Iterative Design and Optimization of Initially Inactive Proteolysis Targeting Chimeras (PROTACs) Identify VZ185 as a Potent, Fast, and Selective von Hippel-Lindau (VHL) Based Dual Degrader Probe of BRD9 and BRD7

Article abstract of DOI:10.1021/acs.jmedchem.8b01413

Developing PROTACs to redirect the ubiquitination activity of E3 ligases and potently degrade a target protein within cells can be a lengthy and unpredictable process, and it remains unclear whether any combination of E3 and target might be productive for degradation. We describe a probe-quality degrader for a ligase-target pair deemed unsuitable: the von Hippel-Lindau (VHL) and BRD9, a bromodomain-containing subunit of the SWI/SNF chromatin remodeling complex BAF. VHL-based degraders could be optimized from suboptimal compounds in two rounds by systematically varying conjugation patterns and linkers and monitoring cellular degradation activities, kinetic profiles, and ubiquitination, as well as ternary complex formation thermodynamics. The emerged structure-activity relationships guided the discovery of VZ185, a potent, fast, and selective degrader of BRD9 and of its close homolog BRD7. Our findings qualify a new chemical tool for BRD7/9 knockdown and provide a roadmap for PROTAC development against seemingly incompatible target-ligase combinations.

Full text of DOI:10.1021/acs.jmedchem.8b01413

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Article
Iterative design and optimization of initially inactive Proteolysis Targeting
Chimeras (PROTACs) identify VZ185 as a potent, fast and selective von
Hippel-Lindau (VHL)-based dual degrader probe of BRD9 and BRD7
Vittoria Zoppi, Scott J Hughes, Chiara Maniaci, ANDREA TESTA, Teresa Gmaschitz, Corinna
Wieshofer, Manfred Koegl, Kristin Riching, Danette L. Daniels, Andrea Spallarossa, and Alessio Ciulli
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01413 • Publication Date (Web): 12 Dec 2018
Downloaded from http://pubs.acs.org on December 12, 2018
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Iterative design and optimization of initially inactive
Proteolysis Targeting Chimeras (PROTACs)
identify VZ185 as a potent, fast and selective von
Hippel-Lindau (VHL)-based dual degrader probe of
BRD9 and BRD7
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1,3†
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Vittoria Zoppi , Scott J. Hughes , Chiara Maniaci , Andrea Testa , Teresa Gmaschitz ,
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Corinna Wieshofer , Manfred Koegl , Kristin M. Riching , Danette L. Daniels , Andrea
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Spallarossa , Alessio Ciulli
1
Division of Biological Chemistry and Drug Discovery, School of Life Sciences, James Black
Centre, Dow Street, University of Dundee, DD1 5EH, Dundee, United Kingdom
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Dipartimento diFarmacia, Sezione di Chimica del Farmaco e del Prodotto Cosmetico, Viale
Benedetto XV 3, 16132 Genova, Italy
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Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life
Sciences, University of Dundee, James Black Centre, Dow Street, Dundee, Scotland, DD1 5EH,
United Kingdom
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Boehringer Ingelheim RCV GmbH & Co KG, 1221, Vienna, Austria
5
Promega Corporation, 2800 Woods Hollow Road, Madison, Wisconsin 53711, United States
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KEYWORDS: PROTACs, protein degradation, E3 ubiquitin ligases, bromodomain, von Hippel-
Lindau, BRD7, BRD9
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ABSTRACT
Developing PROTACs to redirect the ubiquitination activity of E3 ligases and potently degrade a
target protein within cells can be a lengthy and unpredictable process, and it remains unclear
whether any combination of E3 and target might be productive for degradation. We describe a
probe-quality degrader for a ligase-target pair deemed unsuitable: the von Hippel-Lindau (VHL)
and BRD9, a bromodomain-containing subunit of the SWI/SNF chromatin remodeling complex
BAF. VHL-based degraders could be optimized from suboptimal compounds in two rounds by
systematically varying conjugation patterns and linkers, and monitoring cellular degradation
activities, kinetic profiles, and ubiquitination, as well as ternary complex formation
thermodynamics. The emerged structure-activity relationships guided the discovery of VZ185, a
potent, fast and selective degrader of BRD9 and of its close homolog BRD7. Our findings
qualify a new chemical tool for BRD7/9 knockdown, and provide a roadmap for PROTAC
development against seemingly incompatible target-ligase combinations.
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INTRODUCTION
Targeted protein degradation is an emerging strategy to use small molecules to knockdown a
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,2
protein by hijacking the ubiquitin-proteasome system. PROTACs (PROteolysis TArgeting
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Chimeras) are bifunctional degrader molecules composed of a ligand for the target protein and a
3,4
ligand for E3 ligase recruitment, connected by a linker. Upon formation of a ternary complex
5
–7
target:degrader:E3, the protein of interest is ubiquitinated and degraded by the proteasome.
Compared to target blockade, post-translational protein degradation, more closely phenocopies
genetic approaches to target validation, and can lead to a more sustained cellular effect with
more extended duration of action. An attractive feature of PROTACs is their catalytic mode of
action, as any one molecule may perform multiple rounds of target ubiquitination and
8
degradation. A consequence of this feature is that degraders can function at sub-stoichiometric
receptor occupancies, meaning they exhibit degradation activities at concentrations that can be
orders of magnitudes lower than their binary dissociation constants (K ) from the target,
d
9
alleviating the requirement for full target engagement. Moreover, PROTAC molecules can add a
layer of target selectivity beyond that expected from the constitutive binding ligands, thus
5
,10–12
providing highly selective degraders with reduced off-target effect.
The mounting interest in
PROTAC drug discovery is also motivated by the promise to target proteins considered
1
3
“
undruggable” via conventional medicinal chemistry approaches. To date, different target
classes have been successfully degraded, including epigenetic targets such as bromodomain-
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,10,11,14–17
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containing proteins BRD2, BRD3, and BRD4,
BRD9, TRIM24, SIRT2,
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PCAF/GNC5, protein kinases,
nuclear receptors
and E3 ubiquitin ligases to self-
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degrade.
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To fulfill the potential of targeted protein degradation, a general methodology for an efficient
PROTAC design would be desirable. However, the development of active PROTAC degraders is
often a laborious and unguided process. The choice of E3 ligase as well as the selection of target
ligands and their conjugation are all potential optimization variables that expand the chemical
space to be exploited by medicinal chemists. Properties of the linker, such as length, composition
and site of attachment, are known to be important but often their impact on activity vary in a
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target- and context-dependent fashion.
Moreover, small-molecule binders for both the
protein of interest and the E3 ligase are required. Despite the large number of human E3
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ubiquitin ligases postulated to function in cells, only a few have good-quality ligands that have
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4
been successfully used for PROTACs. The most common ligases recruited are the von Hippel-
VHL
CRBN
Lindau (VHL) protein complex CRL2
and the cereblon (CRBN) complex CRL4
. Studies
have shown that PROTACs made of the same target ligand but either VHL or CRBN ligands can
1
1,14,18,22
exhibit different degradation selectivity and efficacy.
In some systems, CRBN-based
degraders show a more active profile than VHL-based molecules. Potential greater flexibility of
CRBN
VHL
the Cullin4 based CRL4
compared to CRL2 has been invoked to suggest more productive
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,32,35
ubiquitination of the accessible lysine residues on the target protein.
These observations
would suggest that the development of VHL-based degraders might require more exploration in
the PROTAC design than those based on CRBN. Even if degradation of a given target protein
can be readily obtained by recruiting one E3 ligase, emerging evidence suggests that it could be
beneficial to develop a parallel chemical series hijacking other E3 ligases. For example, chemical
liabilities on a particular ligase ligand could be readily circumvented by switching to a different
compound. The hijacked E3 ligase expression and intrinsic activity may be context-dependent,
3
6
and vary widely amongst different cell and tissue types. Furthermore, resistance mechanisms
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could potentially arise from the loss of the hijacked E3 ligase, as demonstrated by the correlation
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7
between level of CRBN and response to CRBN-recruiting drugs in multiple myeloma.
Switching the hijacked ligase can thus aid targeted protein degradation. It however remains
unclear whether optimal target-E3 pairs exist, or indeed whether any combination of E3 ligase
and target protein might be tractable.
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Here, we demonstrate the development of probe-quality PROTACs for a ligase-target pair
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previously considered incompatible: VHL and the protein BRD9. BRD9 and its close homolog
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8
BRD7 (85% sequence identity ) are bromodomain-containing subunits of the BAF (BRG-
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/BRM-associated factor) and PBAF (Polybromo-associated BAF) complexes, respectively.
BAF and PBAF represent two variants of the SWI/SNF complex, one of the four mammalian
ATP-dependent chromatin remodeling complexes. The SWI/SNF complexes control gene
expression, DNA replication and DNA repair by modulating access to promoters and coding
41–43
regions of DNA through modification of the degree of compactness of chromatin.
Mounting
evidence from genetics and sequencing of cancer-associated mutations have spurred efforts to
unravel yet largely elusive physiological roles of BAF/PBAF subunits and to develop targeted
3
9
therapeutics in cancer and other human diseases. In particular, BRD9 is overexpressed in
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several malignancies, such as cervical cancer and in non-small cell lung cancer (NSCLC).
In
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contrast, BRD7 gene has been proposed as candidate tumor suppressor gene,
as it regulates
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breast cancer cell metabolism, and acts as negative regulator of aerobic glycolysis essential for
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tumor progression. BRD7 also promotes X-box binding protein 1 (XBP1) nuclear translocation,
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which prevents the development of insulin-resistance disorders. In contrast to these roles, it has
been recently shown that inactivation of the BRD7 gene sensitizes tumor cells to T cell-mediated
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killing, suggesting that knockdown of BRD7 could be an attractive target for cancer
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immunotherapy. Potent and selective inhibitors that bind to the BRD7/9 bromodomains have
recently emerged from structure-guided medicinal chemistry campaigns, including compounds I-
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BRD9, LP99, ketone “compound 28”, BI-7273 and BI-9564, (1a-b, Figure 1), and GNE-
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75. These BRD7/9 inhibitors have been used in cells to help clarify the roles of the BRD7/9
bromodomain in oncogenesis and other disease states. For example, pharmacological studies of
inhibitors 1a and 1b in combination with domain-swap protein engineering revealed that an
active bromodomain of BRD9 is required to sustain MYC transcription and proliferation of
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leukemic cells.
These findings and availability of bromodomain ligands prompted us to
initiate a PROTAC medicinal chemistry campaign to target BRD7 and BRD9 proteins for
degradation.
RESULTS AND DISCUSSION
First generation of BRD7 and BRD9 degraders. We began our investigation by designing a
small set of PROTACs aimed to induce BRD7/9 degradation by recruiting three different E3
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ubiquitin ligases: VHL, CRBN and DCAF15.
We aimed to leverage available E3 ligase
ligands, and to maximize the opportunity for complementary surfaces between the bromodomain
and the ligase within the ternary complex. As BRD7/9 bromodomain ligands, we selected
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compounds 1a-b (Figure 1), on the basis of their high binding affinity, and of their superiority
5
8
as BRD9 chemical probes over other ligands. To design the first generation of degraders, we
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inspected the crystal structure of 1a bound to BRD9 (PDB code: 5EU1) to identify suitable
attachment points and vectors for linker conjugation, important considerations for PROTAC
1
1,29
design as known to greatly influence degradation activities.
The dimethylamine group of the
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molecule was identified as a solvent-exposed group not involved in interactions with the protein.
For synthetic reasons, the dimethylamine group of compound 1a was replaced by a piperazine
group (BrdL1 (1c), Figure 1 and Supporting figure S1), providing a convenient isosteric handle.
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As E3 ligase recruiting moieties, VH032 (2, VHL ligand, Figure 1)
and pomalidomide (3,
CRBN ligand, Figure 1) were selected and modified to afford compounds VHL1 (2a) and 3a,
Figure 1 and Supporting figure S1). The amino terminal group of 2a and 3a were conjugated via
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4
(
amide bond to the linker without perturbing the interaction with the E3 ligases, as previously
8,10,15,22
demonstrated.
To expand the arsenal of E3 ligase ligands being explored, we designed
conjugates containing indisulam (4, Figure 1), a small molecule recently reported to bind to the
DCAF15
E3 ligase DCAF15 and to redirect the activity of the CRL4
complex towards the neo-
59,60
substrate CAPERα (also known as RBM39).
As the binding mode of 4 is not known, we
leveraged information on the activity of a biotinylated photoactive analogue probe to guide our
6
0
conjugation strategy. Accordingly, a para-benzylamine analogue of 4 (derivative 4a, Figure 1
and Supporting figure S1) was designed as conjugatable ligase ligand. To generate a first set of
compounds, we decided to use PEG linkers composed of two or four PEG units to connect the
two warheads (compounds 5-10, Scheme 2).
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N
N
N
R2 R1
O
O
O
N
N
N
O
O
NH
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2
BI-7273 (1a) R =OCH
3
, R =H
BrdL1 (1c)
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BI-9564 (1b) R =H, R =OCH
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OH
OH
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N
N
N
N
H N
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N
H
O
N
H
N
H
O
O
O
S
S
VH032 (2)
VHL1 (2a)
H
2
N
NH
2
O
N
NH
O
O
N
O
NH
NH
O
O
O
O
Pomalidomide (3)
POMA (3a)
Cl
Cl
HN
HN
O
S
O
O
S N
O
H
S N
O
H
O
H
2
N
2
H N
Indisulam (4)
4a
Figure 1. Chemical structures of parent (1a, 1b, 2-4) and modified (1c, 2a-4a) BRD7/9 and E3
ligase ligands. Functional groups selected for conjugation are shown in blue on parent ligands
and in red on modified ligands.
Compounds 5-10 were obtained as reported in Schemes 1 and 2. Briefly, reductive amination
between the commercially available 4-bromo-2,6-dimethoxybenzaldehyde (11) and boc-
piperazine led to the formation of compound 12 which was then cross-coupled with 14 (obtained
by methylation of 13), under the Myaura-Suzuki condition (one-pot two-steps); the cleavage of
tert-butyloxycarbonyl protecting group in acidic conditions afforded 1c in quantitative yield
(
Scheme 1). The primary alcohol functionality of the linkers (15 and 16) was oxidized to
aldehyde group using a Swern reaction and condensed with the terminal secondary amine of 1c
to afford the tert-butyl ester intermediates 17 and 18 that were converted into the corresponding
acids by trifluoroacetic acid (TFA) treatment (Scheme 2). Compounds 19 and 20 were then
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1,65
condensed with the conjugatable E3 ligands (2a and 3a prepared as previously described,
and
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a synthesis in Supplementary info-synthetic procedures) using HATU as coupling reagent
yielding final compounds 5-10.
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Scheme 1. Synthesis of the BRD7/9 ligand 1c
Br
Br
a
O
O
O
O
O
N
O
H
N
N
N
Boc
1
1
12
c,d
O
O
I
O
O
HN
N
b
N
N
N
NH
2
+Cl-
I
1c
1
3
14
Reagents and conditions: (a) 1-Boc-piperazine, NaBH(OAc)
b) NaH, CH I, DMF, 0 ºC, 5 h, yield 95%; (c) step 1: 12, B
dioxane, µw 140 ºC, 40 min; step 2: 14, K CO (aq) are added to step 1, µw 120 ºC, 30min, yield
5%; (d) HCl 4M in dioxane, DCM, rt, 1 h, quantitative yield
3
, THF, rt, overnight, yield 97%;
(
3
2
pin , KOAc, Pd(dppf)Cl , 1,4
2
2
2
3
5
Scheme 2. Synthesis of the first generation of degraders.
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
N
N
O
O
O
O
O
a,b
N
O
HO
O
n
N
n
1
1
5 n=2
6 n=4
O
1
1
7 n=2 32%
8 n=4 34%
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
c
O
N
N
O
O
O
N
OH
n
N
O
19 n=2 quant. yield
0 n=4 quant. yield
2
d
2
a
4a
3
a
OH
O
N
N
O
N
N
O
O
O
O
O
O
N
N
N
H
HN
N
N
H
n
H
N
n
N
Cl
N
O
N
H
S
S
O
O
2
O
9
n=2 34%
10 n=4 37%
O
N
5
6
n=2 44%
n=4 30%
O
NH
O
O
O
N
N
O
N
H
N
O
O
O
N
N
H
n
N
O
7
8
n=2 33%
n=4 54%
Reagents and conditions: (a) oxalyl chloride, DMSO, DCM, then TEA, -78˚C to rt, 2 h; (b) 1c,
NaBH(OAc) , TEA, DMF, rt, overnight; (c) TFA, DCM, rt, 2 h; (d) E3 ligand (2a, 3a or 4a),
3
HATU, HOAt, DIPEA, DMF, rt, 2 h
To assess the degradation activity of the first generation of PROTACs, HeLa cells were treated
with compounds 5-10 at fixed concentration of 1 µM, at 4 and 16 h before harvesting (Figure
2
A). CRBN-based PROTACs 7 and 8 demonstrated strong degradation of BRD9 already after 4
h of treatment, whereas no BRD7 degradation was observed (Figure 2A). In contrast, VHL-based
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degraders 5 and 6 showed weak activity against both BRD7 and BRD9, inducing at most 20%
degradation after 4 h and 30% after 16 h, and even weaker activity was observed with indisulam-
based PROTACs 9 and 10 (Figure 2A). siRNA knockdown experiments (Figure 2B) validated
the specificity of the bands observed by western blot.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. Screening of first generation of degraders. A) Western-blot analysis of BRD9 and
BRD7 levels after treatment of HeLa cells with 1 µM of compounds for 4 and 16 h before
harvesting. Degradation activity is reported below each lane as % of protein abundance relative
to 0.1% DMSO vehicle. B) Western-blot analysis of BRD9 and BRD7 levels after 48 h
transfection with 1.25 nM of siRNA targeting respectively BRD7, BRD9 or negative control
(N.C.) siRNA. Intensity values are quantified as described in the experimental section.
18
While this research was underway, Remillard et al. disclosed the BRD9 degrader dBRD9
(21, Figure 3) confirming the strong degradation activity of our CRBN-recruiting PROTACs.
Compounds 7 and 21 share the same target and E3 ligands, which, however, were differently
modified to attach two distinct linkers (Figure 3). CRBN-based PROTACs, however, can exhibit
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
off-target degradation of non-PROTAC-targets such as IKZF1/3 and GSPT1 due to the
1
8,66
neomorphic activity of the CRBN ligand alone.
N
O
O
O
HN
N
HN
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
O
N
O
O
O
O
O
O
N
O
N
O
N
H
N
H
N
O
O
O
N
O
O
N
H
O
O
O
N
H
N
7
dBRD9 (21)
1
8
Figure 3. Structure of compound 7 and 21.
We therefore turned our attention to VHL-based PROTACs. Encouraged by the degradation,
albeit partial, of both BRD7 and BRD9 induced by our initial VHL-based PROTACs, we
decided to characterize the binding of 5 biophysically and structurally. To determine the binding
mode of the conjugated bromodomain ligand, compound 5 was co-crystallized with the BRD9
bromodomain (BRD9-BD). In the BRD9-BD:5 complex, the bromodomain ligand was clearly
observed within its binding pocket, and its binding mode closely recapitulates that of 1a (rmsd:
0
.569 Å) (Figures 4A,B). The alkylated piperazine is favourably accommodated within the
binding site, with the linker directed toward solvent, as desired (Figures 4A,B).
To characterize biophysically the interplay between VCB (VHL-ElonginC-ElonginB
complex), compound 5 and the BRD9 bromodomain, we applied our previously developed ITC
5,11
assay that measures the thermodynamics and cooperativities of ternary complex formation.
Previous work has demonstrated that VHL is capable of forming a highly stable and cooperative
5
ternary complexes - as seen with MZ1-Brd4BD2 (ref. ) and with the homo-PROTAC dimerizer
29
CM11 (ref. ). Titrations of protein into PROTAC alone revealed a binary binding affinity for
VCB (K = 33 ± 2 nM, Figure 4D and Table 1) within two-fold of that measured on previously
d
5
,11
characterized VHL-based BET degrader MZ1 (K = 70 nM, refs. ). Similarly, the binary
d
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affinity (K = 15 ± 3 nM) and binding enthalpy (ΔH = –12 kcal/mol) for BRD9-BD (Figure 4C
d
5
6
and Table 1) was comparable to that of inhibitor 1a, consistent with the conserved binding
modes of 5 and inhibitor 1a observed crystallographically (Figure 4A). The titrations of VCB
into BRD9-BD:5 complex (Figure 4E) showed that VCB binds more weakly to the binary
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
complex BRD9-BD:5 (K = 73 nM) than to compound 5 alone (K = 33 nM), indicating negative
d
d
cooperativity (α < 1, Table 1). Negative cooperativity has previously been observed with
tetrahydroquinoline-based BET PROTACs, where it was demonstrated that linker length and exit
1
1
vector can significantly influence ternary complex formation. Based on these considerations,
we reasoned to explore variations on the linker as well as the point of derivatization.
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1
1
1
1
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Ternary complex formation and analysis of binding mode for compound 5. A) Co-
crystal structure of BRD9-BD and compound 5. The warhead component of the degrader (purple
and cyan; one from each protomer in the ASU) recapitulates the binding of inhibitor 1a
5
6
(
magenta, PDB code 5EUI) , whereas the alkylated piperazine used to attach the linker is
solvent-exposed and does not form any unfavourable interactions with the protein; B) Fourier
difference map (F – F ) of compound 5 (contoured at 2.5 σ) showing electron density for the
o
c
alkylated piperazine ring; C) BRD9-BD titrated into 5 alone; D) VCB titrated into 5 alone; E)
VCB titrated into BRD9-BD:5 binary complex. VCB binds more weakly to the binary complex
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BRD9-BD:5 (K = 73 nM) compared to compound 5 alone K = 33 nM), indicating negative
d
d
cooperativity.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 1. Thermodynamic parameters of formation of binary and ternary complexes between
VCB, BRD9 bromodomain and compound 5 measured by isothermal titration calorimetry (ITC).
ΔH
ΔG
–TΔS
Syringe
Cell
K
d
(nM)
N
α
(
kcal/mol)
(kcal/mol)
(kcal/mol)
0
.753 ±
BRD9-BD
5
15 ± 3
33 ± 2
73 ± 2
–12.5 ± 0.4
–15.2 ± 0.1
–6.4 ± 0.1
–10.7 ± 0.1
–10.2 ± 0.1
–9.7 ± 0.1
1.8 ± 0.5
4.9 ± 0.1
–3.3 ± 0.1
0
.004
.60 ±
.01
.75 ±
.05
0
5
0
VCB
0
BRD9-BD:5
0.45
0
All ITC titrations were performed at 25 °C. Values reported are the mean ± S.E.M. from at least
three independent measurements (n ≥ 3).
Second generation of BRD7 and BRD9 degraders. To improve ternary complex formation
and degradation activity of PROTACs 5 and 6, we explored the impact of varying their
derivatization point, linker length and composition (compounds 22-31, Table 2). First, to expand
the conjugation pattern between VHL1 and BrdL1 (Figure 1), the number of PEG units was
modified (3 units for 24; 5 units for 22), a more lipophilic 11-atoms chain was inserted
(compound 23) and a different attachment to BrdL1 moiety via amide conjugation was explored
(
compound 25) (Table 2). Furthermore, we explored a conjugation vector via a phenolic position
VHL2 (2b), compound 28 and 31, Table 2 and Supporting figure S1), as previously
(
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1
1
1
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1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
29,67
described.
To improve binary binding affinities to VHL and potentially fine-tune
5
intermolecular interactions in the ternary complex, we replaced the VHL2 acetyl group with
either a cyano-cyclopropyl group (VHL3 (2c), derivatives 27 and 30, Table 2 and Supporting
2
9,62
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
figure S1)
or with a fluoro-cyclopropyl group (VHL4 (2d), derivatives 26 and 29, Table 2 and
6
3
Supporting figure S1). As assessed by crystallographic studies (for 2c, PDB code 5LLI; for 2d,
PDB code 5NVX), these modifications accommodate snugly on the VHL protein surface and
6
2,63
enhance binding affinity.
To allow direct comparison with the compounds from the first
series, compounds 26-28 and 29-31 were designed to contain PEG2 and PEG4 linkers,
respectively.
Compounds 22 and 24 were obtained via the same synthetic route used for the first generation
(
synthesis of compounds 22, 24 and 25 detailed in the Supporting Information). The synthesis of
2
3 (Scheme 3) involved the nucleophilic attack of diethylene glycol (PEG2) to 6-bromo-1-
hexene to afford derivative 33 that was oxidized to carboxylic acid 34 by treatment with TEMPO
and bis-acetoxy iodobenzene (BAIB). After coupling with 2a, Lemieux-Johnson oxidation of the
double bond yielded the aldehyde intermediate that was reacted with amine 1c to yield
compound 23 in 54% yield. A similar synthetic strategy was applied for the synthesis of
degraders 26-31 (Scheme 4). Briefly, after nucleophilic substitution of polyethylene glycol
(
PEG2 and PEG4) with allyl bromide, the alcoholic group of 38 and 39 was activated as
mesylate and reacted with the phenolic group present in the VHL ligand scaffold affording
derivatives 40-45. The alkene moiety was then oxidized and the resulting aldehyde derivative
was directly used for the final reductive amination step with 1c to afford the final products 26-
3
1.
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Table 2. SAR of second generation of PROTACs
BRD7/9 ligand
Linkers
E3 ligase ligands
O
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
N
O
O
N
N
BrdL1
*
Structural features and degradation activity of second-generation panel of BRD7/9 PROTACs.
BRD9
16 h
BRD9
4 h
BRD7
4 h
BRD7
16 h
E3 ligase
ligand
Linker length # of O
Code
Linker
(atoms)
atoms
(%)
(%)
(%)
(%)
O
5
6
O
O
O
8
2
4
50
99
89
78
68
83
2
O
14
102
119
4
O
22
23
24
17
11
11
5
2
3
77
43
93
112
61
104
106
107
54
80
5
VHL1
O
O
O
5
O
O
95
106
3
O
O
25
O
11
3
74
73
95
113
2
O
2
2
2
2
6
7
8
9
VHL4
VHL3
VHL2
VHL4
8
8
2
2
2
4
14
50
99
68
36
63
97
88
90
O
187
107
163
2
8
156
97
95
14
110
O
4
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6
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
3
0
1
VHL3
VHL2
14
14
4
4
91
92
98
89
124
98
114
109
*
Degradation activity is reported as % of total protein remaining after 1 µM compound treatment
relative to 0.1% DMSO vehicle as quantified by western blotting of HeLa cell lysates.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 3. Synthesis of compound 23
O
O
a
O
b
O
HO
H
2
HO
HO
O
2
3
2
34 81%
3
3 49%
c
N
N
O
H
N
O
H
N
O
S
N
O
N
O
S
d,e
O
H
H
N
N
O
N
O
N
O
O
N
O
O
O
23 54%
35 30%
N
OH
OH
Reagents and conditions: (a) NaH, 6-bromo-1-hexene, DMF/THF, rt, overnight; (b) BAIB,
TEMPO, ACN/H O, rt, overnight; (c) 2a, HATU, HOAt, DIPEA, DMF, rt, 2 h; (d) OsO 4 wt.%
2
4
in H O, NaIO , pyridine, dioxane/ H O, rt; (e) 1c, NaBH(OAc) , TEA, DMF, rt, overnight
2
4
2
3
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Scheme 4. Synthesis of compounds 26-31
a
O
O
HO
HO
H
n
n
38 n=2 54%
9 n=4 50%
3
6 n=2
37 n=4
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
b, c
2
b-d
N
O
S
S
N
d,e
O
HO
HN
HN
O
HO
n
N
N
O
O
O
O
N
O
N
N
O
O
n
N
O
HN
NH
R
26 2d, n=2 45%
27 2c, n=2 44%
28 2b, n=4 39%
29 2d, n=4 46%
R
40-45
3
3
0 2c, n=2 17%
1 2b, n=4 17%
Reagents and conditions: (a) NaOH, allyl bromide, dioxane, rt, overnight; (b) MsCl, TEA,
DCM, rt, 3h; (c) VHL ligands 2b-d, K CO , DMF, 70 ºC, overnight; (d) OsO , NaIO , pyridine,
dioxane/ H O, rt; (e) 1c, NaBH(OAc) , TEA, DMF, rt, overnight
2
3
4
4
2
3
All second-generation compounds were profiled for BRD9 and BRD7 degradation after
treatment for 4 h and 16 h (Figure 5A and Supporting figures S2,3). The two VHL-based
PROTACs 5 and 6 were included as reference compounds. Derivative 26 demonstrated marked
and selective (around 90%) depletion of BRD9 over BRD7 already after 4 h treatment in HeLa
cells (Figure 5A, Table 2). By comparing and contrasting the degradation profiles of related
compounds, structure-activity relationships began to emerge (Table 2). As expected, the length
of linkers influenced degradation profiles, with more pronounced BRD9 degradation observed
with shorter linkers for given matched pairs (compare 26 and 29; 27 and 30; 5 with 24, 6, and
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2
2). We also observed that for a given linker length, replacement of an oxygen atom with a
methylene group was beneficial to degradation activity (compare 23 vs 24). Amongst conjugates
derivatized at the phenolic position of the VHL ligand, the nature of the terminal capping group
also influenced degradation potency. Within series of conjugates of fixed linker, compounds
bearing the fluoro-cyclopropyl moiety (VHL4) were more potent degraders than those containing
cyano-cyclopropyl (VHL3) and even more so than acetyl group (VHL2) (26 vs 27 and 28; and
0
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2
9 vs 30 and 31, respectively) (Table 2). These trends are consistent with the binary binding
63
affinity values measured for the corresponding VHL ligands. To account for potential different
protein expression levels, compounds 22-31 were tested across a panel of different cancer cell
lines (Supporting figures S2,3). From these degradation screens, 26 was confirmed as the most
active compound, consistently across all assays and cell lines.
Compound 26 was further characterized by profiling its concentration-dependent activity at
early time points (30 min and 4 h before harvesting; Figure 5C and Supporting figure S4).
PROTAC 26 induced preferential degradation of BRD9 over BRD7, resulting in a half-
degrading concentration (DC ) of 560 nM against BRD9 (Figure 5C and Supporting figure S4).
5
0
More than 60% of degradation of BRD9 was observed at 1 µM after 4 h, while maximal
degradation (Dmax around 80%) was reached at 10 µM. At higher doses, the characteristic ‘hook-
effect’ was observed, consistent with 26 acting preferentially as inhibitor over degrader at high
concentrations. Rapid and selective target degradation was confirmed by profiling cellular
activities over time (Figure 5D and Supporting figure S4). Treatments of HeLa cells at fixed
concentration (1 µM) of 26 at varying time points revealed rapid degradation of BRD9 with an
apparent half-live of 3.5 h, resulting in more than 50% degradation already after 4 h treatment,
and achieving the highest level of depletion after 8 h (Figure 5D).
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Figure 5. Compound 26 induces rapid and profound depletion of BRD9 in cells. A) Western-
blot analysis of BRD9, BRD7 and β-actin after treatment of HeLa cells with 1 µM of compounds
for 4 h before harvesting (data reported in Table 2); B) Chemical structure of 26; C)
Quantification of BRD9 protein levels after treatment of HeLa with six different concentration of
2
6 at 30 min and 4 h before harvesting; D) Quantification of BRD9 protein levels after treatment
with 1 µM of 26 in HeLa at the desired time points. Intensity values, DC and half-live were
50
quantified as described in the experimental section.
To determine to what extent a correlation could be seen between ternary complex formation
and the improved degradation properties of 26, we turned to measurements of thermodynamic
parameters and cooperativity by ITC (Figure 6A and Supporting figure S5). Titrating VCB into
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2
6 alone or BRD9-BD:26 complex indicated a cooperativity α = 1, an over two-fold
improvement over 5. At the binary level compared to 5, we observed negligible difference
between 26 and 5 in binding affinity for BRD9-BD, but a slight loss in affinity for VCB (K = 87
d
0
1
2
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9
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2
3
4
5
6
7
8
9
0
±
5 nM for 26 compared to 33 ± 2 nM for 5) (Supporting Table S1).
To confirm these findings, we repurposed an existing fluorescence polarization (FP) assay that
6
8
involves the competitive displacement of a FAM-labelled HIF-1α peptide. By comparing
PROTAC-induced peptide displacement in the presence and absence of BRD9-BD, it is possible
to determine the cooperativity (Figure 6B,C). FP measurements for compound 5 showed a
rightward-shift in the IC curve in the presence of BRD9-BD - indicating negative cooperativity
50
6
3
- whereas compound 26 showed no shift. Back-calculation of K values, produced affinity
d
values that were comparable to those obtained by ITC (Supporting Table S1 and Supporting
figure S5).
Previous studies have demonstrated that the amount of ternary complex formed is well
5
correlated to the cooperativity of the system, at least in the case of VCB. Therefore, we
employed an AlphaLISA proximity assay to determine the effect of increasing cooperativity on
ternary complex formation (Figure 6D). Indeed, there was a 50% increase in the maximum alpha
intensity measured for VCB:26:BRD9-BD compared to VCB:5:BRD9-BD, suggesting that the
increased cooperativity of 26 compared to 5 resulted in increased population of ternary complex.
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9
0
Figure 6. Improved ternary complex formation by series-2 degraders. A) Overlay of
compound 26 titrations shows no difference between binary (VCB into 26 alone; black) and
ternary (VCB into BRD9-BD:26 binary complex; green) titrations; B) Fluorescence polarization
measurements for compound 5 in the presence (red) and absence (black) of BRD9-BD. The
rightward shift in the presence of BRD9-BD indicates negative cooperativity. Each point is mean
±
SEM of at least two individual experiments performed in triplicate; C) Fluorescence
polarization measurements for compound 26 in the presence (green) and absence (black) of
BRD9-BD. Each point is mean ± SEM of at least two individual experiments performed in
triplicate; D) AlphaLISA assay showing increased ternary complex formation for 26 (green)
compared to 5 (red). Each point is mean ± SEM of at least one experiment performed in
quadruplicate.
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Third generation of BRD7 and BRD9 degraders. The pronounced degradation of BRD9
observed with 26 motivated us to design a third generation of PROTACs with the goal to further
optimize our degrader. On the basis of the SAR previously acquired, the design strategy was to
keep VHL4 moiety fixed, and to focus on systematically varying the linker length and
composition, as well as the substitution and conjugation chemistry at the BRD7/9 warhead. The
focus on the linker concentrated on exploring varying ratios between hydrophilic and lipophilic
portions within the context of three different lengths: 5, 8 and 11 atoms (Table 3). We reasoned
that varying the balance between hydrophilicity and lipophilicity might influence the
conformational equilibria and intrinsic folding propensity of the linker, with direct impact on the
process of ternary complex formation as well as potentially cell permeability. Linkers were
connected to four different analogues of BRD7/9 ligands. Guided by the structure-activity
0
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6
relationships of BRD7/9 bromodomain inhibitors developed by Martin et al., we introduced
two structural variations, single or in combination. Firstly, we arranged the methoxy groups on
the phenyl ring in either meta (BrdL1, 1c) or para (BrdL2, 1d) (Table 3 and Supporting figure
S1) relative to each other. Second, we replaced the piperazine moiety with an azetidine group
(BrdL3, 1e and BrdL4, 1f, Table 3 and Supporting Figure S1). For conjugation between the
bromodomain ligand and the linker, either tertiary amines (via reductive amination reactions) or
amide bonds were contemplated. This design yielded a set of 19 new PROTACs (Table 3).
To circumvent the need to use the hazardous reagent osmium tetroxide previously applied in the
Lemieux-Johnson reaction, some key modifications and optimization to the synthesis were made
(Scheme 5 and Supplementary Info-synthetic procedures). Mesylation of the free hydroxy group
of the linkers allowed nucleophilic attack by the phenolic group of 2d (Scheme 5). The lipophilic
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compound 66, instead, was conjugated to 2d via alkylation (Scheme 5). Then, diacetal functional
group on the linker was hydrolysed to aldehyde under acidic conditions to allow reductive
amination with 1c,d (synthesis of 1d is described in the Supporting Information) warheads,
leading to the formation of compounds 46-54. To conjugate the linker to 1e,f via amide bond,
VHL-linker aldehyde intermediates were oxidized to acid through a Pinnick reaction. Then,
HATU-mediated coupling with 1e,f (synthesis described in the Supporting Information) were
applied to obtain the final compounds 55-64 (Scheme 5 and Supplementary Info- synthetic
procedures).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3. SAR of third generation of PROTACs
BRD7/9 ligand
Linkers
E3 ligase ligands
*
Structural features and degradation activity of third-generation panel of BRD7/9 PROTACs.
Linker
length
BRD9-
HeLa 4 h
(%)
BRD9-
RI-1 2 h
(%)
BRD7-
HeLa 4 h
(%)
BRD7-
RI-1 2 h
(%)
E3
# of O
Code
BrdL
Linker
ligand
atoms
(atoms)
O
2
26
BrdL1
8
2
7
68
7
127
VHL4
4
4
6
7
BrdL1
BrdL2
5
5
1
1
0
3
63
94
8
3
63
98
O
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5
5
5
5
5
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O
4
8
BrdL2
8
2
18
125
126
149
2
4
5
5
9
0
1
BrdL1
BrdL2
11
11
3
3
12
36
105
127
182
206
125
134
O
3
0
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1
2
3
4
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7
8
9
0
BrdL1
5
0
14
100
10
16
(
VZ185)
5
5
5
5
5
5
5
5
6
6
6
2
3
4
5
6
7
8
9
0
1
2
BrdL2
BrdL1
BrdL2
BrdL3
BrdL4
BrdL3
BrdL4
BrdL3
BrdL4
BrdL3
BrdL4
BrdL3
BrdL4
5
8
0
1
1
1
1
2
2
3
3
0
0
1
1
53
46
74
27
53
9
65
69
151
98
65
83
O
5
8
89
336
29
102
125
112
92
5
109
99
O
O
5
158
53
8
95
O
O
2
8
59
6
103
89
280
103
138
25
120
98
11
11
5
O
O
3
41
8
95
85
68
70
O
5
2
110
92
54
131
106
97
63
64
8
9
31
O
O
8
39
94
250
*
Degradation activity is reported as % of total protein remaining after 1 µM compound treatment
relative to 0.1% DMSO vehicle as quantified by western blotting of HeLa and RI-1 cell lysates.
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Scheme 5. General synthetic routes for third-generation compounds
OEt
OEt
F
OH
a,b
O
HO
O
N
H
N
O
65
O
NH
O
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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3
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9
0
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7
8
9
0
O
RO
c
Br
4
X
N
O
S
OR
6
6
6
7 R= Et, X=O 87%
68 R=Me, X=CH2 55%
d
F
OH
OH
O
F
N
N
O
H
O
N
H
N
O
O
NH
O
R1
O
R2
NH
O
N
O
N
e
N
O
X
N
1
c -d
S
H
X
N
N
1
2
S
46 R =OCH , R =H, X=O 16%
3
O
1
2
5
1 R =OCH , R =H, X=CH 24%
3 2
6
7
9 X=O
0 X=CH2
1
2
47 R =H, R =OCH X=O 4%
3
,
1
2
52 R =H, R =OCH X=CH 20%
3
,
2
f
F
OH
F
OH
O
N
H
N
O
O
N
H
N
O
O
N
O
NH
O
NH
O
g
O
R2
R1
H
N
N
1
e-f
X
N
HO
N
N
S
X
O
S
O
O
1
2
5
5 R =OCH , R =H, X=O 12%
3
7
7
1 X=O
2 X=CH2
1
2
6
1 R =OCH , R =H, X=CH 18%
3 2
1
2
56 R =H, R =OCH X=O 17%
3
,
1
2
62 R =H, R =OCH X=CH2 23%
3
,
Reagents and conditions: (a) MsCl, TEA, DCM, rt, 3 h; (b) 2d, K CO , DMF, 70 ºC, overnight;
2
3
(
c) K CO , DMF, 70 ºC, overnight; (d) HCl 1 N, THF (1:1), 50 ºC, 2 h; (e) 1c,d, NaBH(OAc) ,
2 3 3
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5
5
5
5
6
TEA, DMF, rt, overnight; (f) Na HPO , NaClO , 2-methyl-2-butene, t-BuOH, H O, rt, 4 h ; (g)
2
4
2
2
1
e,f, HATU, HOAt, DIPEA, DMF, rt, 2 h
0
1
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3
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Degraders 46-64 were screened at 1 µM in HeLa cells following 4 h and 16 h of treatment
Table 3, Figure 7A and Supporting figure S6). A large proportion of compounds induced
(
efficient degradation of both BRD7 and BRD9 in HeLa cells, with more than 10% total protein
remaining after 4 h treatment (Table 3, Figure 7A). Overall, within matched pairs, greater
degradation activity was observed for compounds containing meta (BrdL1 and BrdL3) over para
(BrdL2 and BrdL4) di-methoxy substituents (as representative examples: 46 vs 47, 26 vs 48, 51
vs 52, Figure 7A). To assess consistency of cellular activity, compound screening was repeated
in RI-1 (DLBCL) cells at 2 h treatment (Table 3, Figure 7B). This cell line was chosen because it
5
8
is sensitive to BRD9 inhibition, it is of clinical relevance, and also expresses both VHL and
CRBN ligases (Supporting figure S7). The compounds that induced greatest protein degradation
activity after 2 h were 46 and 52 (on BRD9), and 51 (on BRD7) (Figure 7B). Compounds 46 and
5
1 differ only by one atom in the centre of the linker, which is oxygen or carbon, respectively
(Table 3). Compound 52 is the analogue of 51 but with BrdL2 instead of BrdL1 (Table 3). 46
and 51 showed enhanced degradation of BRD9 compared to 26 and similar levels of degradation
achieved with CRBN-based degrader 7 in RI-1 cells after 8 h treatment. In addition, 46 and 51
achieved PROTAC-induced degradation of BRD7 in addition to BRD9 (Figure 7C).
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Figure 7. Screening of third generation of degraders. A) Western-blot analysis of BRD9 and
BRD7 levels after treatment of HeLa cells with 1 µM of compounds for 4 h before harvesting; B)
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