Dot1L Inhibitor EPZ-5676: Synthesis, pharmacokinetic and tissue distribution studies in Sprague-Dawley rats
Main Article Content
Abstract
Histone methyl-transferase Dot1L can methylate histone 3 on lysine 79 (H3K79). Herein we present the chemical synthesis of a cis/trans mixture of a potent Dot1L inhibitor known as EPZ-5676 (2). Upon preparing compound 2, we tested the compound in mixed and non-mixed lineage leukemia cell lines. The two MLL-rearranged cell lines were MV4;11 and Molm14; whereas the two non-MLL-rearranged (control) cell lines were Molt4 and Kasumi. We observed anticipated in vitro activity for compound 2 in these four leukemia cell lines; results illustrating that Dot1L inhibition can trigger cancer cell death. In addition, we also tested 2 in a new leukemia cell line known as KO99L; KO99L cells have been shown to over express the membrane amino acid transporter known as L-amino acid type 1 (LAT1). Compound 2 was also observed to decrease cell viability in the KO99L cell; albeit at higher concentrations as compared to the MLL-rearranged cell lines. In addition to in vitro experiments, we also performed in vivo experiments in Sprague-Dawley rats. Intravenous (i.e. orbital sinus dosing) experiments were performed at two different doses (i.e. 1.0 and 2.0 mg/kg). These rat Pharmacokinetic (PK) results indicate that compound 2 has a slow distribution phase, followed by an extended terminal half-life (i.e. 11.2 ± 3.1 h). Furthermore, tissue distribution experiments demonstrate that 2 predominately distributes to kidney, blood and liver, and to a limited extend, was detectable in brain.
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How to Cite
KUMAR, Vijay et al.
Dot1L Inhibitor EPZ-5676: Synthesis, pharmacokinetic and tissue distribution studies in Sprague-Dawley rats.
Medical Research Archives, [S.l.], n. 3, july 2015.
ISSN 2375-1924.
Available at: <https://esmed.org/MRA/mra/article/view/275>. Date accessed: 16 dec. 2024.
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Research Articles
The Medical Research Archives grants authors the right to publish and reproduce the unrevised contribution in whole or in part at any time and in any form for any scholarly non-commercial purpose with the condition that all publications of the contribution include a full citation to the journal as published by the Medical Research Archives.
References
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Basavapathruni, A.; Olhava, E. J.; Daigle, S. R.; Therkelsen, C. A.; Jin, L.; Boriack-Sjodin, P. A. 2014. Nonclinical pharmacokinetics and metabolism of EPZ-5676, a novel DOT1L histone methyltransferase inhibitor. Biopharm Drug Dispos., 35:237-252.
Bernt, K. M.; Zhu, N.; Sinha, A. U.; Vempati, S.; Faber, J.; Krivtsov, A. V. 2011. MLL-rearranged leukemia is dependent on aberrant H3K79 methylation by DOT1L. Cancer Cell., 20:66-78.
Bitoun, E.; Oliver, P. L.; Davies, K. E. 2007. The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling. Hum. Mol. Gen.,16:92-106.
Chang, M. J.; Wu, H.; Achille, N. J.; Reisenauer, M. R.; Chou, C. W.; Zeleznik-Le, N. J. 2010. Histone H3 lysine 79 methyltransferase Dot1 is required for immortalization by MLL oncogenes. Cancer Res., 70:10234-10242.
Chen, L.; Deshpande, A. J.; Banka, D.; Bernt, K. M.; Dias, S.; Buske, C. 2013. Abrogation of MLL-AF10 and CALM-AF10-mediated transformation through genetic inactivation or pharmacological inhibition of the H3K79 methyltransferase Dot1l. Leukemia., 27:813-22.
Chen, C-W.; Kocke, R.P.; Sinha, A.U.; Deshpande, A.J.; Zhu, N.; Eng, R.; Doench, J.G.; Xu, H.; Chu, S.H.; Qi, J.; Wang, X.; Delaney, C.; Bernt, K.M.; Root, D.E.; Hahn, W.C.; Bradner, J.E.; Armstrong, S.A. 2015. DOT1L inhibits SIRT1-mediated epigenetic silencing to maintain leukemic gene expression in MLL-rearranged leukemia. Nature Medicine., 21: 335-343.
Daigle, S. R.; Olhava, E. J.; Therkelsen, C. A.; Majer, C. R.; Sneeringer, C. J.; Song, J. 2011. Selective killing of mixed lineage leukemia cells by a potent small-molecule DOT1L inhibitor. Cancer Cell., 20:53-65.
Daigle, S. R.; Olhava, E. J.; Therkelsen, C. A.; Basavapathruni, A.; Jin, L.; Boriack-Sjodin, P. A. 2013. Potent inhibition of DOT1L as treatment of MLL-fusion leukemia. Blood., 122:1017-1025.
Deshpande, A. J.; Chen, L.; Fazio, M.; Sinha, A. U.; Bernt, K. M.; Banka, D. 2013. Leukemic transformation by the MLL-AF6 fusion oncogene requires the H3K79 methyltransferase Dot1l. Blood., 121:2533-2541.
Dlakic, M. 2001. Chromatin silencing protein and pachytene checkpoint regulator Dot1p has a methyltransferase fold. Trends Biochem Sci.,26:405-407.
Ho, L. L.; Sinha, A.; Verzi, M.; Bernt, K. M.; Armstrong, S. A.; Shivdasani, R. A. 2013. DOT1L-mediated H3K79 methylation in chromatin is dispensable for Wnt pathway-specific and other intestinal epithelial functions. Mol Cell Biol., 33:1735-1745.
Jo, S. Y.; Granowicz, E. M.; Maillard, I.; Thomas, D.; Hess, J. L. 2011. Requirement for Dot1l in murine postnatal hematopoiesis and leukemogenesis by MLL translocation. Blood., 117:4759-4768.
Jones, B.; Su, H.; Bhat, A.; Lei, H.; Bajko, J.; Hevi, S. 2008. The histone H3K79 methyltransferase Dot1L is essential for mammalian development and heterochromatin structure. PLoS Genet., 4:e1000190.
Klaus, C.R.; Iwanowicz, D.; Johnston, D.; Campbell, C.A.; Smith, J.J.; Moyer, M.P.; Copeland, R.A.; Olhava, E.J.; Scott, M.P.; Pollock, R.M.; Daigle, S.R.; Raimondi, A. 2014. DOT1L Inhibitor EPZ-5676 Displays Synergistic Antiproliferative Activity in Combination with Standard of Care Drugs and Hypomethylating Agents in MLL-Rearranged Leukemia Cells. JPET., 350: 646-656.
Mahmoudi, T.; Boj, S. F.; Hatzis, P.; Li, V. S.; Taouatas, N.; Vries, R. G. 2010. The leukemia-associated Mllt10/Af10-Dot1l are Tcf4/beta-catenin coactivators essential for intestinal homeostasis. PLoS Biol., 8:e1000539.
Min, J.; Feng, Q.; Li, Z.; Zhang, Y.; Xu, R. M. 2003.
Structure of the catalytic domain of human DOT1L, a non-SET domain nucleosomal histone methyltransferase. Cell., 112:711-723.
Mueller, D.; Bach, C.; Zeisig, D.; Garcia-Cuellar, M. P.; Monroe, S.; Sreekumar, A. 2007. A role for the MLL fusion partner ENL in transcriptional elongation and chromatin modification. Blood., 110:4445-4454.
Mueller, D.; Garcia-Cuellar, M. P.; Bach, C.; Buhl, S.; Maethner, E.; Slany, R. K. 2009. Misguided transcriptional elongation causes mixed lineage leukemia. PLoS Biol. 7:e1000249.
Ng, H. H.; Feng, Q.; Wang, H.; Erdjument-Bromage, H.; Tempst, P.; Zhang, Y. 2002. Lysine methylation within the globular domain of histone H3 by Dot1 is important for telomeric silencing and Sir protein association. Genes Dev., 16:1518–1527.
Nguyen, A. T.; He, J.; Taranova, O.; Zhang, Y. 2011(a). Essential role of DOT1L in maintaining normal adult hematopoiesis. Cell Res.,21:1370-1373.
Nguyen, A. T.; Taranova, O.; He, J.; Zhang, Y. 2011(b). DOT1L, the H3K79 methyltransferase, is required for MLL-AF9-mediated leukemogenesis. Blood., 117:6912-6922.
Nguyen, A. T.; Xiao, B.; Neppl, R. L.; Kallin, E. M.; Li, J.; Chen, T. 2011(c). DOT1L regulates dystrophin expression and is critical for cardiac function. Genes Dev., 25:263-274.
Nguyen, A. T.; Zhang, Y. 2011(d). The diverse functions of Dot1 and H3K79 methylation. Genes Dev., 25:1345-58. doi: 10.1101/gad.2057811
Okada, Y.; Feng, Q.; Lin, Y.; Jiang, Q.; Li, Y.; Coffield, V. M. 2005. hDOT1L links histone methylation to leukemogenesis. Cell., 121:167-78.
Olhava, J.E.; Chesworth, R.; Kuntz, W.K.; Richon, M.V.; Pollock, M.R. 2012. International Application Published Under The Patent Cooperation Treaty (PCT). WO 2012/075381 A1.
Olhava, J.E.; Chesworth, R.; Kuntz, W.K.; Richon, M.V.; Pollock, M.R.; Daigle, R.S. 2014(a). International Application Published Under The Patent Cooperation Treaty (PCT). WO 2014/039839 A1.
Onder, T. T.; Kara, N.; Cherry, A.; Sinha, A. U.; Zhu, N.; Bernt, K. M. 2012. Chromatin-modifying enzymes as modulators of reprogramming. Nature., 483:598-602.
Park, J. H.; Cosgrove, M. S.; Youngman, E.; Wolberger, C.; Boeke, J. D. 2002. A core nucleosome surface crucial for transcriptional silencing. Nat Genet.,32:273-279.
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. Leukemia., in press. Dec 8. doi: 10.1038/leu.2014.338. [Epub ahead of print] PMID: 25482130.
Sawada, K.; Yang, Z.; Horton, J. R.; Collins, R. E.; Zhang, X.; Cheng, X. 2004. Structure of the conserved core of the yeast Dot1p, a nucleosomal histone H3 lysine 79 methyltransferase. J Biol Chem., 279:43296-43306.
Segawa, H.; Fukasawa, Y.; Miyamoto, K.; Takeda, E.; Endou, H.; Kanai, Y. 1999. Identification and functional characterization of a Na+-independent neutral amino acid transporter with broad substrate selectivity. J Biol Chem., 274:19745-19751.
Steger, D. J.; Lefterova, M. I.; Ying, L.; Stonestrom, A. J.; Schupp, M.; Zhuo, D. 2008. DOT1L/KMT4 recruitment and H3K79 methylation are ubiquitously coupled with gene transcription in mammalian cells. Mol Cell Biol., 28:2825-2839.
van Leeuwen, F.; Gafken, P. R.; Gottschling, D. E. 2002. Dot1p modulates silencing in yeast by methylation of the nucleosome core. Cell., 109:745-56.
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.
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., 1514:291-302.
Yang, L.; Lin, C.; Jin, C.; Yang, J. C.; Tanasa, B.; Li, W. 2013. lncRNA-dependent mechanisms of androgen-receptor-regulated gene activation programs. Nature., 500:598-602.
Yu, W.; Chory, E. J.; Wernimont, A. K.; Tempel, W.; Scopton, A.; Federation, A. 2012. Catalytic site remodelling of the DOT1L methyltransferase by selective inhibitors. Nat Commun., 3:1288.
Yu, W.; Smil, D.; Li, F.; Tempel, W.; Fedorov, O.; Nguyen, K. T. 2013. Bromo-deaza-SAH: a potent and selective DOT1L inhibitor. Bioorg Med Chem., 21:1787-1794.
Basavapathruni, A.; Olhava, E. J.; Daigle, S. R.; Therkelsen, C. A.; Jin, L.; Boriack-Sjodin, P. A. 2014. Nonclinical pharmacokinetics and metabolism of EPZ-5676, a novel DOT1L histone methyltransferase inhibitor. Biopharm Drug Dispos., 35:237-252.
Bernt, K. M.; Zhu, N.; Sinha, A. U.; Vempati, S.; Faber, J.; Krivtsov, A. V. 2011. MLL-rearranged leukemia is dependent on aberrant H3K79 methylation by DOT1L. Cancer Cell., 20:66-78.
Bitoun, E.; Oliver, P. L.; Davies, K. E. 2007. The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling. Hum. Mol. Gen.,16:92-106.
Chang, M. J.; Wu, H.; Achille, N. J.; Reisenauer, M. R.; Chou, C. W.; Zeleznik-Le, N. J. 2010. Histone H3 lysine 79 methyltransferase Dot1 is required for immortalization by MLL oncogenes. Cancer Res., 70:10234-10242.
Chen, L.; Deshpande, A. J.; Banka, D.; Bernt, K. M.; Dias, S.; Buske, C. 2013. Abrogation of MLL-AF10 and CALM-AF10-mediated transformation through genetic inactivation or pharmacological inhibition of the H3K79 methyltransferase Dot1l. Leukemia., 27:813-22.
Chen, C-W.; Kocke, R.P.; Sinha, A.U.; Deshpande, A.J.; Zhu, N.; Eng, R.; Doench, J.G.; Xu, H.; Chu, S.H.; Qi, J.; Wang, X.; Delaney, C.; Bernt, K.M.; Root, D.E.; Hahn, W.C.; Bradner, J.E.; Armstrong, S.A. 2015. DOT1L inhibits SIRT1-mediated epigenetic silencing to maintain leukemic gene expression in MLL-rearranged leukemia. Nature Medicine., 21: 335-343.
Daigle, S. R.; Olhava, E. J.; Therkelsen, C. A.; Majer, C. R.; Sneeringer, C. J.; Song, J. 2011. Selective killing of mixed lineage leukemia cells by a potent small-molecule DOT1L inhibitor. Cancer Cell., 20:53-65.
Daigle, S. R.; Olhava, E. J.; Therkelsen, C. A.; Basavapathruni, A.; Jin, L.; Boriack-Sjodin, P. A. 2013. Potent inhibition of DOT1L as treatment of MLL-fusion leukemia. Blood., 122:1017-1025.
Deshpande, A. J.; Chen, L.; Fazio, M.; Sinha, A. U.; Bernt, K. M.; Banka, D. 2013. Leukemic transformation by the MLL-AF6 fusion oncogene requires the H3K79 methyltransferase Dot1l. Blood., 121:2533-2541.
Dlakic, M. 2001. Chromatin silencing protein and pachytene checkpoint regulator Dot1p has a methyltransferase fold. Trends Biochem Sci.,26:405-407.
Ho, L. L.; Sinha, A.; Verzi, M.; Bernt, K. M.; Armstrong, S. A.; Shivdasani, R. A. 2013. DOT1L-mediated H3K79 methylation in chromatin is dispensable for Wnt pathway-specific and other intestinal epithelial functions. Mol Cell Biol., 33:1735-1745.
Jo, S. Y.; Granowicz, E. M.; Maillard, I.; Thomas, D.; Hess, J. L. 2011. Requirement for Dot1l in murine postnatal hematopoiesis and leukemogenesis by MLL translocation. Blood., 117:4759-4768.
Jones, B.; Su, H.; Bhat, A.; Lei, H.; Bajko, J.; Hevi, S. 2008. The histone H3K79 methyltransferase Dot1L is essential for mammalian development and heterochromatin structure. PLoS Genet., 4:e1000190.
Klaus, C.R.; Iwanowicz, D.; Johnston, D.; Campbell, C.A.; Smith, J.J.; Moyer, M.P.; Copeland, R.A.; Olhava, E.J.; Scott, M.P.; Pollock, R.M.; Daigle, S.R.; Raimondi, A. 2014. DOT1L Inhibitor EPZ-5676 Displays Synergistic Antiproliferative Activity in Combination with Standard of Care Drugs and Hypomethylating Agents in MLL-Rearranged Leukemia Cells. JPET., 350: 646-656.
Mahmoudi, T.; Boj, S. F.; Hatzis, P.; Li, V. S.; Taouatas, N.; Vries, R. G. 2010. The leukemia-associated Mllt10/Af10-Dot1l are Tcf4/beta-catenin coactivators essential for intestinal homeostasis. PLoS Biol., 8:e1000539.
Min, J.; Feng, Q.; Li, Z.; Zhang, Y.; Xu, R. M. 2003.
Structure of the catalytic domain of human DOT1L, a non-SET domain nucleosomal histone methyltransferase. Cell., 112:711-723.
Mueller, D.; Bach, C.; Zeisig, D.; Garcia-Cuellar, M. P.; Monroe, S.; Sreekumar, A. 2007. A role for the MLL fusion partner ENL in transcriptional elongation and chromatin modification. Blood., 110:4445-4454.
Mueller, D.; Garcia-Cuellar, M. P.; Bach, C.; Buhl, S.; Maethner, E.; Slany, R. K. 2009. Misguided transcriptional elongation causes mixed lineage leukemia. PLoS Biol. 7:e1000249.
Ng, H. H.; Feng, Q.; Wang, H.; Erdjument-Bromage, H.; Tempst, P.; Zhang, Y. 2002. Lysine methylation within the globular domain of histone H3 by Dot1 is important for telomeric silencing and Sir protein association. Genes Dev., 16:1518–1527.
Nguyen, A. T.; He, J.; Taranova, O.; Zhang, Y. 2011(a). Essential role of DOT1L in maintaining normal adult hematopoiesis. Cell Res.,21:1370-1373.
Nguyen, A. T.; Taranova, O.; He, J.; Zhang, Y. 2011(b). DOT1L, the H3K79 methyltransferase, is required for MLL-AF9-mediated leukemogenesis. Blood., 117:6912-6922.
Nguyen, A. T.; Xiao, B.; Neppl, R. L.; Kallin, E. M.; Li, J.; Chen, T. 2011(c). DOT1L regulates dystrophin expression and is critical for cardiac function. Genes Dev., 25:263-274.
Nguyen, A. T.; Zhang, Y. 2011(d). The diverse functions of Dot1 and H3K79 methylation. Genes Dev., 25:1345-58. doi: 10.1101/gad.2057811
Okada, Y.; Feng, Q.; Lin, Y.; Jiang, Q.; Li, Y.; Coffield, V. M. 2005. hDOT1L links histone methylation to leukemogenesis. Cell., 121:167-78.
Olhava, J.E.; Chesworth, R.; Kuntz, W.K.; Richon, M.V.; Pollock, M.R. 2012. International Application Published Under The Patent Cooperation Treaty (PCT). WO 2012/075381 A1.
Olhava, J.E.; Chesworth, R.; Kuntz, W.K.; Richon, M.V.; Pollock, M.R.; Daigle, R.S. 2014(a). International Application Published Under The Patent Cooperation Treaty (PCT). WO 2014/039839 A1.
Onder, T. T.; Kara, N.; Cherry, A.; Sinha, A. U.; Zhu, N.; Bernt, K. M. 2012. Chromatin-modifying enzymes as modulators of reprogramming. Nature., 483:598-602.
Park, J. H.; Cosgrove, M. S.; Youngman, E.; Wolberger, C.; Boeke, J. D. 2002. A core nucleosome surface crucial for transcriptional silencing. Nat Genet.,32:273-279.
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. Leukemia., in press. Dec 8. doi: 10.1038/leu.2014.338. [Epub ahead of print] PMID: 25482130.
Sawada, K.; Yang, Z.; Horton, J. R.; Collins, R. E.; Zhang, X.; Cheng, X. 2004. Structure of the conserved core of the yeast Dot1p, a nucleosomal histone H3 lysine 79 methyltransferase. J Biol Chem., 279:43296-43306.
Segawa, H.; Fukasawa, Y.; Miyamoto, K.; Takeda, E.; Endou, H.; Kanai, Y. 1999. Identification and functional characterization of a Na+-independent neutral amino acid transporter with broad substrate selectivity. J Biol Chem., 274:19745-19751.
Steger, D. J.; Lefterova, M. I.; Ying, L.; Stonestrom, A. J.; Schupp, M.; Zhuo, D. 2008. DOT1L/KMT4 recruitment and H3K79 methylation are ubiquitously coupled with gene transcription in mammalian cells. Mol Cell Biol., 28:2825-2839.
van Leeuwen, F.; Gafken, P. R.; Gottschling, D. E. 2002. Dot1p modulates silencing in yeast by methylation of the nucleosome core. Cell., 109:745-56.
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.
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., 1514:291-302.
Yang, L.; Lin, C.; Jin, C.; Yang, J. C.; Tanasa, B.; Li, W. 2013. lncRNA-dependent mechanisms of androgen-receptor-regulated gene activation programs. Nature., 500:598-602.
Yu, W.; Chory, E. J.; Wernimont, A. K.; Tempel, W.; Scopton, A.; Federation, A. 2012. Catalytic site remodelling of the DOT1L methyltransferase by selective inhibitors. Nat Commun., 3:1288.
Yu, W.; Smil, D.; Li, F.; Tempel, W.; Fedorov, O.; Nguyen, K. T. 2013. Bromo-deaza-SAH: a potent and selective DOT1L inhibitor. Bioorg Med Chem., 21:1787-1794.