Concise Article
MedChemComm
1
3
overexpressed according to previously published procedures,
J., 2000, 19, 6141; C. Dhalluin, J. E. Carlson, L. Zeng, C. He,
A. K. Aggarwal and M.-M. Zhou, Nature, 1999, 399, 491.
6 T. Kouzarides, EMBO J., 2000, 19, 1176.
was then injected three times through the column, at a ow rate
of 50 mL min . The column was subsequently evaluated with
ꢂ1
bromosporine. In practice, from a 50 mM stock solution of
bromosporine in DMSO were prepared solutions at concentra-
tions of 15, 7.5, 3.75, and 1.875 mM, both in PBS and in 100 mM
ammonium acetate buffer. Each of these solutions was then
infused in duplicate following the HPLC method described in
the previous section, and the amount of active loaded protein
7 S. Muller, P. Filippakopoulos and S. Knapp, Expert Rev. Mol.
Med., 2011, 13, e29.
8 P. Filippakopoulos, S. Picaud, M. Mangos, T. Keates,
J.-P. Lambert, T. Barsyte-Lovejoy, I. Felletar, R. Volkmer,
S. Muller, T. Pawson, A.-C. Gingras, C. H. Arrowsmith and
S. Knapp, Cell, 2012, 149, 214.
(
B ) and affinity constant (K ) values calculated as explained
9 E. Nicodeme, K. L. Jeffrey, U. Schaefer, S. Beinke, S. Dewell,
C.-W. Chung, R. Chandwani, I. Marazzi, P. Wilson, H. Coste,
J. White, J. Kirilovsky, C. M. Rice, J. M. Lora, R. K. Prinjha,
K. Lee and A. Tarakhovsky, Nature, 2010, 468, 1119.
t
d
1
previously. In these conditions, Kd and B were respectively
t
found to be 1.48 ꢃ 0.42 mM and 2.07 ꢃ 0.23 nmol, satisfyingly
both in PBS and in 100 mM ammonium acetate buffer. The
same method was employed with the analogue of bromosporine 10 M. A. Dawson, R. K. Prinjha, A. Dittmann, G. Giotopoulos,
2
1 and gave similar B
t
and a K
d
of 8.35 ꢃ 1.25 mM. In order to
M. Bantscheff, W.-I. Chan, S. C. Robson, C.-W. Chung,
C. Hopf, M. M. Savitski, C. Huthmacher, E. Gudgin,
D. Lugo, S. Beinke, T. D. Chapman, E. J. Roberts,
P. E. Soden, K. R. Auger, O. Mirguet, K. Doehner,
R. Delwel, A. K. Burnett, P. Jeffrey, G. Drewes, K. Lee,
B. J. P. Huntly and T. Kouzarides, Nature, 2011, 478, 529.
show the reproducibility of this assay, the same experiment was
performed aer six months on the same column, and no change
in K or B was detected within experimental error. Additionally,
during the six-month period in which the column was used
recurrently, the breakthrough volume for a 50 mM sample of
d
t
bromosporine was found to be constant at 324 ꢃ 8 mL, vali- 11 P. Anand, J. D. Brown, C. Y. Lin, J. Qi, R. Zhang, P. Calderon
dating the stability of the immobilised bromodomain protein
on the column as well as the reproducibility of the technique.
When not in use, the column was stored at 4 C in a buffer
Artero, M. Amer Alaiti, J. Bullard, K. Alazem, K. B. Margulies,
T. P. Cappola, M. Lemieux, J. Plutzky, J. E. Bradner and
S. M. Haldar, Cell, 2013, 154, 569.
ꢀ
solution (either PBS or 100 mM ammonium acetate) containing 12 L. Zeng, J. Li, M. Muller, S. Yan, S. Mujtaba, C. Pan, Z. Wang
0
.1% sodium azide.
and M.-M. Zhou, J. Am. Chem. Soc., 2005, 127, 2376.
3 P. Filippakopoulos, J. Qi, S. Picaud, Y. Shen, W. B. Smith,
O. Fedorov, E. M. Morse, T. Keates, T. T. Hickman,
I. Felletar, M. Philpott, S. Munro, M. R. McKeown,
Y. Wang, A. L. Christie, N. West, M. J. Cameron,
B. Schwartz, T. D. Heightman, N. La Thangue,
C. A. French, O. Wiest, A. L. Kung, S. Knapp and
J. E. Bradner, Nature, 2010, 468, 1067.
4 C.-W. Chung and E. Nicodeme, WO2011054843A1, 2011.
5 B. K. Albrecht, J.-C. Harmange, A. Cote and A. M. Taylor,
WO2012174487A2, 2012.
6 http://www.thesgc.org/chemical-probes/bromosporine/.
7 O. Fedorov, H. Lingard, C. Wells, O. P. Monteiro, S. Picaud,
T. Keates, C. Yapp, M. Philpott, S. G. Martin, I. Felletar,
B. Marsden, P. Filippakopoulos, S. Muller-Knapp, S. Knapp
and P. E. Brennan, J. Med. Chem., 2014, 57, 462.
1
Thermal shi assay
Thermal melting experiments were carried out using a Mx3005p
Real Time PCR machine (Stratagene) as previously described.
28
Acknowledgements
1
1
We would like to thank the EPSRC grant EP/F069685/1 (LG and
NN), Novartis (RJI), the BP Endowment (SVL) for nancial
support and Dr Richard Turner for technical assistance.
1
1
Notes and references
1
L. Guetzoyan, N. Nikbin, I. R. Baxendale and S. V. Ley, Chem.
Sci., 2013, 4, 764.
B. Desai, K. Dixon, E. Farrant, Q. Feng, K. R. Gibson,
W. P. van Hoorn, J. Mills, T. Morgan, D. M. Parry,
18 C. Steilmann, M. C. O. Cavalcanti, M. Bartkuhn, J. Pons-
K u¨ hnemann, H.-C. Schuppe, W. Weidner, K. Steger and
A. Paradowska, Reproduction, 2010, 140, 435.
2
M. K. Ramjee, C. N. Selway, G. J. Tarver, G. Whitlock and 19 S. P. Cleary, W. R. Jeck, X. Zhao, K. Chen, S. R. Selitsky,
A. G. Wright, J. Med. Chem., 2013, 56, 3033; W. Czechtizky,
J. Dedio, B. Desai, K. Dixon, E. Farrant, Q. Feng,
T. Morgan, D. M. Parry, M. K. Ramjee, C. N. Selway,
G. L. Savich, T.-X. Tan, M. C. Wu, G. Getz, M. S. Lawrence,
J. S. Parker, J. Li, S. Powers, H. Kim, S. Fischer, M. Guindi,
A. Ghanekar and D. Y. Chiang, Hepatology, 2013, 58, 1693.
T. Schmidt, G. J. Tarver and A. G. Wright, ACS Med. Chem. 20 C. Kadoch, D. C. Hargreaves, C. Hodges, L. Elias, L. Ho,
Lett., 2013, 4, 768; M. C. Bryan, C. D. Hein, H. Gao, X. Xia,
J. Ranish and G. R. Crabtree, Nat. Genet., 2013, 45, 592.
H. Eastwood, B. A. Bruenner, S. W. Louie and 21 M. Baumann, I. R. Baxendale, S. V. Ley, N. Nikbin, C. D. Smith
E. M. Doherty, ACS Comb. Sci., 2013, 15, 503.
and J. P. Tierney, Org. Biomol. Chem., 2008, 6, 1577.
22 K. Huard, S. W. Bagley, E. Menhaji-Klotz, C. Pr ´e ville,
J. A. Southers Jr, A. C. Smith, D. J. Edmonds, J. C. Lucas,
M. F. Dunn, N. M. Allanson, E. L. Blaney, C. N. Garcia-
Irizarry, J. T. Kohrt, D. A. Griffith and R. L. Dow, J. Org.
Chem., 2012, 77, 10050.
3
4
K.-I. Kasai and S.-I. Ishii, J. Biochem., 1975, 77, 261.
S. R. Haynes, C. Dollard, F. Winston, S. Beck, J. Trowsdale
and I. B. Dawid, Nucleic Acids Res., 1992, 20, 2603.
D. J. Owen, P. Ornaghi, J.-C. Yang, N. Lowe, P. R. Evans,
P. Bellario, D. Neuhaus, P. Filetici and A. A. Travers, EMBO
5
This journal is © The Royal Society of Chemistry 2014
Med. Chem. Commun., 2014, 5, 540–546 | 545