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Bioorganic & Medicinal Chemistry Letters 28 (2018) 2493–2497

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Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier.com/locate/bmcl

Development of the first small molecule histone deacetylase 6 (HDAC6) T

Ka Yanga, Yanling Songa,b, Haibo Xiea, Hao Wua, Yi-Ting Wua,c, Eric D. Leistena,

Weiping Tanga,d,
School of Pharmacy, University of Wisconsin-Madison, Madison, WI 53705, USA
Department of Pharmaceutical Engineering, Shenyang University of Chemical Technology, Liaoning 110042, China (current address)
Department of Chemistry, National Cheng Kung University, Tainan 701, Taiwan (current address)
Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53706, USA


Keywords: Histone deacetylases (HDACs) decrease the acetylation level of histones and other non-histone proteins. Over
HDAC expression of HDACs have been observed in cancers and other diseases. Targeted protein degradation by “hi-
Degrader jacking” the natural ubiquitin-proteasome-system (UPS) recently emerged as a novel technology to “knock-out”
PROTAC endogenous disease-causing proteins. We applied this strategy to the development of the first small molecule
degraders for zinc-dependent HDACs by conjugating non-selective HDAC inhibitors with E3 ubiquitin ligase
ligands. Through cell-based assays, we discovered novel bifunctional molecules (dHDAC6) that could selectively
degrade HDAC6. Further mechanistic studies indicated that HDAC6 was selectively removed by the UPS.

Introduction cytoplasm.18,19 Unlike other HDACs that target histones in the nuclei,
HDAC6 is primarily responsible for the deacetylation of cytoplasmic
Histone deacetylases (HDACs) are epigenetic regulators or erasers proteins including α-tubulin20 and heat shock protein 90 (HSP90).19 In
that can alter gene expression and play crucial roles in cancers and addition, HDAC6 also regulates the turnover of misfolded and poly-
many other diseases.1 The 18 human HDACs can be divided to two ubiquitinated proteins.21 Abnormal expression of HDAC6 has been re-
distinct families: 11 of them use zinc as the cofactor while 7 of them lated to cancer development, progression and migration.22 For ex-
rely on the nicotinamide adenine dinucleotide (NAD) cofactor. Most ample, overexpression of HDAC6 in oral squamous cell carcinoma,
HDACs catalyze the hydrolysis of the Nε-acetyl group from lysine re- acute myeloid leukemia, ovarian cancer, and hepatocellular carcinomas
sidues on histones inside the nucleus. These HDACs generally repress is clinically significant since it indicates advanced disease progression,
gene transcription by modulating chromatin structures in the nucleus. higher rates of metastatic events, and lower survival rates.23–25
In addition, certain HDAC members can also target cellular non-histone Targeted protein degradation (TPD) by heterobifunctional small
proteins, including DNA binding proteins, transcriptional regulators, molecules or proteolysis targeting chimeras (PROTACs) has emerged as
chaperone proteins, and structural proteins.2 Abnormal changes in a promising therapeutic strategy for the removal of various over-
HDAC expression have been implicated in many types of cancers.3 expressed disease-causing proteins.26–28 TPD takes advantage of the
Several HDAC inhibitors have been approved for the treatment of cell’s own ubiquitination-proteasome system (UPS), one of the major
cancers.1,4,5 All of the approved HDAC inhibitors, however, non-selec- pathways for protein degradation,29 by bringing an E3 ubiquitin ligase
tively target various HDACs and exhibit significant toxicity.6 Develop- in close proximity to the protein of interest (POI) for ubiquitination and
ment of selective HDAC inhibitors is one of the current focuses to re- proteasome degradation through a bifunctional small molecule or
duce the toxicity and achieve wider therapeutic indexes for a variety of PROTAC, which can bind simultaneously to the POI and the E3 ligase.
cancers.7 Various bifunctional HDAC inhibitors have also been devel- Early bifunctional molecules recruited E3 ligases SCFβ-TRCP and MDM2
oped for more targeted therapy by conjugating HDAC inhibitors with to degrade MetAp-230 and an androgen receptor,31 respectively. Both
other functional molecules, such as estrogen receptor ligands,8 an- bifunctional molecules, however, exhibited relatively low cellular po-
drogen receptor ligands,9 topoisomerase inhibitors,10 and others.11–17 tency. Recently, more potent bifunctional small molecules were de-
HDAC6 is a class IIb HDAC and is mainly expressed in the veloped to recruit E3 ubiquitin ligases cIAP1,32–36 cullin-RING E3

Corresponding author at: School of Pharmacy, University of Wisconsin-Madison, Madison, WI 53705, USA.
E-mail address: weiping.tang@wisc.edu (W. Tang).

Received 7 May 2018; Received in revised form 25 May 2018; Accepted 29 May 2018
Available online 30 May 2018
0960-894X/ © 2018 Elsevier Ltd. All rights reserved.
K. Yang et al. Bioorganic & Medicinal Chemistry Letters 28 (2018) 2493–2497

ubiquitin ligase cereblon (CRLCRBN or CRBN),37–43 or von Hippel-

Lindau (VHL)39,40,44–48 for the degradation of various proteins. The first
degrader of sirtuin enzyme, a family of NAD-dependent HDACs, was
reported in early 2018.43 To the best our knowledge, no degrader of
zinc-dependent HDACs has been reported to date, and whether or not
this family of HDAC enzymes are viable targets for TPD remains to be
There are several potential advantages for enzyme degraders over
enzyme inhibitors.26 First, inhibition of certain cellular pathways often
increases the expression of the target protein, which is mediated by a
feedback mechanism, and this often leads to pharmacological in-
sufficiency. The degradation activity can then negate the effects of
protein overexpression. Second, the phenotype, or very often the cause,
of the diseases is the overexpression of the target protein. The more
appropriate correction of the disease state is to tune down the level of
this protein until it reaches its physiological level instead of inhibiting
the enzymatic functions. Finally, inhibitors require stoichiometric drug
binding to the target in order to modulate the protein function. In
contrast, TPD by small molecule degraders is catalytic, providing fa-
vorable pharmacology.
We previously developed a strategy that can provide quick access to
a library of diverse HDAC inhibitors, as shown in Scheme 1A.49 A series
of HDAC biasing reagents 1 was prepared and coupled with a diverse
range of aldehydes to generate a large collection of HDAC inhibitors 2.
The resulting products have sufficient purity and can be directly tested
in relevant biological assays. This strategy has yielded a number of
useful chemical probes. For example, compound 2a (WT161), one Condition: a) propargylamine hydrochloride, DIPEA, DMF,
90 °C, 30% yield; b) CuSO4, TBTA, sodium ascorbate, H2O/t-
member of the library, is a highly selective HDAC6 inhibitor and is very
BuOH (1:5), room temperature, 60–70% yield; c) HOAc, DMSO,
effective for the treatment of bortezomib-resistant multiple myeloma 65 °C; d) HOAc, EtOH, 65 °C, 95% yield for 9c.
(MM) in cells and mouse models, when it is combined with borte-
zomib.50 Structurally related analogues such as 3a and 3b are currently Scheme 2. Synthesis of HDAC degraders.
in human clinical trials for the treatment of various cancers.51–53 Within
these trials 3a and 3b are combined with other therapeutics, such as synapsin-1 punctae along dendrites in cultured neurons and enhanced
thalidomide and its analogues (e.g. 4a–4c), which are ligands of the memory in mouse models,58 and compound 2c suppressed aggressive
CRLCRBN E3 ubiquitin ligase.28,54–57 Other members of the library have thyroid cancer in cells and in animal models.59,60 We envision that this
also shown promising results. Compound 2b increased the density of strategy can also facilitate the development of various small molecule
HDAC degraders.
We herein report the development of the first small molecule
HDAC6 degraders by conjugating non-selective HDAC inhibitor 2, de-
rived from our previous strategy, to a thalidomide-type E3 ligase ligand
with various linkers. The profiling of the resulting products in cell-
based assays revealed that our bifunctional small molecules selectively
promoted the degradation of HDAC6 through the UPS.
The synthesis of our HDAC degraders is summarized in Scheme 2.
An alkyne functional group was introduced to pomalidomide analogue
6 by a SNAr reaction between known fluoro-thalidomide 5 and pro-
pargyl amine according to literature procedures.41 Aldehydes 7a–7d
containing an azide functional group and one to several polyethylene
glycol units were also prepared according to literature methods.61,62 A
copper-catalyzed cycloaddition between alkyne 6 and azides 7a–7d
afforded the corresponding pomalidomide-conjugated azides 8a–8d
with varying linker lengths. Condensation of 8a–8d with HDAC-biasing
reagent 1a49 in DMSO yielded products 9a–9d as the bifunctional
molecules. The resulting DMSO solutions were analyzed by LC-MS and
determined to be of sufficient purity (> 95%) for further biological
SAHA (Vorinostat) is one of the FDA-approved HDAC inhibitors for
the treatment of advanced primary cutaneous T-cell lymphoma. SAHA
has potent inhibitory potency against HDAC1, HDAC2, HDAC3 and
HDAC6.6,63 Compound 2c was previously prepared as a pan-HDAC
inhibitor.59,60 SAHA and compound 2c were used as HDAC inhibitor
controls for our following studies.
We first incubated bifunctional molecules 9a–9d at 5 µM in MCF-7
cells. After 12 h, the whole cell lysate was generated for SDS-PAGE and
Scheme 1. A) A previously reported strategy for the generation of diverse
immunoblot. By examining the amount of HDAC1 and HDAC2 as
HDAC inhibitors; B) CRBN E3 ubiquitin ligase ligand.

K. Yang et al. Bioorganic & Medicinal Chemistry Letters 28 (2018) 2493–2497

Fig. 1. Profiling of HDAC degraders. A) Immunoblot of whole cell lysate after MCF-7 cells were treated with 5 µM of 9a-9d for 12 h; B) Immunoblot of whole cell
lysate after MCF-7 cells were treated with 9c at various concentrations (5 nM to 10 µM) for 12 h; C) Band intensity of blot B calculated by Image J, and bar graph
plotted and curve fitted by GraphPad Prism.

typical examples of class I HDACs, and HDAC4 and HDAC6 as typical efficiency of TPD of HDAC6 protein.
examples of class II HDACs, we observed a considerably lower level of Co-treatment of 2 µM of compound 9c with either 5 µM MG132, a
HDAC6 compared to others (Fig. 1A). Meanwhile, the amount of HDAC known proteasome inhibitor, or 5 µM pomalidomide, the CRBN ligand,
proteins remained the same when cells were treated with SAHA. abolished the effect of compound 9c for the degradation of HDAC6
Especially encouraging was the fact that these bifunctional molecules, (Fig. 2B). The tubulin acetylation level was also almost the same as
with relatively high molecular weights, were able to cross the cell DMSO treatment in these cases. These results indicated the essential
membrane to degrade certain HDAC members. Among the four bi- role of the ubiquitin-proteasome pathway for the TPD of compound 9c.
functional molecules with various linker lengths, compound 9c showed First of all, proteasome activity was required for compound 9c medi-
a higher activity for the degradation of HDAC6 than the other three ated HDAC6 turnover since the inhibition of the proteasome by MG132
compounds at a 5 µM concentration. This data indicated that the linker abolished the effect of compound 9c. Second, the recruitment of the
length has significant impact on the efficiency of TPD, which is con- CRBN E3 ligase was also critical for compound 9c mediated degrada-
sistent with previous reports for the targeted degradation of other tion of HDAC6 because pomalidomide competitively binds CRBN and
proteins.41,64 The selective degradation of HDAC6 over other HDACs is removed the effect of compound 9c. In addition, the significantly dif-
unexpected, but not surprising, because it has been reported that se- ferent levels of acetylated tubulin by the treatment with 9c alone and
lective degraders can be developed by tethering non-selective kinase by 9c-pomalidomide or 9c-MG132 co-treatment suggested that HDAC6
inhibitors or BRD ligands with a E3 ubiquitin ligase ligand.40,44 Com- degradation not inhibition by 9c was primarily responsible for raising
pound 9c (dHDAC6) was then resynthesized in ethanol and purified for the level of acetylated α-tubulin, at least in the case of the 12 h treat-
further studies. ment.
To probe the effective concentration range, incubation of cells with A similar effect was also observed when pan-HDAC inhibitor 2c, the
5 nM to 10 µM of 9c resulted in the degradation of HDAC6 in a dose “warhead” of HDAC6 degrader 9c, was used as the competitive ligand
dependent manner. (Fig. 1B) The degradation occurred at concentra- for HDACs (Fig. 2C). In the presence of 2 µM of compound 9c, the
tions as low as 41 nM and reached the maximal effect between 123 nM addition of 1 µM of 2c is sufficient to rescue HDAC6 from TPD. Low-
and 370 nM. No obvious “Hook effect” was observed at higher con- ering the concentration of 2c to 100 nM, however, is not sufficient to
centration.65 The concentration at which half-maximal degradation was abolish compound 9c-mediated degradation of HDAC6. On the other
achieved (DC50) and the maximum percentage of degradation (Dmax) hand, a large degree of tubulin acetylation by compound 9c was still
are 34 nM and 70.5% respectively, based on the blot intensity (Fig. 1C). observed. In addition to the level of acetylated tubulin, we also ex-
Research has established that HDAC6 regulates the acetylation of α- amined the level of acetylated histone 3 (H3) to evaluate the selectivity
tubulin.20 Knockdown of HDAC6 by siRNA or inhibition by small mo- of compound 9c. Compound 9c at a 2 µM concentration clearly ex-
lecule HDAC6 inhibitors can upregulate the level of acetylated α-tu- hibited less of an effect than compound 2c alone at the same con-
bulin.50,66 Indeed, increased acetylated α-tubulin levels were observed centration for the increase of acetylated H3. The effect of compound 9c
at 1.1 µM and above (Fig. 1B). The increased acetylated α-tubulin level alone for the increased of acetylated H3 is likely due to inhibition of
should be the result of inhibition and degradation of HDAC6. class I HDAC(s) in the nucleus. Our results indicate that bifunctional
To further study the mechanism of action of our HDAC6 selective molecule 9c has dual functions: selective degradation of HDAC6 and
degraders, we conducted a series of experiments. A time-course ex- inhibition of HDACs.
periment revealed that significant degradation of HDAC6 and upregu- We also tested compound 9c in the MM.1S cell line. After a 6 h
lation of tubulin acetylation occurred around 2 h after treating MCF-7 treatment, the maximal effect of HDAC6 degradation was observed at
cells with 2 µM of compound 9c (Fig. 2A), which suggested the high 80 nM or higher concentrations (Fig. 3). The increase in acetylated

K. Yang et al. Bioorganic & Medicinal Chemistry Letters 28 (2018) 2493–2497

Fig. 2. Mechanistic studies of HDAC6 degraders. Immunoblot of whole cell lysate of cells: A) MCF-7 cells were treated with 2 µM of 9c or vehicle (DMSO) for various
amounts of time (0–8 h); B) MCF-7 cells were pre-treated with 5 µM of MG132 or 5 µM of Pomalidomide 4b or vehicle (DMSO) for 1 h and then treated with 2 µM of
9c or vehicle (DMSO) for 12 h; C) MCF-7 cells were pre-treated with various concentrations (1 µM or 100 nM) of 2c or vehicle (DMSO) for 1 h and then treated with
2 µM of 9c or vehicle (DMSO) for 12 h.

A. Supplementary data

Supplementary data associated with this article can be found, in the

online version, at http://dx.doi.org/10.1016/j.bmcl.2018.05.057.


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