J.; Haferkamp, A.; Hoppe-Seyler, F.; Hohenfellner, M. 2010. Enhancer of zeste homolog 2 (EZH2) expression is an independent prognostic factor in renal cell carcinoma. BMC Cancer, 10:524.

Warburg, O. 1956. On the origin of cancer cells. Science, 123:309-314.

Wempe, M. F.; Lightner, J. W.; Miller, B.; Iwen, T. J.; Rice, P. J.; Wakui, A.; Anzai, N.; Jutabha, P.; Endou, H. 2012. Potent human uric acid transporter 1 inhibitors: in vitro and in vivo metabolism and pharmacokinetic studies. Drug Design, Development and Therapy, 6:323-339.

Yan, J.; Ng, S-B.; Tay, J. L-S.; Lin, B.; Koh, T.L.; Tan, J.; Selvarajan, V.; Liu, S-C; Bi, C.; Wang, S.; Choo, S-N.; Shimizu, N.; Huang, G.; Yu, Q.; Chng W-J. 2013. EZH2 overexpression in natural killer/T-cell lymphoma confers growth advantage independently of histone methyltransferase activity. Blood, 121:4512-4520.

Yanagida, O.; Kanai, Y.; Chairoungdua, A.; Kim, D. K.; Segawa, H.; Nii, T.; Cha, S. H.; Matsuo, H.; Fukushima, J.; Fukasawa, Y.; Tani, Y.; Taketani, Y.; Uchino, H.; Kim, J. Y.; Inatomi, J.; Okayasu, I.; Miyamoto, K.; Takeda, E.; Goya, T.; Endou, H. 2001. Human L-type amino acid transporter 1 (LAT1): characterization of function and expression in tumor cell lines. Biochim Biophys Acta, 2:291-302.

Zeidler, M.; Kleer, C. G. 2006. The Polycomb group protein Enhancer of Zeste 2: its links to DNA repair and breast cancer. J Mol Hist., 37:219–223.

DOI: http://dx.doi.org/10.18103/mra.v0i3.273


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