Evaluation of class i HDAC isoform selectivity of largazole analogues

Article abstract of DOI:10.1016/j.bmcl.2014.07.006

Largazole is a potent class I selective histone deacetylase (HDAC) inhibitor. The majority of largazole analogues to date have modified the thiazole-thiazoline and the warhead moiety. In order to elucidate class I-specific structure-activity relationships, a series of analogues with modifications in the valine or the linker region were prepared and evaluated for their class I isoform selectivity. The inhibition profile showed that the C2 position of largazole has an optimal steric requirement for efficient HDAC inhibition and that substitution of the trans-alkene in the linker with an aromatic group results in complete loss of activity. This data will aid the design of class I isoform selective HDAC inhibitors.

Full text of DOI:10.1016/j.bmcl.2014.07.006

Bioorganic & Medicinal Chemistry Letters 24 (2014) 3728–3731
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Evaluation of class I HDAC isoform selectivity of largazole analogues
Bumki Kim a, Heekwang Park a, Lilibeth A. Salvador b,c, Patrick E. Serrano a, Jason C. Kwan b,
,
Sabrina L. Zeller a, Qi-Yin Chen b,d, Soyoung Ryu a, Yanxia Liu b,d, Seongrim Byeon a, Hendrik Luesch b,d,
,
⇑
Jiyong Hong a,e,
⇑
a Department of Chemistry, Duke University, Durham, NC 27708, United States
b Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville, FL 32610, United States
c Marine Science Institute, College of Science, University of the Philippines, Diliman, Quezon City 1100, Philippines
d Center for Natural Products, Drug Discovery and Development (CNPD3), University of Florida, Gainesville, FL 32610, United States
e Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Largazole is a potent class I selective histone deacetylase (HDAC) inhibitor. The majority of largazole
analogues to date have modified the thiazole–thiazoline and the warhead moiety. In order to elucidate
class I-specific structure–activity relationships, a series of analogues with modifications in the valine
or the linker region were prepared and evaluated for their class I isoform selectivity. The inhibition profile
showed that the C2 position of largazole has an optimal steric requirement for efficient HDAC inhibition
and that substitution of the trans-alkene in the linker with an aromatic group results in complete loss of
activity. This data will aid the design of class I isoform selective HDAC inhibitors.
Received 20 May 2014
Revised 27 June 2014
Accepted 1 July 2014
Available online 9 July 2014
Keywords:
Largazole
HDAC inhibitor
Ó 2014 Elsevier Ltd. All rights reserved.
Class I histone deacetylase
Isoform selectivity
Structure–activity relationship
Largazole (1, Fig. 1), isolated from a cyanobacterium of the Symp-
loca genus, possesses highly differential growth inhibitory activity
towards cancer cells.1 This selectivity is attributed to its very potent
class I histone deacetylase (HDAC) inhibitory activity.2,3 Because of
its potency, class I selectivity, and in vivo efficacy,4 largazole (1)
possesses great potential as an anti-cancer agent.5 Additionally,
largazole (1) has shown a variety of other biological activities, such
as the in vitro and in vivo induction of osteoblast differentiation bio-
markers,6 the sensitization of EBV+ tumor cells to the anti-herpes
drug ganciclovir (GCV),7 the inhibition of ubiquitin activating
enzyme, E1,8 and the in vitro and in vivo induction of apoptosis in
hepatic stellate cells (HSC) in liver fibrosis models.9 These promising
biological activities led to 11 total syntheses2,3,10–18 and a wide
variety of analogues of largazole (1).2–4,10,12,13,16,18–27
Figure 1. Structure of largazole (1) and largazole thiol (2).
chelating groups have resulted in significant decreases in inhibitor
potency.2,10,13,21,25 While the active site of HDACs is highly con-
served, sequence variety in the cap region is relatively high.28 It
is believed that the interactions between this hydrophobic cap
region and the macrocycle of largazole (1) influence its class selec-
tivity.5,29 However, changes in the thiazole–thiazoline unit of lar-
gazole (1) have not resulted in any significant improvement in its
potency or isoform selectivity.18,21–23,26
To date, the majority of largazole analogues that have been
synthesized and studied have altered the warhead or the thia-
zole–thiazoline moiety. For example, previous attempts to replace
the thiol moiety of largazole thiol (2, Fig. 1) with other Zn2+
In contrast, relatively little work has been done on the valine
subunit at the C2 position or the nature of the linker as only ali-
phatic linkers have been studied. Furthermore, the limited work
on these analogues has mostly focused on anti-proliferative activ-
ity rather than HDAC isoform selectivity. Here we report the class I
⇑
Corresponding authors. Tel.: +1 919 660 1545; fax: +1 919 660 1605 (J.H.);
tel.: +1 352 273 7738; fax: +1 352 273 7741 (H.L.).
E-mail addresses: jiyong.hong@duke.edu (J. Hong), luesch@cop.ufl.edu
(H. Luesch).
Current address: Pharmaceutical Sciences Division, University of Wisconsin-
Madison, Madison, WI, USA.
http://dx.doi.org/10.1016/j.bmcl.2014.07.006
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

B. Kim et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3728–3731
3729
Figure 2. Structure of analogues 3–6.
Figure 3. Structure of analogues 7–11.
isoform selectivity profile of several C2 and linker analogues of lar-
gazole (1) to provide valuable insights for future isoform selective
analogue design.
expected that the addition of an N-Boc group in 11 would block
the hydrogen bond donating ability of the imidazole. The synthesis
of 7–11 very closely followed that of 5 and 6.31
Previous structure–activity relationships have shown that the
valine residue at the C2 position of largazole (1) is able to with-
stand certain variations without significant loss in HDAC inhibitory
activity.4,16,18,19,21 Therefore, compounds 3–6 were prepared by
replacing the valine residue with aromatic (Phe, Tyr), basic (His),
and acidic (Asp) amino acids to investigate the effect of different
chemical functionalities on the class I HDAC isoform selectivity
(Fig. 2).30 Compounds 3–6 have been shown to retain anti-prolifer-
ative activity in HCT116 colon cancer cells.4
The inhibition profile showed that 3–6 are very weak HDAC8
inhibitors, implying that the largazole scaffold has an intrinsic
preference towards HDACs 1, 2, and 3 over HDAC8 (Table 1). Also,
while 3–5 were comparable in potency to largazole thiol (2),
As summarized in Table 2, compounds 7 and 8 showed
decreased activity for all HDACs tested. However, the longer alkyl
chain analogue 8 showed a higher potency than the shorter alkyl
chain analogue 7 for HDACs 1, 2, and 3. The same trend was
observed for the corresponding N-Boc protected analogues, as 10
was more potent than 9 for all HDACs tested. This suggests that
there is an optimal steric requirement at the C2 position for effi-
cient HDAC inhibitory activity. The N-Boc protected histidine ana-
logue 11 showed very little change in potency or selectivity,
demonstrating that the hydrogen bonding of the histidine might
be a non-essential interaction in HDAC inhibition.
There are two phenylalanine residues (Phe150 and Phe205) in
the active site of HDAC1.32 To utilize
p–p stacking interactions
aspartic acid analogue
6 experienced a significant reduction
for the improvement of HDAC1 selectivity, we designed linker ana-
logues 12–14 via docking studies33 on a previously reported
homology model of HDAC1 (Fig. 4).32 Compounds 12–14 were
prepared34 and evaluated for their class I HDAC inhibition. Unfor-
tunately, 12–14 showed no activity against class I HDACs. This
complete lack of HDAC inhibition could be due to steric effects of
bulky aromatic groups in the linker preventing the adoption of
docking poses predicted in silico.
in activity. It is notable that there was an overall decrease in
HDAC2 inhibition and that histidine analogue 5 showed minor
selectivity towards HDAC1 over HDACs 2 and 3 (7- and 5.5-fold,
respectively).
Based on these observations, a second set of analogues was
designed to isolate possible interactions between HDAC1 and the
imidazole ring of 5 (Fig. 3). Compounds 7–10 mimicked the posi-
tion of the two different nitrogens present in histidine. We
Table 1
Class I HDAC isoform selectivity of 3–6
Compound
R
IC50 (nM)
HDAC2
IC50 (HDAC2)/IC50 (HDAC1)
IC50 (HDAC3)/IC50 (HDAC1)
HDAC1
0.40
HDAC3
0.70
HDAC8
102
2a
3
0.90
1.70
2.3
5.9
1.8
2.3
0.29
0.21
0.68
0.38
NIb
NIb
4
5
1.10
5.2
1.8
HO
HN
HO
0.20
39
1.40
150
1.10
100
NIb
NIb
7.0
3.9
5.5
2.6
N
6
O
a
Data from Ref. 5.
No inhibition up to 1 lM.
b

3730
B. Kim et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3728–3731
Table 2
Class I HDAC isoform selectivity of 7–11
Compound
R
IC50 (nM)
HDAC2
IC50 (HDAC2)/IC50 (HDAC1)
IC50 (HDAC3)/IC50 (HDAC1)
HDAC1
0.40
HDAC3
0.70
HDAC8
102
2a
0.90
2.3
1.8
NIb
NIb
NIb
790
3.8
1.9
1.4
1.5
1.4
1.8
1.8
1.9
H2N
H2N
BocHN
BocHN
7
8
5.50
3.30
2.30
0.96
21.00
6.30
3.10
1.40
7.80
6.00
4.20
1.80
9
10
BocN
11
0.62
1.10
1.10
540
1.8
1.8
N
a
Data from Ref. 5.
No inhibition up to 1 lM.
b
the histidine analogue 5 does not play an essential role in HDAC
inhibition. Most importantly, the C2 position has an optimal steric
requirement that needs to be met for efficient HDAC inhibition.
Compounds 7–11 support this insight as the shorter alkyl chain
showed weaker inhibition for all class I HDACs. This is further sup-
ported by the weak inhibitory activity of the aspartic acid analogue
6, and the relatively strong inhibition of 2–5 and 11. The bulkier
groups in these analogues did not significantly reduce the
inhibitory activity, suggesting that even larger groups could be
installed at this position with minimal loss in HDAC inhibition
activity. The trends presented here further extend the existing
structure–activity relationship of largazole (1), and will aid in the
design process of class I isoform selective HDAC inhibitors.
Notes: H.L. is a co-founder of Oceanyx Pharmaceuticals, Inc.,
which is negotiating licenses for largazole-related patents and
patent applications.
Acknowledgments
This work supported by the National Institutes of Health
(National Cancer Institute, R01CA138544). We are grateful to the
North Carolina Biotechnology Center (North Carolina Biotechnol-
ogy Center; Grant No. 2008-IDG-1010) for funding of the NMR
instrumentation and to the National Science Foundation (NSF)
MRI Program (Award ID No. 0923097) for funding mass spectrom-
etry instrumentation. P.E.S. was supported by the Chemistry and
Applications of Smart Molecules and Materials REU program
funded by the NSF and DoD (NSF Award #1062607).
Supplementary data
Figure 4. Structure of analogues 12–14.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.
07.006.
In summary, we have prepared and tested a variety of C2 and
linker analogues and probed them for their class I HDAC inhibition.
Our results provide the following insights into the structure–
activity relationship of largazole (1). First, the largazole scaffold
has an innate preference for HDACs 1, 2, and 3, as every analogue
prepared only weakly inhibits HDAC8. Second, the addition of
aromatic groups to the linker region is detrimental to HDAC
inhibition. Third, the hydrogen bonding of the imidazole ring in
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