Structural biasing elements for in-cell histone deacetylase paralog selectivity

Article abstract of DOI:10.1021/ja0341440

We use the structural dissection of two 1,3-dioxanes with in-cell histone deacetylase (HDAC) paralog selectivity to identify key elements for selective HDAC inhibitors. We demonstrate that o-aminoanilides are inactive toward HDAC6 while apparently inhibit

Full text of DOI:10.1021/ja0341440

Published on Web 04/16/2003
Structural Biasing Elements for In-Cell Histone Deacetylase
Paralog Selectivity
Jason C. Wong, Roger Hong, and Stuart L. Schreiber*
Department of Chemistry and Chemical Biology, HarVard Institute of Chemistry and Cell Biology, and the
Howard Hughes Medical Institute, HarVard UniVersity, 12 Oxford Street, Cambridge, Massachusetts 02138
Received January 13, 2003; E-mail: sls@slsiris.harvard.edu
Histone deacetylases (HDACs) are the key enzymatic compo-
nents of protein complexes responsible for deacetylation of histone
and nonhistone protein substrates.1 To date, 11 human HDACs have
been cloned and partially characterized.2 There is growing evidence
that the acetylation state of proteins, and thus this HDAC enzyme
family, plays an important role in the modulation of a number of
biological processes, including transcription,3 microtubule structure
and function,4 and the cell cycle.5 Small molecule inhibitors of the
HDACs such as trichostatin A (TSA) and trapoxin B (TPX) (Figure
1), while not paralog- or family member-selective, have already
played an important role in discovering HDACs2a and in elucidating
HDAC function.2a,6 Therefore, the development of paralog-selective
small molecule HDAC inhibitors will be a valuable tool for the
characterization of HDAC paralog-specific biological functions.6a
Figure 1. Trichostatin A, trapoxin B, tubacin 1, and histacin 2.
Here we report on the structural origins of in-cell paralog selectivity
demonstrated by two 1,3-dioxanes, tubacin 1 and histacin 2 (Figure
1), which were generated by diversity-oriented synthesis7 and
identified in a cytoblot screen.8
1 and 2 were originally classified as selective inducers of tubulin
acetylation and histone acetylation, respectively, in A549 cells.8
Recent work by Hubbert and co-workers9 demonstrating that
HDAC6 is a tubulin deacetylase suggested to us that small
molecules that selectively induce tubulin acetylation would likely
be paralog-selective HDAC6 inhibitors. Extensive biological char-
acterization of 1 has demonstrated that this is indeed the case.10 It
follows that selective inducers of histone acetylation such as 2 are,
at the very least, HDAC6-insensitive inhibitors, if not potentially
selective inhibitors of another HDAC paralog.
Figure 2. Structures of suberoylanilide hydroxamic acid (SAHA), pimeloyl-
anilide orthoaminoanilide (PAOA), and several analogues.
We were interested in identifying the structural basis for the in-
cell paralog selectivity demonstrated by 1 and 2. In particular, we
were intrigued by the structural similarity between 1 and an HDAC
inhibitor that is currently being evaluated clinically for the treatment
of cancer, suberoylanilide hydroxamic acid (SAHA) (Figure 2).11
In contrast to 1, treatment of A549 cells with 5 µM SAHA markedly
increased both levels of acetylated tubulin and acetylated histone
as determined by immunofluorescence microscopy (Figure 3). This
result demonstrates that the SAHA substructure alone is insufficient
for HDAC6 paralog selectivity in cells and validates the use of
1,3-dioxane diversity as a selectivity generating element.7
These findings led us to wonder whether an analogous structural
dissection of 2 would yield similar conclusions about the origin of
its HDAC selectivity. Thus, pimeloylanilide o-aminoanilide (PAOA)
5 was synthesized in four steps from pimelic acid and tested in
A549 cells. We were surprised to find that 5 demonstrates the same
selectivity as 2 (Figure 4), indicating that in this case the dioxane
scaffold is not necessary for selective induction of histone acetyl-
ation, but that the o-aminoanilide element alone may be sufficient.
The selectivity of 5 was confirmed by treating A549 cells with 5
for 5 h, harvesting and lysing the cells, and then analyzing the
lysates by Western blot (Figure 5).
Figure 3. Immunofluorescence microscopy detection in A549 cells of
acetylated histones (row a); acetylated histones and Hoechst nuclear staining
(row b); acetylated tubulin and Hoechst nuclear staining (row c). Cells were
treated for 14 h with the indicated compound (columns).
To explore the role of the o-aminoanilide as a potential HDAC
paralog selectivity element further, analogues of 5 with various
hydrocarbon linker lengths were synthesized and evaluated in A549
cells by immunofluorescence microscopy (Figure 6). As the linker
length was increased from three to five methylene units, a
corresponding increase in histone acetylation was observed. Fur-
thermore, none of the analogues detectably induced tubulin acetyl-
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J. AM. CHEM. SOC. 2003, 125, 5586-5587
10.1021/ja0341440 CCC: $25.00 © 2003 American Chemical Society

C O M M U N I C A T I O N S
Table 1. Activity of Compounds toward Induction of Acetylated
Tubulin and Acetylated Histone in A549 Cellsa
induction EC , µM
50
compound
Ac-tubulin
Ac-histone
1 (tubacin)
SAHA
2 (histacin)
3
2.9 ( 0.9
1.8 ( 0.5
NDI
NDI
NDI
217 ( 96
3.6 ( 0.4
34 ( 5
2534 ( 985
249 ( 48
36 ( 5
4
5 (PAOA)
6
NDI
NDI
181 ( 47
a95% confidence interval reported. NDI ) no detectable induction over
the tested concentration range.
inhibiting deacetylases that act upon histone substrates. This finding
has important clinical implications for the development of HDAC
inhibitor-based treatments that do not interfere with microtubule
dynamics associated with HDAC6.4 We have also shown that, in
contrast to 1, the SAHA substructure alone is insufficient for in-
cell HDAC6 paralog selectivity. This finding validates our use of
1,3-dioxane chemical diversity to identify paralog-selective HDAC
inhibitors.7
Figure 4. Immunofluorescence microscopy detection in A549 cells of
acetylated histones (row a); acetylated histones and Hoechst nuclear staining
(row b); acetylated tubulin and Hoechst nuclear staining (row c). Cells were
treated for 14 h with the indicated compound (columns).
Acknowledgment. We thank Dr. Gregory Copeland for the
synthesis of suberoylanilide hydroxamic acid. J.C.W. was supported
by an NSF predoctoral fellowship. R.H. was supported by an HRCP
undergraduate summer fellowship. S.L.S. is an investigator at the
Howard Hughes Medical Institute.
Figure 5. Western blot detection of acetylated tubulin and histone H3 levels
in A549 cells after 5 h treatment with the indicated compound. (1) DMSO;
(2) TSA (300 nM); (3) PAOA (20 µM); (4) PAOA (50 µM); (5) PAOA
(100 µM).
Supporting Information Available: Experimental procedures and
spectral data for all relevant compounds (PDF). This material is
available free of charge via the Internet at http://pubs.acs.org.
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Figure 6. Immunofluorescence microscopy detection in A549 cells of
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not only biases small molecules toward HDAC inhibition12 but also
renders such inhibitors inactive toward HDAC6 in cells.
Finally, to quantify the selectivity of tubacin, histacin, and their
analogues in cells, acetylated tubulin and histone induction were
measured for each compound over a range of concentrations by
luminescence detection in a cytoblot format (Table 1).13
In summary, we have used the structural dissection of two 1,3-
dioxanes with in-cell HDAC paralog selectivity to identify key
elements for selective HDAC inhibitors. We have demonstrated
that o-aminoanilides are inactive toward HDAC6 while apparently
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(13) See Supporting Information.
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