Journal of Medicinal Chemistry
ARTICLE
poorly defined electron density. All refinement steps were performed
using REFMAC35 in the CCP4 program suite. The final model com-
prising three molecules of 3MBT domain, two molecules of 14, and
solvent molecules including glycerol and sulfate molecules refined to an
Rwork of 19.1% and Rfree of 24.2%. Data collection and structure
refinement statistics are summarized in the Supporting Information,
Table 1. Structure figures were prepared using PyMOL.36 Atomic
coordinates and structure factors for 3MBT/14 complex have been
deposited in the Protein Data Bank with accession code 3P8H.
domains; MBTD1, MBT domain containing 1; PHF13, PHD finger
protein 13; CBX7, chromobox 7; FAM, carboxyfluorescein; FP, fluor-
escence polarization
’ REFERENCES
(1) Berger, S. L.; Kouzarides, T.; Shiekhattar, R.; Shilatifard, A. An
operational definition of epigenetics. Genes Dev. 2009, 23 (7), 781–783.
(2) Bernstein, B. E.; Meissner, A.; Lander, E. S. The mammalian
epigenome. Cell 2007, 128 (4), 669–681.
(3) Nirmalanandhan, V. S.; Sittampalam, G. S. Stem cells in drug
discovery, tissue engineering, and regenerative medicine: emerging
opportunities and challenges. J. Biomol. Screening 2009, 14 (7), 755–768.
(4) Gelato, K. A.; Fischle, W. Role of histone modifications in
defining chromatin structure and function. Biol. Chem. 2008, 389 (4),
353–363.
’ ASSOCIATED CONTENT
S
Supporting Information. ITC binding curves, cocrystal
b
structure refinements, and experimental procedures including
spectra for all final compounds. This material is available free of
(5) (a) Liu, F.; Chen, X.; Allali-Hassani, A.; Quinn, A. M.; Wasney,
G. A.; Dong, A.; Barsyte, D.; Kozieradzki, I.; Senisterra, G.; Chau, I.;
Siarheyeva, A.; Kireev, D. B.; Jadhav, A.; Herold, J. M.; Frye, S. V.;
Arrowsmith, C. H.; Brown, P. J.; Simeonov, A.; Vedadi, M.; Jin, J.
Discovery of a 2,4-diamino-7-aminoalkoxyquinazoline as a potent and
selective inhibitor of histone lysine methyltransferase G9a. J. Med. Chem.
2009, 52 (24), 7950–7953. (b) Copeland, R. A.; Solomon, M. E.;
Richon, V. M. Protein methyltransferases as a target class for drug
discovery. Nature Rev. Drug Discovery 2009, 8 (9), 724–732. (c) Liu, F.;
Chen, X.; Allali-Hassani, A.; Quinn, A. M.; Wigle, T. J.; Wasney, G. A.;
Dong, A.; Senisterra, G.; Chau, I.; Siarheyeva, A.; Norris, J. L.; Kireev,
D. B.; Jadhav, A.; Herold, J. M.; Janzen, W. P.; Arrowsmith, C. H.; Frye,
S. V.; Brown, P. J.; Simeonov, A.; Vedadi, M.; Jin, J. Protein lysine
methyltransferase G9a inhibitors: design, synthesis, and structure activ-
ity relationships of 2,4-diamino-7-aminoalkoxy-quinazolines. J. Med.
Chem. 2010, 53 (15), 5844–5857.
Accession Codes
†The coordinates and structure factors of the cocrystal structure
of the L3MBTL1À14 complex have been deposited in the
’ AUTHOR INFORMATION
Corresponding Author
*Phone: 919-843-5486. Fax: 919-843-8465. E-mail: svfrye@e-
mail.unc.edu.
’ ACKNOWLEDGMENT
(6) Filippakopoulos, P.; Qi, J.; Picaud, S.; Shen, Y.; Smith, W. B.;
Fedorov, O.; Morse, E. M.; Keates, T.; Hickman, T. T.; Felletar, I.;
Philpott, M.; Munro, S.; McKeown, M. R.; Wang, Y.; Christie, A. L.;
West, N.; Cameron, M. J.; Schwartz, B.; Heightman, T. D.; La Thangue,
N.; French, C. A.; Wiest, O.; Kung, A. L.; Knapp, S.; Bradner, J. E.
Selective inhibition of BET bromodomains. Nature 2010, 468, 1067–
1073.
(7) Kireev, D.; Wigle, T. J.; Norris-Drouin, J.; Herold, J. M.; Janzen,
W. P.; Frye, S. V. Identification of nonpeptide malignant brain tumor
(MBT) repeat antagonists by virtual screening of commercially available
compounds. J. Med. Chem. 2010, 53 (21), 7625–7631.
(8) (a) Campagna-Slater, V.; Arrowsmith, A. G.; Zhao, Y.; Schapira,
M. Pharmacophore Screening of the Protein Data Bank for Specific
Binding Site Chemistry. J. Chem. Inf. Model. 2010, 50, 358–367. (b)
Campagna-Slater, V.; Schapira, M. Finding Inspiration in the Protein
Data Bank to Chemically Antagonize Readers of the Histone Code. Mol.
Inf. 2010, 29, 322–331.
This work is supported by NIH grant number RC1GM090732
and the Ontario Research Fund and the Structural Genomics
Consortium, a registered charity (no. 1097737) that receives
funds from the Canadian Institutes for Health Research, the
Canada Foundation for Innovation, Genome Canada through
the Ontario Genomics Institute, GlaxoSmithKline, Karolinska
Institute, the Knut and Alice Wallenberg Foundation, the
Ontario Innovation Trust, the Ontario Ministry for Research
and Innovation, Merck & Co. Inc., the Novartis Research
Foundation, the Swedish Agency for Innovation Systems, the
Swedish Foundation for Strategic Research, and the Wellcome
Trust. Postdoctoral fellowships for J.M.H. and T.J.W. from the
Carolina Partnership are gratefully acknowledged. We thank Dr.
Shiamalee Perumal (G.E.) for helpful discussions concerning
ITC, Dr. Keduo Qian and Professor K. H. Lee for HRMS
support, Dr. Krzysztof Krajewski and Professor Brian Strahl for
support with peptide synthesis, Farrell MacKenzie for supply of
D355A L3MBTL1 construct, Dr. Chuanbing Bian for expression
and purification of PHF13, Mani Ravichandran for expression
and purification of FLAG-CBX7, Dr. Hui Ouyang for expression
and purification of L3MBTL1 (MBT repeat containing residues
200-522) for crystallization, and Dr. Wolfram Tempel for
assistance with data collection.
(9) Simple Modular Architecture Research Tool (SMART) Database,
2010.
(10) Adams-Cioaba, M. A.; Min, J. Structure and function of histone
methylation binding proteins. Biochem. Cell Biol. 2009, 87 (1), 93–105.
(11) Bonasio, R.; Lecona, E.; Reinberg, D. MBT domain proteins in
development and disease. Semin. Cell Dev. Biol. 2010, 21 (2), 221–230.
(12) (a) Li, H.; Fischle, W.; Wang, W.; Duncan, E. M.; Liang, L.;
Murakami-Ishibe, S.; Allis, C. D.; Patel, D. J. Structural basis for lower
lysine methylation state-specific readout by MBT repeats of L3MBTL1
and an engineered PHD finger. Mol. Cell 2007, 28 (4), 677–691. (b)
Min, J.; Allali-Hassani, A.; Nady, N.; Qi, C.; Ouyang, H.; Liu, Y.;
MacKenzie, F.; Vedadi, M.; Arrowsmith, C. H. L3MBTL1 recognition
of mono- and dimethylated histones. Nature Struct. Mol. Biol. 2007, 14
(12), 1229–1230.
(13) Kalakonda, N.; Fischle, W.; Boccuni, P.; Gurvich, N.; Hoya-
Arias, R.; Zhao, X.; Miyata, Y.; Macgrogan, D.; Zhang, J.; Sims, J. K.;
Rice, J. C.; Nimer, S. D. Histone H4 lysine 20 monomethylation
promotes transcriptional repression by L3MBTL1. Oncogene 2008, 27
(31), 4293–4304.
’ ABBREVIATIONS USED
Rb, retinoblastoma protein; MBT, malignant brain tumor; L3MBTL1,
lethal(3) malignant brain tumor protein 1; ITC, isothermal titration
calorimetry; H4, histone 4; PWWP, proline-tryptophan-tryptophan-
proline; PHD, plant homeodomain; KMe1, monomethyl-lysine;
H4K20Me1, histone H4 lysine 20 monomethyl; E2F, E2
transcription factor; SMYD2, SET and MYND domain-containing
protein 2; SFMBT1, SCM-related gene containing four MBT
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dx.doi.org/10.1021/jm200045v |J. Med. Chem. 2011, 54, 2504–2511