PROTAC-mediated crosstalk between E3 ligases

Article abstract of DOI:10.1039/c8cc09541h

Small-molecule heterobifunctional degraders can effectively control protein levels and are useful research tools. We assembled proteolysis targeting chimeras (PROTACs) from a cereblon (CRBN) and a von-Hippel-Lindau (VHL) ligase ligand and demonstrated a PROTAC-induced heterodimerization of the two E3 ligases leading to unidirectional and efficient degradation of CRBN.

Full text of DOI:10.1039/c8cc09541h

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and M. Gütschow, Chem. Commun., 2019, DOI: 10.1039/C8CC09541H.
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DOI: 10.1039/C8CC09541H
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PROTAC-mediated crosstalk between E3 ligases
Christian Steinebach,‡a Hannes Kehm,‡b Stefanie Lindner,b Lan Phuong Vu,a Simon Köpff,b Álvaro
López Mármol,c Corinna Weiler,a Karl G. Wagner,c Michaela Reichenzeller,b,d Jan Krönke*,b and
Michael Gütschow*,a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Rsc.li/chemcomm
Small-molecule heterobifunctional degraders can effectively
control protein levels and are useful research tools. We assembled
proteolysis targeting chimeras (PROTACs) from a cereblon (CRBN)
and a von-Hippel-Lindau (VHL) ligase ligand and demonstrated a
PROTAC-induced heterodimerization of the two E3 ligases leading
to unidirectional and efficient degradation of CRBN.
Previous work:
N
OH
H
O
N
S
O
O
H
N
O
N
N
O
N
H
4
S
O
O
O
N
H
O
HO
N
O
CM11
E3 ubiquitin ligases belong to a large protein family vitally
involved in proteostasis. Ubiquitin-dependent protein
degradation relies on the orchestrated crosstalk of ubiquitin-
activating (E1), conjugating (E2) and ligating enzymes (E3).
Among the variety of human E3 ligases, cereblon (CRBN) and
von-Hippel-Lindau (VHL) have received particular attention for
the development of proteolysis targeting chimeras (PROTACs).
Both ligases belong to the family of cullin-RING E3 ubiquitin
ligases (CRLs).1,2 CRBN serves as the substrate adaptor for the
CRL4 E3 ubiquitin ligase (CRL4CRBN) and is the primary target of
immunomodulatory imide drugs (IMiDs).3 IMiDs enhance the
binding of the lymphoid transcription factors Ikaros (IKZF1)
O
N
HN
O
O
HN
O
NH
O
O
NH
15a
N
O
O
This work:
OH
O
N
Linker
N
H
HN
O
O
O
S
N
HN
O
H
O
N
N
O
and Aiolos (IKZF3) to CRL4CRBN
, thereby facilitating the
Fig. 1 Homo- and heterobifunctional E3 ligase degrader.
proteasomal degradation of these neo-substrates.4 VHL is part
of the CRL2VHL complex and mediates the degradation of its
substrate HIF-1α.5 PROTAC technology targeting several
therapeutically relevant proteins has gained enormous
momentum6 and has recently also been introduced for the
self-degradation of the E3 ligases VHL and CRBN by homo-
PROTACs. These are bivalent molecules and consist of two
identical, linker-connected ligands (Fig. 1).7,8
In this work, we designed a series of heterodimeric molecules,
out of which each PROTAC can tether two different ligases, i.e.
CRBN and VHL. The formation of heteroternary (1:1:1)
complexes of both ligases and the PROTAC can lead to the
cross-ubiquitination and subsequent depletion of both or to a
favoured degradation of either ligase. In the second case, one
ligase would act as the hijacked enzyme and the other would
represent the target for degradation. The crosstalk of two
ligases as part of a heteroternary complex would be influenced
e.g. by the presentation of a target’s lysine residues and their
availability for the cullin domain of the hijacked enzyme.
PROTACs are expected to be useful chemical probes to
elucidate ligase specificities in such an induced cellular
scenario. CRBN, the primary IMiD target, is a protein of strong
clinical relevance.9 Thus, small molecules able to execute the
chemical knockdown of CRBN represent valuable tool
compounds for inducing a cellular state of inactivated CRBN.
Assuming a preferred ubiquitination of CRBN, the envisaged
a. Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der
Immenburg 4, 53121 Bonn, Germany.
b. Department of Internal Medicine III, University Hospital Ulm, Albert-Einstein-Allee
23, 89081 Ulm, Germany.
c. Pharmaceutical Institute, Pharmaceutical Technology, University of Bonn,
Gerhard-Domagk-Straße 3, 53121 Bonn, Germany.
d. DKFZ, Molecular Genetics, Im Neuenheimer Feld 260, 69120 Heidelberg, Germany
† Electronic Supplementary Information (ESI) available: Supplementary Table S1,
Supplementary Figures S1-S10; biological, chemical and physicochemical methods;
synthetic schemes and procedures; structures, 1H NMR, 13C NMR and MS data.
See DOI: 10.1039/x0xx00000x
‡ These authors contributed equally.
This journal is © The Royal Society of Chemistry 2018
Chem. Commun., 2018, 00, 1-3 | 1
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hetero-PROTACs might be efficient degraders of this ligase. In Table 1 Synthesized orthogonally protected N − C linkers
View Article Online
contrast to CRL4CRBN addressing homo-PROTACs, productive
ternary complexes can be formed even at low CRBN
concentrations and complete cereblon degradation can be
expected. Treatment with hetero-PROTACs could impair binary
target-PROTAC complexes and thus the accompanying,
unwanted degradation of neo-substrates. By following these
considerations and using the example of CRBN and VHL, we
adjusted the classical paradigm of heterobifunctional PROTACs
and established a novel strategy of targeting two different
ligases and selectively degrading one of them.
The chemical crosslink between the CRBN- and VHL-ligand
necessitated orthogonally protected amino (N) to carboxylic
acid (C) linkers. We developed a new approach to obtain the
desired linker structures by assembling three building blocks,
i.e. dihaloalkanes, diols and aliphatic bromocarboxylic acids
(Scheme 1). The reaction sequence of the synthesis was crucial
insofar the base-promoted Williamson ether formations had to
be executed prior to the introduction of the N-terminal
carbamate moiety through Gabriel synthesis and Cbz-
protection. We obtained eight unique linkers (L1-L8, ESI†) of
different lengths and composition (Table 1) equipped with
terminal moieties compatible with the subsequent steps of the
linear synthesis. The consecutive hemi-deprotection at the N-
terminus and a nucleophilic aromatic substitution of 4-
fluorothalidomide derivatives led to linker-conjugated CRBN
ligands. Subsequent deprotection of the tert-butyl ester and
coupling to a VHL ligand10 yielded the envisaged eight CRBN-
VHL hetero-PROTACs (Table S1, ESI†).
DOI: 10.1039/C8CC09541H
Structure
Codea
O
2-2-2
CbzHN
O
O
O
O
O
2-2-2-2
2-2-2-5
O
O
CbzHN
CbzHN
O
O
O
O
O
O
O
CbzHN
CbzHN
CbzHN
CbzHN
CbzHN
O
6
O
5
6-5-5
6-6-6
O
4
O
O
6
O
6
O
5
O
O
4
O
4
O
O
4-4-4-6
6-(2)5-6
6-(4)3-6
4
5
O
O
6
O
2
O
O
O
O
2
O
2
2
2
5
O
O
6
O
4
O
O
O
4
4
5
a Represents the number of carbon atoms between heteroatoms.
and Fig. S2, ESI†). Thus, it could be concluded that these
compounds are cell permeable, able to form heteroternary
complexes and to induce target ubiquitination. CRBN protein
levels were quantified and normalized to those of tubulin
(Table 2). CRBN-6-5-5-VHL, CRBN-6-6-6-VHL, and CRBN-4-4-4-
6-VHL were identified as particularly potent CRBN degraders
(Fig. 2). Noteworthy, they showed a negligible effect on the
degradation of the neo-substrates IKZF1 and IKZF3 (Fig. 2) and
were superior to the CRBN homo-PROTAC 15a (Fig. 1)8 with
respect to CRBN depletion and avoidance of neo-substrate
degradation. CRBN-6-5-5-VHL had a DC50 value of 1.5 nM, and
The synthesized compounds, at concentrations lower than
10 µM, did not cause toxic effects on K562 cells and the
myeloma cell line MM1S (Fig. S1, ESI†). To investigate the
capability of the PROTACs for ligase degradation, MM1S cells
were treated with each compound and western blot analyses
were performed with CRBN and VHL specific antibodies. No
reduction of the VHL protein level was observed after a 24 h
treatment with any of the compounds at a concentration
range between 10 nM and 100 nM (Fig. 2 and Fig. S2, ESI†).
However, CRBN levels were decreased in most cases (Fig. 2
treatment with
1 µM for 24 hours induced complete
degradation of CRBN, whereas for up to 10 µM no changes in
the VHL protein levels were observed (Fig. S3, ESI†). In a time
course experiment with 100 nM PROTAC, a more effective
CRBN depletion by CRBN-6-5-5-VHL was observed as
compared to 15a (Fig. S4, ESI†). These data exemplified the
desirable features of the CRBN-VHL hetero-PROTACs as non-
toxic, powerful and selective PROTACs for CRBN.
HO
O
OH
m
n o
Three CRBN-VHL hetero-PROTACs with 8-14 linker atoms were
much weaker CRBN degraders, in comparison to the other
compounds listed in Table 2. To draw structure-degradation
relationships, polar surface area, lipophilicity and linker length
have been taken into account. Activity was assessed for our
PROTACs with TPSA values breaking beyond 140 Å2, confirming
that this limit is not valid for cellular PROTAC activity.11 For
example, a high TPSA value in CRBN-6-(2)5-6-VHL was well
tolerated. Partition coefficients were experimentally
determined by an HPLC method. As expected, a higher C/O
ratio of linker atoms resulted in a higher elogP value.
Noteworthy, within this small series of related compounds, a
relationship between PROTAC activity and elogP value was
obvious and active compounds exhibited elogP values larger
than 4.5 (Table 2).
O
Cl
O
I
Br
6
OH
p
O
Linker
O
N
O
H
Cereblon
ligand
VHL
ligand
OH
O
N
Linker
N
H
HN
O
O
R
X
O
S
N
H
N
O
N
N
O
Scheme 1 Synthesis of CRBN-VHL hetero-PROTACs.
2 Chem. Commun., 2018, 00, 1-3
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Table 2 Overview on physicochemical properties anVdiedweAgrtricaledaOtniloinne
DOI: 10.1039/C8CC09541H
potency of CRBN-VHL hetero-PROTACs
Linker
atoms
c
Compound
TPSAa
elogPb DCRBN
CRBN-2-2-2-VHL
CRBN-2-2-2-2-VHL
CRBN-2-2-2-5-VHL
CRBN-6-5-5-VHL
CRBN-6-6-6-VHL
CRBN-4-4-4-6-VHL
CRBN-6-(2)5-6-VHL
CRBN-6-(4)3-6-VHL
8
226
235
235
226
226
235
263
244
3.7
3.8
4.0
5.1
5.3
5.0
4.7
5.5
> 80%
> 80%
> 80%
< 10%
< 10%
< 10%
< 20%
< 20%
11
14
18
20
21
28
28
Fig. 2 CRBN-VHL hetero-PROTACs induce degradation of CRBN, but not of VHL.
MM1S cells were treated for 24 h at the indicated concentrations (µM). For
comparison, pomalidomide (POM) and the CRBN homo-PROTAC 15a8 were used.
The CRBN non-binding negative control (-)CRBN-6-5-5-VHL is not capable of
CRBN degradation.
The distance between the two terminal moieties is a critical
factor for PROTAC effectiveness.12 An apparent lower limit cut-
off of 12 linear non-H linker atoms was recommended.11,13 In
this study, a linker length of 8 to 14 atoms appears less
consistent with the formation of a productive ternary complex.
We considered the linker length and the lipophilicity, rather
than the polar surface area to be activity-determining factors
for our CRBN-VHL hetero-PROTACs.
a
b
Topological polar surface area given in Ų. Experimental partition coefficient.
c CRBN degradation indicated as remaining CRBN levels after 24 h treatment with
100 nM of each compound.
Several CRBN-6-5-5-VHL analogues have been designed to
serve as negative controls (Fig. S5, ESI†). We incorporated D-
tert-leucine instead of the L-isomer and reversed the stereo-
chemistry at C-4 of the hydroxyproline unit, a modification
attenuating the affinity to VHL.7 This compound, unable to
simultaneously bind VHL and CRBN, lost CRBN-degrading
capacity, but exhibited IMiD activity and provoked IKZF1 and
IKZF3 degradation. At the CRBN addressing part, the
glutarimide portion was either N-methylated or its critical C=O
group was removed.14 In the fourth negative control, both
ligase binding moieties were rendered inoperative. The three
latter analogues did not affect CRBN, IKZF1 or IKZF3 levels.
Next, monomeric ligands as competitors of ternary complex
formation were included. Pomalidomide (POM) served as the
component for CRBN-targeting homo- and hetero-PROTACs (Fig. 1).
Co-treatment of an excess of POM with 15a or CRBN-6-5-5-VHL
caused an impaired capability for CRBN degradation (Fig. 3, lane 4
vs 5 and lane 6 vs 7). Similarly, the VHL ligand VH298,15 whose
structural features have been employed for the homo-PROTAC
CM11 and our CRBN-VHL hetero-PROTACs (Fig. 1), prevented
the CM11-induced degradation of the 30 kDa VHL isoform (Fig.
3, lane 10 vs 11). Co-treatment of CRBN-6-5-5-VHL and VH298
abrogated CRBN degradation and depleted IKZF1 and IKZF3 (lane 6
vs 8). In line with the chemical competition, genetic VHL knockdown
prevented CRBN degradation in CRBN-6-5-5-VHL-treated cells (Fig.
S6, ESI†). These data clearly indicated that the CRBN degradation
relies on requisite heteroternary complexes consisting of VHL (E3
ligase), hetero-PROTAC (degrader) and CRBN (target).
Next, we examined whether CRBN-VHL hetero-PROTACs cause
resistance of multiple myeloma cells to IMiDs. A single treatment of
100 nM CRBN-6-5-5-VHL had sustainable effects on MM1S cells, as
CRBN levels below 50% were maintained for more than 48 h (Fig.
S8, ESI†). Consistently, CRBN-6-5-5-VHL was capable to protect or
rescue MM1S cells from IMiD toxicity by preventing induction of
IKZF1 and IKZF3 degradation (Fig. 4, Fig. S9, ESI†). The broad
applicability of CRBN-6-5-5-VHL was further confirmed by
examining CRBN levels in different cell lines (Fig. S10, ESI†).
E3 ligases comprise a highly diverse group of enzymes with
around 700 members. Their general biology and chemistry are
complex and not well understood. Although drugging E3
ligases has been considered a particularly promising approach
in medicinal chemistry,6,16 the development of low molecular
To address the expected involvement of the ubiquitin-
proteasome system, MM1S cells were incubated with the
proteasome inhibitor MG132 and the neddylation-activating
enzyme inhibitor MLN4924. Both inhibitors prevented CRBN
degradation induced by either CRBN-6-5-5-VHL or 15a (Fig. S7,
ESI†) demonstrating that it occurred through the ubiquitin-
proteasome pathway.
Fig. 3 Impact of PROTACs on CRBN degradation can be abrogated by excess
competitive ligands. MM1S cells were treated with ligands 10 µM POM, 100 µM
VH298, 100 nM PROTAC or combinations of those for 24 h
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Fig. 4 Impact of CRBN-6-5-5-VHL, POM or lenalidomide (LEN) on cell viability in the
IMiD-sensitive cell line MM1S. Cell viability was analyzed after 96 h. Arrow indicates
that cells were pre-treated with PROTACs (100 nM) or IMiDs (1 µM) for 3 h, followed by
combined treatment period for 4 days. Error bars express the standard errors of the
mean from three biological replicates. ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.
weight inhibitors is complicated as E3 ligases do not provide a
well-defined and catalytically active pocket.1,17 The knowledge
about protein-protein interactions between E3 ligases and
their substrates can be utilized in the design of small molecule
ligands, as successfully demonstrated in case of VHL.18 In our
approach towards CRBN-VHL hetero-PROTACs, we have
assembled degrader molecules with ligands exhibiting similar
affinities for their corresponding ligases.10,19 Unexpectedly, we
observed degradation of only one E3 ligase, i.e. CRBN, but not VHL,
possibly caused by an unrequited ubiquitin transfer. The VHL form
in the CRL2-VHL ubiquitin ligase complex might be protected from
ubiquitination; similar observations were made with the VHL homo-
PROTACs.7 This implies that the CRL2-VHL may be utilized to also
effectively target other E3 ligases than CRBN without being
ubiquitinated and degraded itself. Having in mind the diversity of
the E3 ligase family, a variety of appropriate ligand combinations
might be implemented for future hetero-PROTACs.20 Such tailored
compounds are expected to degrade elected ligases and possess
potential therapeutic value.
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DOI: 10.1039/C8CC09541H