PROTAC-mediated degradation of class i histone deacetylase enzymes in corepressor complexes

Article abstract of DOI:10.1039/d0cc01485k

We have identified a proteolysis targeting chimera (PROTAC) of class I HDACs 1, 2 and 3. The most active degrader consists of a benzamide HDAC inhibitor, an alkyl linker, and the von Hippel-Lindau E3 ligand. Our PROTAC increased histone acetylation levels and compromised colon cancer HCT116 cell viability, establishing a degradation strategy as an alternative to class I HDAC inhibition.

Full text of DOI:10.1039/d0cc01485k

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DOI: 10.1039/D0CC01485K
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PROTAC-Mediated Degradation of Class I Histone Deacetylase
Enzymes in Corepressor Complexes
Joshua P. Smalley,a Grace E. Adams,b Christopher J. Millard,c Yun Song,c James K.S. Norris,a John
Received 00th January 20xx,
Accepted 00th January 20xx
W.R. Schwabe,*c Shaun M. Cowley*b and James T. Hodgkinson*a
DOI: 10.1039/x0xx00000x
We have identified a proteolysis targeting chimera (PROTAC) of
class I HDACs 1, 2 and 3. The most active degrader consists of a
benzamide HDAC inhibitor, an alkyl linker, and the von Hippel-
Lindau E3 ligand. Our PROTAC increased histone acetylation levels
and compromised colon cancer HCT116 cell viability, establishing a
degradation strategy as an alternative to class I HDAC inhibition.
O
O
O
N
N
N
H
H
H
NH2
N
N
NH2
N
H
N
CI-994
MGCD0103
O
N
H
N
H
O
NH2
Targeting enzymes involved in the regulation of epigenetic
modifications have provided therapeutic drugs in treating
cancer and have potential in treating other diseases including
neurological disorders and cardiovascular disease.1 The Histone
Deacetylase (HDAC) family of enzymes, often termed epigenetic
erasers, function by removing the acetyl moiety from histone
tails thereby modifying chromatin structure and gene
expression.2 Currently five HDAC inhibitors have been approved
for clinical use to treat T-cell lymphoma and multiple myeloma
with other compounds in clinical trials.3 18 HDAC enzymes have
been identified in humans, 11 with a divalent zinc cation in the
catalytic site and 7 sirtuins whose activity is NAD+ dependent.4
Inhibitors of the Zn2+-dependent enzymes, such as Valproic acid
or SAHA (Zolinza), are currently used in the clinic to treat
cutaneous T-cell lymphoma (CTCL) and bipolar disorder.
However, these drugs exhibit limited selectivity and this has
been attributed as a cause of their debilitating side-effects in
patients.5
O
MS-275
Figure 1. Benzamide based HDAC inhibitors. CI-994 demonstrates selective
inhibition for HDAC 1, 2 & 3, inhibits cell proliferation, induces apoptosis and has
anti-tumor activity. MS-275 is a HDAC 1 & 3 selective inhibitor with anti-tumor
activity currently in phase III clinical trials. MGCD0103 is a HDAC 1, 2, 3 inhibitor in
clinical trials.
function as singular entities, but exist in vivo as catalytic
subunits in much larger multi-protein corepressor complexes,
including Sin3, NuRD, CoREST, MiDAC and NCoR.4 These
complexes play an essential role in targeting the HDAC enzyme
to specific regions in the genome and demonstrate distinct cell
type specific functions.7
The Proteolysis Targeting Chimera (PROTAC) technology has
been receiving considerable attention as a novel strategy to
target difficult-to-drug proteins of interest (POI). PROTACs are
heterobifunctional molecules that couple a ligand for the POI
with a ligand for an E3 ligase such that the POI becomes
polyubiquitinated and degraded by the proteasome.8 One of the
exciting observations regarding PROTACs is their ability to
induce selectivity that has otherwise proved very challenging.9
One hypothesis for PROTAC-induced selectivity is the necessity
of a protein-protein interaction between the POI and the E3
ligase required for degradation.10 Towards this endeavor we
wanted to generate PROTACs specifically for class I HDAC 1, 2 &
3, present in corepressor complexes.
HDAC 1, 2 and 3 are localized in the nucleus, constitute
approximately 50% of total cellular deacetylase activity and are
the most prominent HDACs in gene expression.6 They do not
a. Leicester Institute of Structural and Chemical Biology and Department of
Chemistry, University of Leicester, University Road, Leicester, LE1 7RH, UK,
E-mail: JTHodgkinson@le.ac.uk
b. Address here. Department of Molecular and Cell Biology, University of Leicester,
Leicester LE1 9HN, UK, E-mail: smc57@leicester.ac.uk
c. Leicester Institute of Structural and Chemical Biology and Department of
Molecular and Cell Biology, University of Leicester, Leicester LE1 9HN, UK, E-mail:
john.schwabe@le.ac.uk
For our PROTAC design we chose benzamide-based HDAC
inhibitors that demonstrate selectivity for HDAC 1, 2 & 3. CI-994
has reported Ki values of 0.41ꢀμM for HDAC 1, 0.75ꢀμM for HDAC
3, and approximately 100ꢀμM for HDAC 8 (Fig 1).11 This inhibitor
† Dr Y. Song present address: Diamond Light Source Ltd, Oxfordshire, OX11 0DE, UK
Electronic Supplementary Information (ESI) available: [Full protocols, assays and 1H
and 13C NMR data]. See DOI: 10.1039/x0xx00000x
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values of 1-4 were determined side-by-side with CI-994 as a
positive control. As a negative control we also synthesized Boc
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DOI: 10.1039/D0CC01485K
H2
N
Zn2+
protected CI-994, compound 5. Compound 5 is not capable of
chelating zinc in the HDAC active site and should be incapable
of HDAC inhibition. We observed that CI-994 had an IC50 value
of approx. 0.5 µM consistent with previous reported values,11
and 5 demonstrated no HDAC inhibition (Fig 3A). The putative
PROTACs 1 and 3 with shorter linker lengths all engaged HDAC1
in the CoREST complex with IC50 values directly comparable to
CI-994, while the longer linker lengths, 2 and 4, still maintained
inhibition but at reduced levels compared to CI-994 and
compounds 1 and 3 (Fig 3A). We proceeded to assess the effects
of these compounds on HDAC activity in cells. In a previous
study we demonstrated that acetylated Histone 3 Lysine 56
(H3K56Ac) is a direct substrate of HDAC 1 in embryonic stem
(ES) cells.17 To examine the ability of 1-4 to reduce HDAC 1 and
2 activity we began by measuring H3K56 acetylation using
quantitative western blotting in ES cells (Fig 3B). CI-994 and the
pan-HDAC inhibitor, Panobinostat, were used as positive
controls and H3K56 acetylation increased as expected, 15-20
fold compared to non-treated cells (Fig 3B). Intriguingly, only
the compounds 2 and 4 with longer linker lengths, which were
less potent in vitro inhibitors against the CoREST complex,
caused an increase in histone acetylation (Fig 3B), with 4
resulting in a 10 fold increase at 10 µM (see supporting info Fig
S2).
O
HN
O
HN
Linker
O
N
O
O
H
O
H
NH2
N
N
N
O
n
H
NH
O
O
n = 1, 1
n = 7, 2
O
HO
S
O
N
O
N
O
N
H
N
H
HN
NH2
N
O
n
H
O
n = 1, 3
n = 7, 4
Figure 2. The benzamide acetyl group is surface exposed in a crystal structure
bound to the HDAC
2 catalytic active site (PDB: 4LY1). Four preliminary
heterobifunctional molecules were synthesised. PROTAC 1 and 3 with a six carbon
alkyl linker and either the VHL or Cereblon E3 ligand. PROTAC 2 and 4 with a twelve
carbon alkyl linker and either the VHL or Cereblon E3 ligand.
exhibits phenotypes related to HDAC 1, 2 & 3 activity, inhibition
of cell proliferation, induction of apoptosis and broad anti-
tumor activity in vitro and in vivo.11,12 CI-994 has been in clinical
trials for its anti-tumor properties and analogous benzamides,
MS-275 and MGCD0103, are currently in phase III clinical trials
for breast cancer and non-small cell lung cancer (Fig 1).3 More
recently CI-994 has also been reported for its neuroprotective
effects in mice following spinal cord injury, the treatment of
cognitive disorders, and reducing atrial fibrillation.13
Figure 3. A) AMC-fluorescence histone deacetylase inhibition assay with the LSD1-
CoREST-HDAC complex in vitro. B) Histone 3 Lysine 56 acetylation (H3K56Ac) levels
in E14 mouse embryonic stem cells after 24h; Negative = untreated, CI-994 =
40µM, Panobinostat
supplementary info).
= 30 nM (For fold change in acetylation levels see
We functionalized CI-994 from the acetyl group of the phenyl
moiety (Fig 2, see supplementary information for complete
synthesis) as the acetyl group of an analogous benzamide
inhibitor is protruding from the catalytic active site and surface
exposed in a crystal structure bound to HDAC 2.14 Hence,
functionalization at this position should maintain HDAC binding.
A short alkyl linker length was prepared (six carbon) and a
longer linker length prepared (twelve carbon) as in previous
PROTAC studies it has been shown the linker length can play an
essential role in inducing degradation.15 Alkyl linkers were
chosen to hasten synthesis. Two different E3 ligands were also
chosen; the von Hippel-Lindau (VHL) ligand and the cereblon
ligand, since successful protein-protein engagement with an E3
ligase is a critical step in promoting degradation.10
We evaluated compounds 1-4 in vitro using an established
fluorescent deacetylase assay with a purified ternary LSD1-
CoREST-HDAC1 complex.16 This complex was used as an
exemplar of an HDAC multi-protein complex to help determine
if heterobifunctional molecules can still engage with the HDAC
enzyme when incorporated into a multi-protein entity. The IC50
2 | J. Name., 2012, 00, 1-3
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With confirmation that 2 and 4 decrease HDAC activity both in
vitro and in cells, we proceeded to quantify HDAC 1, 2 & 3
protein abundance. In ES cells approx. 50% degradation was
observed, however, degradation was even more prominent in
human colon cancer cell line HCT116. After a 24 hour treatment
degradation was observed in a dose-dependent manner with 4
in HCT116 (Fig 4A). PROTAC 4, VHL-based, was a more effective
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DOI: 10.1039/D0CC01485K
degrader than the cereblon-based PROTAC
2
(see
supplementary info Fig S5). HDAC 1 & 2 underwent near
complete degradation at 10 µM with 4, while HDAC 3 levels
were also decreased, but to a lesser extent. At 1 µM of 4
approximately 50% degradation was observed for HDAC 1, 2 &
3, whilst even at 10 nM of 4 HDAC 1 & 2 levels were still reduced
below that of controls. Levels of Histone 3 Lysine 9 acetylation
(H3K9Ac), another established residue for HDAC activity,18 were
also determined in HCT116 cells after a 24 hour treatment with
4. At 10µM of 4 acetylation levels were highly elevated
compared to the DMSO control (Fig 4B), consistent with
decreased HDAC 1, 2 & 3 levels at this concentration (Fig 4A).
Figure 5. Flow cytometry was used to determine the viability of HCT116 cells
following treatment with compound 4 and CI-994 for the indicated times and
concentrations. Error bars represent standard deviation of n=3 biological
replicates
Figure 4. HDAC 1, 2 & 3 degradation occurs in a dose dependent manner with 4.
A) Western blot showing protein levels of HDAC1, 2 & 3 and α-tubulin in HCT116
cells following 24 hours of the indicated treatments. Numerical values represent
percentage of protein compared to DMSO control = 100% B) Histone H3 Lys9
acetylation levels in HCT116 cells following 24 hours of the indicated treatment.
C) The inactive diastereoisomer VHL ligand in 6 does not induce degradation. CI-
994 = 40µM, Panobinostat = 30 nM
To confirm HDAC degradation was occurring via a VHL mediated
proteasome degradation pathway we synthesised 6 with the
inactive diastereoisomer VHL ligand (Fig 4C). No degradation of
HDAC 1 & 2 was observed with 6 when compared side-by-side
with PROTAC 4 confirming degradation is occurring via a VHL-
mediated E3-ubiquitin ligase pathway.
As the most effective of our four PROTAC compounds, we
tested the effects of 4 on cell cycle and cell viability of HCT116
cell lines using flow cytometry. After a 48 hour treatment we
observed significant cell death at 10µM of 4 (Fig 5). At 40µM cell
death after 48 hours was at similar levels to CI-994 (78% vs 73%
respectively). Although the effect of 4 on cell viability was
comparable to CI-994 it is important to note that the IC50 of 4
against the LSD1-CoREST-HDAC1 complex in vitro was 16.8 µM
compared to 0.5 µM for CI-994 (Fig 3A), with near complete
HDAC 1, 2 & 3 degradation being observed with 4 in this cell line
at 10 µM. Hence, the effects of 4 at 10 µM are most likely due
to the absence of HDAC 1, 2 & 3 by a degradation mechanism
rather than enzyme inhibition.
The PROTAC approach has been applied to a number of protein
targets, yet, importantly, not all proteins are as amenable to
degradation. This has been observed with PROTACs based on
non-selective pan-kinase inhibitors.19 Non-complex forming,
cytoplasmic localized HDAC 6 has also recently been identified
as a preferential target for degradation with hydroxamic acid
based PROTACs.20 In this study, with the synthesis of four
heterobifunctional molecules, we have demonstrated the
feasibility of targeted degradation of HDAC 1, 2 & 3. These
enzymes are localized in the nucleus and present in large multi-
protein corepressor complexes. PROTAC 4 produces near
complete degradation of HDAC 1, 2 & 3, with an associated
increase in global acetylation levels and a loss of cell viability in
colon cancer cells. Compounds 1 and 3 despite being a lower
molecular weight and more potent HDAC inhibitors in vitro than
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
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14 B.E L. Lauffer, R. Mintzer, R. Fong, S. Mukund, C. Tam, I.
2 and 4 against the CoREST complex, failed to alter histone
acetylation levels in cells by inhibition or degradation. It is
tempting to speculate that the increased hydrophobicity of
PROTACs 2 and 4 enhances their cell permeability or nucleus
localisation in cells. This may account for the discrepancy in
HDAC activities in vitro compared to in cells between 1 & 3 and
PROTACs 2 & 4. It seems likely that the linker length and
physiochemical properties of Class I HDAC PROTAC linkers will
have a profound effect on their activity in cells. Class I HDAC
degraders, such as reported in this manuscript, offer an
important alternative strategy to inhibition in targeting HDAC
corepressor complexes. Such degraders have potential in the
development of novel therapeutics in cancer and other diseases
related to Class I HDACs.
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Zilberleyb, B. Flicke, A. Ritscher, G. Fedorowicz, R. Vallero,
DOI: 10.1039/D0CC01485K
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Conflicts of interest
There are no conflicts to declare.
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