CHEMMEDCHEM
FULL PAPERS
[7] B. S. Mann, J. R. Johnson, M. H. Cohen, R. Justice, R. Pazdur, Oncologist
Experimental Section
Synthesis: Detailed synthetic procedures and analytical data for all
compounds can be found in the Supporting Information.
[8] S. J. Whittaker, M.-F. Demierre, E. J. Kim, A. H. Rook, A. Lerner, M. Duvic,
J. Scarisbrick, S. Reddy, T. Robak, J. C. Becker, A. Samtsov, W. MacCulloch,
[9] F. Cherblanc, N. Chapman-Rothe, R. Brown, M. J. Fuchter, Future Med.
[10] M. G. J. Baud, T. Leiser, F.-J. Meyer-Almes, M. J. Fuchter, Org. Biomol.
[11] M. G. J. Baud, T. Leiser, P. Haus, S. Samlal, A. C. Wong, R. J. Wood, V. Pet-
rucci, M. Gunaratnam, S. Hughes, L. Buluwela, F. Turlais, S. Neidle, F.-J.
[12] P. Di Fruscia, K.-K. Ho, S. Laohasinnarong, M. Khonghow, S. H. B. Kroll,
S. A. Islam, M. J. E. Sternberg, K. Schmidtkunz, M. Jung, E. W.-F. Lam,
[13] B. Peck, C. Y. Chen, K. K. Ho, P. Di Fruscia, S. S. Myatt, R. C. Coombes,
HDAC assays: Thiols were obtained from their corresponding di-
sulfides as previously reported,[10,11] and were assayed immediately
after reduction. HDAC assays were performed as previously report-
ed.[10,11] DMSO, Pluronic, TCEP, and trypsin (bovine pancreas) were
purchased from Sigma–Aldrich, and Boc-Lys(Ac)-AMC as well as
Boc-Lys(TFA)-AMC from Bachem (Switzerland). The recombinant
human histone deacetylases HDAC1–4, HDAC6, and HDAC8 were
obtained from BPS Bioscience (USA). All reactions were performed
in black half area 96-well microplates (Greiner bio-one, Germany)
according to the general procedure described by Wegener et al.[35]
with some minor modifications. The reaction buffer contains
50 mm KH2PO4/K2HPO4, 15 mm Tris·HCl pH 8, 250 mm NaCl, 0.001%
(v/v) Pluronic, and 0.005% BSA. The buffer components were pur-
chased from Merck (Germany), Roth (Germany), and Sigma–Aldrich.
A serial dilution of test compounds was pre-incubated with 7.4 nm
HDAC1, 53.6 nm HDAC2, 241 nm HDAC3, 3.9 nm HDAC4, 2.8 nm
HDAC6, or 23.6 nm HDAC8 for 20 min at 21ꢁ18C in the dark. The
enzyme reaction was initiated by the addition of Boc-Lys(Ac)-AMC
(for HDAC1: 80 mm, HDAC2 and HDAC3: 40 mm, HDAC6: 100 mm) or
Boc-Lys(TFA)-AMC (for HDAC4: 40 mm, HDAC8: 30 mm) substrate.
The reaction mixture was incubated at 308C in the dark and
stopped after 60 min by the addition of a mixture of 67 mm trypsin
and 200 nm SAHA. The fluorescence of AMC serves as an indirect
measure of HDAC activity. The kinetics of AMC release was mea-
sured on a PolarStar fluorescence plate reader (BMG) using an exci-
tation wavelength of 340 nm and an emission wavelength of
460 nm. Complete cleavage of deacetylated Boc-Lys-AMC by tryp-
sin was achieved after ~10–15 min. The fluorescence intensity of
the plateau was averaged over at least 5 min and normalized with
respect to percent enzyme activity. Finally, the normalized fluores-
cence intensities were plotted versus the concentration of test
compounds and fitted to a four-parameter logistic model[36] to cal-
culate the IC50 values.
PiÇa, J. T. Gautschi, G. Y. S. Wang, M. L. Sanders, F. J. Schmitz, D. France,
S. Cornell-Kennon, L. C. Sambucetti, S. W. Remiszewski, L. B. Perez, K. W.
Bair, P. Crews, J. Org. Chem. 2003, 68, 3866–3873; c) E. QuiÇoꢂ, P. Crews,
Tetrahedron Lett. 1987, 28, 3229–3232; d) A. D. Rodriguez, R. K. Akee,
[15] J. Garcꢃa, G. Franci, R. Pereira, R. Benedetti, A. Nebbioso, F. Rodriguez-
Barrios, H. Gronemeyer, L. Altucci, A. R. de Lera, Bioorg. Med. Chem.
2011, 19, 3637–3649.
[16] A. Nebbioso, R. Pereira, H. Khanwalkar, F. Matarese, J. Garcia-Rodriguez,
M. Miceli, C. Logie, V. Kedinger, F. Ferrara, H. G. Stunnenberg, A. R.
[17] D. Kim, I. S. Lee, J. H. Jung, C. O. Lee, S. U. Choi, Anticancer Res. 1999,
19, 4085–4090.
[19] J. Shin, H. S. Lee, Y. Seo, J. R. Rho, K. W. Cho, V. J. Paul, Tetrahedron 2000,
[20] J. N. Tabudravu, V. G. H. Eijsink, G. W. Gooday, M. Jaspars, D. Komander,
[21] G. M. Nicholas, L. L. Eckman, S. Ray, R. O. Hughes, J. A. Pfefferkorn, S.
Docking: All ligands were docked in HDAC2 (PDB ID: 3MAX)[30]
using Glide.[37] The protein structure was prepared using the Pro-
tein Preparation Wizard[38] from Schrçdinger. His145 was protonat-
ed. Ligands were prepared (LigPrep)[39] and docked (Glide)[37] in
their deprotonated forms (thiolate). No constraint was applied to
the system. Docking poses were subjected to one round of
Prime[40] minimization, then analyzed visually with PyMOL, and dis-
tances were calculated in PyMOL.[41]
[23] Y. H. Jiang, E.-Y. Ahn, S. H. Ryu, D.-K. Kim, J.-S. Park, H. J. Yoon, S. Yoo, B.-
7818; b) C. T. A. Evelo, A. A. M. G. Spooren, R. A. G. Bisschops, L. G. M.
Acknowledgements
This work was supported by the Association for International
Cancer Research (08-0407).
[26] M. Krayer, M. Ptaszek, H.-J. Kim, K. R. Meneely, D. Fan, K. Secor, S. Lind-
[28] P. Jones, S. Altamura, P. K. Chakravarty, O. Cecchetti, R. De Francesco, P.
Gallinari, R. Ingenito, P. T. Meinke, A. Petrocchi, M. Rowley, R. Scarpelli, S.
[29] L. W. Deady, R. A. Shanks, A. D. Campbell, S. Y. Choii, Aust. J. Chem.
[30] J. C. Bressi, A. J. Jennings, R. Skene, Y. Wu, R. Melkus, R. De Jong, S.
Keywords: epigenetics · HDAC inhibitors · isoform selectivity ·
ligand design · psammaplin A
[4] S. Crabb, M. Howell, H. Rogers, M. Ishfaq, A. Yurek-George, K. Carey, B.
Pickering, P. East, R. Mitter, S. Maeda, P. Johnson, P. Townsend, K. Shin-
[31] P. J. Watson, L. Fairall, G. M. Santos, J. W. B. Schwabe, Nature 2012, 481,
335–340.
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2013, 8, 149 – 156 155