Homo-PROTACs for the Chemical Knockdown of Cereblon

Article abstract of DOI:10.1021/acschembio.8b00693

The immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide, all approved for the treatment of multiple myeloma, induce targeted ubiquitination and degradation of Ikaros (IKZF1) and Aiolos (IKZF3) via the cereblon (CRBN) E3 ubiquitin ligase. IMiD-based proteolysis-targeting chimeras (PROTACs) can efficiently recruit CRBN to a protein of interest, leading to its ubiquitination and proteasomal degradation. By linking two pomalidomide molecules, we designed homobifunctional, so-called homo-PROTACs and investigated their ability to induce self-directed ubiquitination and degradation. The homodimerized compound 15a was characterized as a highly potent and efficient CRBN degrader with only minimal effects on IKZF1 and IKZF3. The cellular selectivity of 15a for CRBN degradation was confirmed at the proteome level by quantitative mass spectrometry. Inactivation by compound 15a did not affect proliferation of different cell lines, prevented pomalidomide-induced degradation of IKZF1 and IKZF3, and antagonized the effects of pomalidomide on multiple myeloma cells. Homobifunctional CRBN degraders will be useful tools for future biomedical investigations of CRBN-related signaling and may help to further elucidate the molecular mechanism of thalidomide analogues.

Full text of DOI:10.1021/acschembio.8b00693

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Article
Homo-PROTACs for the Chemical Knockdown of Cereblon
Christian Steinebach, Stefanie Lindner, Namrata D. Udeshi, Deepak C. Mani,
Hannes Kehm, Simon Köpff, Steven A. Carr, Michael Gütschow, and Jan Kronke
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00693 • Publication Date (Web): 17 Aug 2018
Downloaded from http://pubs.acs.org on August 18, 2018
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Homo-PROTACs for the Chemical Knockdown of Cereblon
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Christian Steinebach,‡,# Stefanie Lindner,†,# Namrata D. Udeshi,§ Deepak C. Mani,§ Hannes Kehm,†
Simon Köpff,† Steven A. Carr,§ Michael Gütschow,*,‡ and Jan Krönke*,†
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‡Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany
†Department of Internal Medicine III, University Hospital Ulm, Albert-Einstein-Allee 23, 89081 Ulm, Germany
§Proteomics, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA
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Graphical Abstract
ABSTRACT: The immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and
pomalidomide, all approved for the treatment of multiple myeloma, induce targeted ubiquitination and
degradation of Ikaros (IKZF1) and Aiolos (IKZF3) via the cereblon (CRBN) E3 ubiquitin ligase.
IMiDꢀbased proteolysis targeting chimeras (PROTACs) can efficiently recruit CRBN to a protein of
interest leading to its ubiquitination and proteasomal degradation. By linking two pomalidomide
molecules, we designed homobifunctional, soꢀcalled HomoꢀPROTACs and investigated their ability to
induce selfꢀdirected ubiquitination and degradation. The homodimerized compound 15a was
characterized as a highly potent and efficient CRBN degrader with only minimal effects on IKZF1 and
IKZF3. The cellular selectivity of 15a for CRBN degradation was confirmed at the proteome level by
quantitative mass spectrometry. Inactivation by compound 15a did not affect proliferation of different
cell lines, prevented pomalidomideꢀinduced degradation of IKZF1 and IKZF3 and antagonized the
effects of pomalidomide on multiple myeloma cells. Homobifunctional CRBN degraders will be
useful tools for future biomedical investigations on CRBNꢀrelated signaling and may help to further
elucidate the molecular mechanism of thalidomide analogs.
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INTRODUCTION
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In the past two decades, a paradigm shift from predominant inhibition of a protein target by
occupancyꢀbased drugs to the modulation of the target’s intracellular protein levels has occurred. As
known from natural products, endogenous ligands are able to regulate substrate recruitment to
ubiquitin ligase complexes. The plant hormones auxin and jasmonate represent prominent examples of
E3 ligase modulation.1,2 The IMiDs thalidomide, lenalidomide, and pomalidomide, all approved for
the treatment of multiple myeloma, were found to bind CRBN,3–5 a substrate adaptor for the CRL4 E3
ubiquitin ligase (CRL4CRBN), thereby facilitating the ubiquitination and the subsequent proteasomal
degradation of substrates. All IMiDs enhance the binding of the lymphoid transcription factors Ikaros
(IKZF1) and Aiolos (IKZF3) to CRL4CRBN, leading to an increased ubiquitination and degradation.6–10
Depletion of IKZF1 and IKZF3 results in growth inhibition in multiple myeloma cells. This mode of
action of IMiDs is further supported by crystal structures that provided insights into the binding mode
of IMiDs to CRL4CRBN and the function of cereblon as a substrate adaptor.3,11,12
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The ability of smallꢀmolecules like IMiDs to target a protein for degradation is an exciting
implication for modern drug development. Another approach for chemicalꢀinduced ubiquitination and
degradation of diseaseꢀcausing proteins are heterobifunctional molecules, which are also known as
proteolysis targeting chimeras (PROTACs).13,14 To direct an E3 ubiquitin ligase to a protein of interest
(POI) and induce the ubiquitination and subsequent proteasomal degradation of the POI, PROTACs
consist of a target binding moiety and an E3 ligase recruiting molecule connected via a variable linker.
Simultaneous binding to the target protein and a ubiquitin ligase leads to a transient ternary complex
of the ligase, the POI, and the bifunctional PROTAC itself. The induced proximity of the target and E3
ligase enables a ubiquitin transfer to the POI, which can then be recognized and degraded by the
proteasome system.15–17 IMiDs represent an excellent starting point for the assembly of
heterobifunctional compounds, which includes the CRBNꢀbinding substructure and a linkerꢀconnected
ligand of the corresponding POI.
A particular advantage of this technology is that chimeric molecules act catalytically and are
therefore more potent than the ligands from which they derive. Once ubiquitin labeling of the POI is
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completed, the PROTAC dissociates and is capable of forming a new ternary complex. Thus, a very
low PROTAC concentration is sufficient for the degradation of the target protein.18
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Current chemicalꢀinduced knockdown strategies are mainly focused on substrate adaptors of E3
ligase complexes, i.e. VonꢀHippelꢀLindau (VHL) and CRBN. By addressing VHL or CRBN, the
degradation of specific targets, such as proteins of the bromoꢀ and extraꢀterminal domain (BET) and
nonꢀBET families, sirtuin 2, the estrogenꢀrelated receptor alpha, the BCRꢀABL fusion protein,
dihydroorotate dehydrogenase, CDK9, and other kinases have been demonstrated.19–35
Here we describe the synthesis of homobifunctional PROTACs that utilize CRBN as the
hijacked degrader and, at the same time, as the protein targeted for degradation. In such Homoꢀ
PROTACs, identical linkerꢀconnected moieties are incorporated into a single molecule. This design
accomplishes a chemicalꢀinduced CRBN knockout.
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RESULTS AND DISCUSSION
Synthesis of Homo-PROTACs.
The preparation of phthalimidoꢀglutarimides (Figure 1) was performed by condensation of
phthalic anhydrides 1ꢀ3 with Bocꢀprotected 2ꢀaminoꢀglutarimide 4 in refluxing glacial acidic acid in
the presence of sodium acetate.36,37 The reaction conditions led to the successive deprotection, ringꢀ
opening, and recyclization and furnished the intermediate compounds 6 and 11 as well as thalidomide
(9). The other monocyclic building block, compound 5, obtained through methylation of 4, was
similarly used for the preparation of intermediates 7 and 12 and Nꢀmethylꢀthalidomide (10). This was
also accessible by Nꢀmethylation of thalidomide (9), and by an ultrasoundꢀsupported Mitsunobu
reaction38 of 9 and methanol. Here, sonication was advantageous to circumvent steric constraints and
to account for the relatively low acidity of the imide. The mild Mitsunobu conditions appear to be
generally useful for the Nꢀmethylation at the CRBNꢀbinding portion of PROTACs in order to produce
negative control compounds. As their structures are part of our final linkerꢀconnected dimers,
pomalidomide (13) and Nꢀmethylꢀpomalidomide (14) were required as control compounds. They were
obtained by a palladiumꢀcatalyzed hydrogenation of their corresponding 4ꢀnitro precursors 11 and 12.
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R1
R1
O
O
R2
O
O
O
R2
N
N
a
O
N
O
+
O
5
6
HN
O
O
O
6 R1 = F, R2 = H
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R1 = F
2 = H
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4
R
b
R1 = F, R2 = Me
2 R1 = H
5 R2 = Me
7
c
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8 R1 = NH(CH2)2O-(CH2)2-
3 R1 = NO2
O(CH2)2NHBoc, R2 = H
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R1 = H, R2 = H
O
9
d
10 R1 = H, R2 = Me
11 R1 = NO2, R2 = H
O
N
R3
N
O
O
R1 = NO2, R2 = Me
HN
12
Linker
e
13 R1 = NH2, R2 = H
NH
O
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O
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N
-
R
2 = R3 = H
14 R1 = NH2, R2 = Me
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15a e
N
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g
R2 = H, R3 = Me
R2 = R3 = Me
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O
f
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Figure 1. Synthesis of thalidomide derivatives and PROTACs. Reagents and conditions: (a) NaOAc,
AcOH, reflux, h; (b) MeI, K2CO3, DMF, ))), rt, h; (c) tertꢀbutyl Nꢀ[2ꢀ[2ꢀ(2ꢀ
6
2
aminoethoxy)ethoxy]ethyl] carbamate, DIPEA, DMF, 90 °C, 10 h; (d) DIAD, PPh3, MeOH, THF, ))),
rt, 1 h; (e) Pd/C, H2, DMF, rt, 8–24 h; (f) α,ωꢀdiamine, DIPEA, DMSO, 90 °C, 18 h; (g) 1. TFA,
CH2Cl2, 40 °C, 2 h; 2. 7, DIPEA, DMF, 90 °C, 10 h.
4ꢀFluoroꢀthalidomide (6) allowed the synthetic access to introduce a linker at position 4 leading
to alkylated pomalidomide derivatives. Such a linker attachment, forming a secondary amine, has been
established in PROTAC design.27,31,35 Accordingly, the synthesis of five homodimeric PROTACs was
finalized by reacting 6 with different α,ωꢀdiamines in a 2:1 molar ratio. Initially, DMF was used as a
solvent but was found to be less appropriate. In the presence of a tertiary amine and at high
temperature, DMF undergoes thermal decomposition to produce dimethylamine,36 which competes in
the aromatic nucleophilic substitution, generating the undesired 4ꢀdimethylaminoꢀthalidomide.
Instead, performing the reaction in DMSO led to the final CRBN HomoꢀPROTACs 15aꢀe (Table 1).
Similar, their structures were confirmed by MS and NMR data. Due to the mirror plane of 15aꢀe,
symmetryꢀequivalent nuclei show identical resonances in H and 13C NMR spectra. PROTACs 15aꢀe
1
all contain two pomalidomide moieties connected via a linker of various lengths between 5 to 13 linear
atoms, introducing varying hydrophobicity into the PROTAC molecules. It is known that the linker
length is critical for the activity of PROTACs and that the nature of the linker needs to be fineꢀtuned
over the course of their structural optimization.26,39
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Table 1. HomoꢀPROTACs with the corresponding linker substructures and their biological activities.
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CK1α
degradation
No. of linear
linker atoms
CRBN
degradation
IKZF1
degradation
Cmpd.
Linker
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O
O
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O
O
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15b
15c
15d
15e
16a
8
10
12
13
5
++
+
+
+
++
ꢀ
+
++
++
++
+
ꢀ
ꢀ
O
O
ꢀ
O
O
O
ꢀ
O
ꢀ
O
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++
ꢀ
n.d.f
O
O
O
O
O
O
O
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n.d.
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8
+
±
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+
ꢀ
ꢀ
19
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+
THALb
10
n.a.e
n.a.
n.a.
n.a.
n.a.
ꢀ
+
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
POMc
14
ꢀ
ꢀ
++
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
LENd
ꢀ
ꢀ
++
+
aHeterodimeric PROTAC. bThalidomide (9). cPomalidomide (13). dLenalidomide. enot applicable. fnot determined.
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Compound 16 represents a heterodimer with pomalidomide and
Nꢀmethylꢀpomalidomide as
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9
terminal moieties. Its synthesis was not feasible by a Mitsunobu reaction or methylation of the
homodimeric analog 15a. The synthesis was accomplished in a linear approach by first performing the
nucleophilic substitution of 6 with BocNH(CH2)2O(CH2)2O(CH2)2NH2 followed by deprotection and
the second nucleophilic substitution of 7. Applying the condition to produce 15aꢀe, the homodimeric
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compound 17 was synthesized from the methylated building block
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and
H2N(CH2)2O(CH2)2O(CH2)2NH2 in a 2:1 reaction. The introduction of one or two methyl group(s) at
the glutarimide portion is expected to abolish the binding of two CRBN molecules at the same time
and hence to form ternary complexes which are required for PROTACꢀmediated selfꢀdegradation.
Pomalidomide-based Homo-PROTACs induce degradation of CRBN.
In order to determine the effect of different HomoꢀPROTACs, we performed western blot
analyses of the multiple myeloma cell line MM1S that expresses endogenous CRBN and its IMiDꢀ
induced neoꢀsubstrates IKZF1, IKZF3, and casein kinase 1A1 (CK1α). After treatment with
compounds 15aꢀe at nanomolar concentrations for 16 hours, we observed a reduction of CRBN
protein levels (Figure 2). In contrast, the IMiDs thalidomide, pomalidomide, and lenalidomide had no
effect on CRBN protein levels, but induced degradation of IKZF1, consistent with previous
observations.6,7,9,12
All of our CRBN HomoꢀPROTACs also induced a doseꢀdependent decrease of IKZF1 protein
levels in MM1S cells. However, we observed clear differences in the impact on IKZF1 degradation:
compounds 15a and 15e with linker sizes of 8 and 5 linear atoms, respectively, had only minor effects
on IKZF1 protein levels, while compounds 15b, 15c, and 15d with linker sizes of 10ꢀ13 atoms led to a
pronounced degradation of IKZF1 (Figure 2A–C). None of the homodimers affected the level of
CK1α, a CRBN neoꢀsubstrate that is specifically targeted for degradation by lenalidomide.8 None of
the compounds produced a change in protein levels of CUL4A, which is part of the CRL4CRBN E3
ligase complex (Figure 2D).
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Figure 2. Pomalidomideꢀbased HomoꢀPROTACs induce degradation of CRBN. (A) The multiple
myeloma cell line MM1S was treated with compound 15a, (B) compound 15e and (C) compounds
15b, 15c, 15d for 16 hours at the indicated concentrations. (D) Direct comparison of pomalidomideꢀ
derived PROTACs at a concentration of 100 nM, 1 µM pomalidomide (POM), and 10 µM
lenalidomide (LEN).
Compound 15a with a linker length of 8 atoms was identified to be the most potent CRBN
degrader. Accordingly, we maintained the size of the linker, but modified the chemical attachment to
the phthalimide core and included two analogues of 15a. The structures of 18 and 19 (Figure 3A) were
accessible via selective Mitsunobu alkylation40 and carboxamide formation, respectively (see
Supplementary Figure 9 for the synthetic routes). Both PROTACs exhibited a weaker capability for
the chemically induced CRBN degradation (see Figure 3B and Supplementary Figure 1A) and were
not further investigated. These data indicate in this case the advantage of a linker attachment via
a
secondary amine structure. The following biological evaluations have thus been performed with 15a,
the most advantageous HomoꢀPROTAC of this study.
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Figure 3. (A) Structures of analogous HomoꢀPROTACs with different attachments of the linker. (B)
Comparison of the induction of CRBN, IKZF1, IKZF3 and CK1α degradation by 15a, 18, 19 and
pomalidomide, each at a concentration of 100 nM.
Compound 15a induces CRBN-mediated ubiquitination and proteasomal degradation.
HomoꢀPROTAC 15a decreased CRBN protein levels in a timeꢀ and doseꢀdependent manner. An
effect could be observed at a concentration as low as 10 nM after incubation for 16 hours (Figure 4A).
Maximum CRBN degradation was achieved with a concentration of 100 nM to 1 µM. Further increase
of the concentration to 100 µM abrogated the degradation of CRBN, which is likely explained by the
resulting saturation of each CRBN molecule to form binary complexes. This finding is in agreement
with the “hook effect” observed for other PROTACs at higher concentrations, whereby the formation
of the ternary complexes is limited due to predominant univalent saturation of the excess bifunctional
ligand compared to protein partners.41–44 At the concentration range of the “hook effect”, a strong
degradation of IKZF1 was observed (Figure 4A). The decrease of CRBN protein levels was observed
within 15 minutes of treatment with 100 nM of compound 15a and reached its maximum at 6 hours
(Figure 4B and Supplementary Figure 1B). The CRBN protein halfꢀlife was reduced to approximately
30 minutes with 15a as compared to more than 8 hours in control cells treated with cycloheximide
(CHX) to block protein translation (see Supplementary Figure 3). The effect of PROTAC 15a on
CRBN persisted for at least 96 hours (see Supplementary Figure 2A–B). After wash out of compound
15a, CRBN depletion persisted for several hours and slowly recovered to approximately 50% after 24
hours (see Supplementary Figure 1C).45 Compound 15a strongly decreased CRBN protein levels in all
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multiple myeloma (U266, RPMIꢀ8226, OPMꢀ2) and nonꢀmultiple myeloma cell lines (HEK293T,
K562, OCIꢀAML5) tested (see Supplementary Figure 2C).
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Introduction of a methyl group at the imino nitrogen of the glutarimide ring of pomalidomide or
thalidomide (leading to compounds 14 and 10, respectively) prevented binding to CRBN and
abrogated degradation of IKZF1 (see Supplementary Figure 2D). The same modification on one of the
pomalidomide moieties in a homodimeric PROTAC (leading to compound 16, Figure 1, bottom)
abrogated degradation of CRBN by preventing the simultaneous binding of two CRBN molecules and
the induction of their proximity (Figure 4C). Coꢀimmunoprecipitation with antiꢀFLAG in HAꢀ and
FLAGꢀtagged CRBN expressing HEK293T cells revealed that treatment with compound 15a induced
binding of two CRBN molecules (Figure 4D). These results further support that pomalidomideꢀbased
HomoꢀPROTACs induce CRBN ubiquitination and degradation by the formation of ternary complexes
of a 2:1 stoichiometry with two CRBNs and one PROTAC, rather than by autoꢀubiquitination or
binding to another ubiquitin ligase. In contrast to 15a, compound 16 caused a strong degradation of the
IKZF1 protein, suggesting that a 2:1 ternary complex, formed from 15a and two CRBN molecules, has
a reduced capability to bind IKZF1. Accordingly, compound 17 with both imino nitrogens being
methylated caused neither CRBN nor IKZF1 depletion (data not shown).
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We next treated HEK293T cells transfected with a vector expressing FLAGꢀtagged CRBN.
Homodimer 15a resulted in effective degradation of ectopically expressed CRBN. In contrast, 15a had
no effect on the ectopically expressed CRBNY384A/ W386A mutant that does not bind IMiDs (Figure 4E).4
This finding demonstrates that binding of the pomalidomideꢀhomodimers to the IMiD binding site is
essential for induction of CRBN degradation.
To determine whether compound 15a degrades CRBN via the ubiquitinꢀproteasome pathway, we
preꢀincubated MM1S cells with the proteasome inhibitor MG132 and the neddylation activating
enzyme (NAE) inhibitor MLN4924, which inhibits neddylationꢀdependent cullinꢀtype ubiquitin
ligases including CRL4CRBN. Both, MG132 and MLN4924, prevented compound 15aꢀmediated
degradation of CRBN (Figure 4F). Pretreatment with excess thalidomide (1000×) or lenalidomide
(100×) inhibited compound 15aꢀmediated CRBN degradation consistent with a competition for the
IMiD binding region of CRBN (Figure 4F).
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Figure 4. Compound 15a induces ubiquitination and proteasomal degradation of CRBN. (A) The
multiple myeloma cell line NCIꢀH929 was treated with compound 15a for 16 hours. (B) Time course
of 100 nM compound 15a treatment in MM1S cells. (C) Effect of compound 16 that has a methyl
group at one glutarimide ring predicted to abrogate binding to CRBN on MM1S cells. (D) AntiꢀFLAG
immunoprecipitation in HEK293T cells expressing HAꢀ and FLAGꢀCRBN. Cells were treated with
100 nM 15a or control for 30 minutes. MLN4924 (10 µM) was added to prevent 15aꢀmediated
degradation of CRBN. (E) HEK293T cells expressing FLAGꢀtagged normal CRBN or the YW/AA
mutation that prevents binding of IMiDs were treated with the indicated concentrations of compound
15a for 16 hours. (F) MM1S cells were treated with vehicle, 10 µM MG132, 10 µM MLN4924, 100
µM thalidomide, or 10 µM lenalidomide 3 hours before addition of 100 nM compound 15a. (G)
Ubiquitination analysis of FLAGꢀtagged CRBN in HEK293T cells. Cells were treated with compound
15a for 3 hours.
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We then performed an immunoblot ubiquitination analysis in HEK293T cells expressing
FLAGꢀCRBN. After treatment with compound 15a for 3 hours, we observed a higher degree of CRBN
ubiquitination as compared to untreated cells (Figure 4G).
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The effects of 15a on the entire proteome were assessed by applying quantitative mass
spectrometry (MS) using tandem mass tag (TMT) labeling. MM1S cells were treated in two biological
replicates with 100 nM of compound 15a and pomalidomide, respectively, or DMSO as a control. In
total, 9448 proteins were identified by two or more peptides and quantified across all samples. CRBN
showed the highest degree of degradation in cells treated with compound 15a for 3 hours (log2foldꢀ
change (FC) = ꢀ0.82) and 24 hours (log2FC = ꢀ1.0) (see Supplementary Figure 7A and 8A). In
contrast, pomalidomide had no effect on CRBN protein levels (see Supplementary Figure 7B and 8B).
Consistent with previous studies,6ꢀ10,46 after pomalidomide treatment for 3 hours, the most downꢀ
regulated proteins were the IMiDꢀinduced CRBN neoꢀsubstrates IKZF1 (log2FC = ꢀ1.11) and IKZF3
(log2FC = ꢀ1.24). In comparison, compound 15a treatment for 3 hours had only weak effects on IKZF1
(log2FC = ꢀ0.24) and IKZF3 (log2FC = ꢀ0.26). Offꢀtarget degradation by pomalidomideꢀderived
PROTACs was considered an undesired IMiD effect. It has been found through proteomics analysis
that a heterobifunctional PROTAC, acting as a multiꢀkinase degrader, also depleted IKZF1 to some
degree, suggesting that pomalidomide partially retains its IMiD functions despite linker attachment.47
Compound 15a did not cause degradation of other members of the CRL4 ligase including DDB1,
CUL4A, RBX1 or any other ubiquitin ligases.
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PROTAC 15a antagonizes the effects of IMiDs on multiple myeloma cells.
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IMiDs mediate their antiꢀproliferative effect on multiple myeloma cells through induced
degradation of the lymphoid transcription factors IKZF1 and IKZF3 via the CRL4CRBN E3 ubiquitin
ligase.7,9,10 In addition, IMiDs disrupt the chaperone function of CRBN for MCTꢀ1 and BSG proteins,
what contributes to the antiꢀproliferative effects.48
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Since our HomoꢀPROTACs are potent inducers of CRBN degradation, we tested their impact on
cell viability. Pomalidomide, as well as PROTAC 15a, had no effect on the IMiDꢀinsensitive AML
cell line OCIꢀAML5, even at concentrations up to 10 µM (Figure 5A). In the pomalidomideꢀsensitive
multiple myeloma cell lines MM1S, NCIꢀH929 (Figure 5A), LPꢀ1, KMS27, and RPMI 8226 (see
Supplementary Figure 4), compound 15a had little or no effect on cell proliferation at concentrations
of up to 1 µM, demonstrating that multiple myeloma cells tolerate degradation of CRBN to very low
levels. Such low CRBN levels are achieved after treatment with nanomolar concentrations of our
HomoꢀPROTACs (see Figure 2A, as well as 4A). Consistent with these observations, genetic
inactivation of CRBN by CRISPR/Cas9 gene editing with lentiviralꢀdelivered CRBNꢀspecific small
guide RNAs (sgRNAs) targeting either the Nꢀterminus or the IMiDꢀbinding site had no effect on
MM1S cell proliferation, in contrast to sgRNAs targeting the essential gene RNA polymerase II
subunit A (PolR2A) (see Supplementary Figure 6 and Supplementary Table 1).
At concentrations of 10 µM, compound 15a inhibited the proliferation of MM1S cells (Figure
5A), which can be explained by the increasing degradation of IKZF1 protein compared to that of
CRBN. Consistently, compounds 15c (with an extended linker) and 16 (with one glutarimide structure
methylated), which had a more pronounced effect on IKZF1 protein levels but little or no effect on
CRBN, were more toxic to multiple myeloma cell lines (see Supplementary Figures 5B–C). These
finding might reflect that compounds of reduced ability to form 2:1 ternary complexes tend to act as
IMiDs rather than CRBN degrader.
It was then tested whether our PROTACs could antagonize the activity of pomalidomide and
lenalidomide on multiple myeloma cell lines. In MM1S cells, degradation of CRBN by pretreatment
with compound 15a abrogated pomalidomideꢀinduced degradation of IKZF1 (Figure 5B), which is
consistent with the genetic inactivation of CRBN.7,9,48 Since induced degradation of IKZF1 and IKZF3
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by CRBN is one of the main mechanisms by which IMiDs inhibit multiple myeloma cell growth, and
in the light of IMiD resistance gained by genetic CRBN depletion,49 we tested whether chemical
inactivation of CRBN could rescue multiple myeloma cell lines from pomalidomide and lenalidomide.
For this purpose, the IMiDꢀsensitive multiple myeloma cell lines MM1S and NCIꢀH929 were
pretreated for 3 hours with compound 15a, followed by the addition of lenalidomide or pomalidomide.
After treatment for 4 days, we found that the growth inhibition induced by either pomalidomide or
lenalidomide was blocked (see Figure 5C, as well as Supplementary Figure 4B). Even treatment with
compound 15a after addition of lenalidomide or pomalidomide resulted in reduced activity of both
IMiDs. Similarly, CRISPR/Cas9ꢀmediated inactivation of CRBN by sgRNAs targeting the Nꢀterminus
or IMiDꢀbinding region of CRBN conferred IMiD resistance to CRISPRꢀmodified cells (see
Supplementary Figure 6). This demonstrates that chemicalꢀinduced inactivation of CRBN by the
HomoꢀPROTACs is as potent as genetic inactivation to abrogate the effect of IMiDs in multiple
myeloma cells.
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Figure 5. Impact of compound 15a on cell proliferation and the activity of lenalidomide and
pomalidomide. (A) The pomalidomideꢀinsensitive acute myeloid leukemia cell line OCIꢀAML5 and
the pomalidomideꢀsensitive multiple myeloma cell lines MM1S and NCIꢀH929 were treated with the
indicated concentrations of compound 15a or pomalidomide. Cell viability was analyzed after 4 days
in triplicate. (B) MM1S cells were treated with 100 nM compound 15a for 6 hours or 1 µM
pomalidomide for 3 hours without or with compound 15a pretreatment for 3 hours. (C) MM1S and
NCIꢀH929 cells were treated with 100 nM or 1 µM compound 15a alone, 3 hours before (pre) or after
(post) addition of 1 µM pomalidomide. Cell viability was measured after 4 days. Error bars express the
mean ± SEM from 3 biological replicates. *** p<0.001.
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Discussion
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Here we describe the design, synthesis and biological evaluation of novel, pomalidomideꢀbased
HomoꢀPROTACs. Since our homobifunctional PROTACs are capable of simultaneously interacting
with two CRBN molecules, ternary complexes can be formed (Figure 6A). The induced homoꢀ
dimerization of CRBN results in subsequent ubiquitination and proteasomal degradation,
demonstrating for the first time, a chemically induced knockꢀdown of CRBN. In particular, the Homoꢀ
PROTAC 15a exhibited, at low concentrations, a longꢀlasting effect on the intracellular CRBN level.
Our mutation and competition experiments strongly indicate binding of the PROTACs at the IMiD
binding site of CRBN. Immunoblotting and global proteome analyses revealed that PROTAC 15a
specifically induced degradation of CRBN and had only weak effects on pomalidomide neoꢀsubstrates
IKZF1 and IKZF3 and no effect on the other members of the CRL4 ligase family, including DDB1
and CUL4A which are in close proximity to CRBN.
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By investigating the influence of the incorporated linkers, we found that the extent of CRBN
degradation depends on the nature of the linker and of its attachment to the E3 ligase binder. It also
affected the protein levels of the pomalidomideꢀinduced CRBN neoꢀsubstrate IKZF1. This implies that
the addition of a linker to the phthalimide part does not necessarily interfere with binding of CRBN to
neoꢀsubstrates such as IKZF1 and IKZF3 which has also been observed with other IMiDꢀbased
PROTACs.24,47 Thus, the CRBNꢀPROTAC complexes (see Figure 6A and 6B) offer a de novo
interface12 for neoꢀsubstrate recruitment (see Figure 6D and 6C). For this reason, remodeling of the
CRBN binding surface by small molecule binders needs to be considered as an unwanted sideꢀeffect in
the development of IMiDꢀbased PROTACs.
Direct proteinꢀprotein interactions (PPIs) of CRBN with neoꢀsubstrates were also observed in
the presence of the CRBN modulators CCꢀ885 and CCꢀ220. Their chemoꢀneomorphic activities
triggered by new interaction hotspots on the surface of CRBN recruit GSPT1, IKZF1, or IKZF3 to the
CRL4CRBN ubiquitin ligase.50ꢀ53 Moreover, recent models for PROTAC action postulate stabilizing
PPIs between the ligase and the POI. Hence, a positive cooperativity of a ternary ligaseꢀPROTACꢀPOI
system might explain the efficacy of a bifunctional molecule.24,54,55 The surface of CRBN is known to
be amenable for PPIs.24 In the case of our HomoꢀPROTACs, we hypothesized that they may facilitate
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proteinꢀprotein interaction between two cereblon molecules. Consistently, the incorporated short linker
in 15a contributes to a closer proximity of two CRBN molecules, beneficial PPIs, and an efficient
CRBN knockdown (Figure 2A). Thus, we were able to achieve the desired PROTAC activity at
concentrations below the “hook effect” range, when the equilibria (Figure 6) are shifted to the left.
Presumably, a tight ternary 2:1 complex (Figure 6A) is not capable of binding a neoꢀsubstrate (Figure
6D) and the unwanted IKZF1 degradation is prevented (see Figure 2A, as well as 4A). In contrast, the
expanded pomalidomide PROTACs (15bꢀd) preserve the capacity of neoꢀsubstrate binding and
degradation. In line with this, the heterodimeric PROTAC 16, which cannot form complexes with two
CRBN units (see Figure 6A and 6D), provoked a strong IKZF1 degradation. Similar effects on target
degradation induced by bifunctional molecules have been reported: a VHLꢀaddressing PROTAC with
a short linker led to a much stronger degradation of HER2 than an analog with a slightly longer
linker.45,56
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Figure 6. Interactions of CRBN with HomoꢀPROTACs. Equilibria between possible complexes of
CRBN (green, the three distinct domains are indicated),12 PROTACs (red) and IKAROS neoꢀ
substrates (blue) are shown
Very recently, the generation of HomoꢀPROTACs for the VHL E3 ligase was described.57
These HomoꢀPROTACs were assembled by linking two VHLꢀbinding molecules. Like our
homobifunctional PROTACs, the VHLꢀHomoꢀPROTACs caused specific ubiquitination and
proteasomal degradation at nanomolar levels. Noteworthy, only the long VHL isoform was degraded.
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The short isoform, which is preferentially part of the CRL2ꢀVHL complex, was not affected.
Consequently, the VHLꢀHomoꢀPROTACs had no effect on the CRL2ꢀVHL substrate HIFꢀα.
For the VHLꢀHomoꢀPROTACs, an increased linker size resulted in more effective degradation
of VHL.57 In the present study, we found that an extended linker length led to an alleviated CRBN
degradation, for reasons that have been discussed above. These data confirm that the appropriate
choice of the linker remains one of the most important aspects of PROTAC design and more research
into the nature of the linker is required since it largely affects ternary complex formation and stability
required for potent degradation.24,55,58
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CRBN is a multifunctional protein that is involved in different cellular processes and diseases.
For example, a CRBN mutation is associated with human autosomal recessive nonsyndromic mental
retardation.59 Genetic inactivation of CRBN confers resistance to sepsis and prevents highꢀfatꢀdietꢀ
induced obesity and insulin resistance in mice,60–62 implying that pharmacological inhibition of CRBN
may have clinical applications. The multiprotein structure of E3 ligases and the lack of
pharmaceutically targetable regions on the protein surface have so far prevented the generation of
specific inhibitors for these enzymatic complexes. Our homodimeric pomalidomideꢀPROTACs
operate as potent inhibitors of the CRL4CRBN E3 ligase, insofar as they initiate the selfꢀdegradation of
the adapter protein CRBN.
In the past years, CRBN has drawn attention as the target of the IMiDs thalidomide,
lenalidomide, and pomalidomide that are a mainstay in the treatment of multiple myeloma. These
drugs mediate their activity in cancer by modulating the specificity of the CRL4CRBN E3 ligase to
degrade the neoꢀsubstrates IKZF1, IKZF3, and CK1α.3,6–12 Genetic inactivation of CRBN has been
shown to cause resistance to IMiDs in cell lines and inactivating CRBN mutations were detected in
IMiDꢀresistant multiple myeloma patients.49,63 Both, the chemicalꢀinduced CRBN degradation and
approaches of genetic knockdown of CRBN confer resistance to IMiDs. We show that chemical
knockdown of CRBN by our HomoꢀPROTAC 15a did not affect multiple myeloma cell proliferation,
abrogated the degradation of IKZF1 and IKZF3 and conferred pomalidomide and lenalidomide
resistance in different multiple myeloma cells. Our data therefore highlight the essential role of CRBN
in IMiD activity and further support that the main effect of IMiDs in multiple myeloma relies in
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induced degradation of neoꢀsubstrates by CRBN rather than blocking the physiological function of
CRBN. In the light of the clinical relevance of CRBN as a target for IMiDs and PROTACs, our
compounds represent valuable tools to induce a cellular state of inactivated CRBN. The low toxicity of
15a also implies that this homodimeric PROTAC seems not to have a profound global effect on the
ubiquitinꢀproteasome system since multiple myeloma cells are strongly dependent on cellular protein
quality control mechanisms.64,65
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Unlike the antiꢀcancer properties of IMiDs, the exact mechanism for many of their pleiotropic
effects, including immune modulation, teratogenicity, and antiꢀangiogenesis, are not completely
understood. These effects may result from degradation of neoꢀsubstrates but alternatively also from
inhibition of the endogenous function of CRBN. Hence, our tool compounds will be useful to
discriminate whether an IMiD effect depends on CRBNꢀmediated targeted degradation of neoꢀ
substrates or from blocking CRBN activity. Recently, IMiDs gained increased importance due to the
establishment of the PROTAC technology.31,34 Some of the IMiDꢀbased PROTACs have promising
preclinical antiꢀtumor activity but may have unwanted effects similar to those of their parent IMiDs.
Thus, further knowledge on the function of CRBN and the identification of its downstream protein
substrates is needed.66
Chemical inactivation of a target protein offers several advantages over classical genetics, such
as reversibility, the rapid and direct impact on the posttranslational protein level, as well as the general
applicability to a broad range of cell and tissue types.67 The typically low selectivity of chemical
probes can be overcome by a sophisticated design of such compounds. The HomoꢀPROTACs reported
herein provide a further example and will be useful tools to induce CRBN degradation, to unravel
endogenous substrates and physiological roles of CRBN and to provide additional insights into the
molecular mechanism of IMiDs.
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METHODS
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Chemistry. Chemical synthesis and physicochemical data for all compounds are provided in the
Supporting Information.
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Cell lines. Cell lines MM1S, U266, KMS27, LPꢀ1, NCIꢀH929, OPMꢀ2, K562, OCIꢀAML5, and
HEK293T were obtained from American Type Culture Collection (ATCC) or the Deutsche Sammlung
von Mikroorganismen und Zellkulturen (DSMZ). Cells were cultured in RPMI 1640 (Biochrom) or
DMEM (Thermo Scientific) supplemented with 10ꢀ20% heatꢀinactivated fetal bovine serum (FBS)
(Biochrom) and 1% penicillin, streptomycin, and Lꢀhumidified incubator under 5% CO2. Cells have
been authenticated by STR profile analyses and tested for mycoplasma contamination.
Ectopic CRBN expression. For overexpression of CRBN, an RSF91 expression vector (gift of C.
Baum, Hannover Medical School) containing normal FLAGꢀtagged CRBN or mutant CRBN
Y384A/W386A (YW/AA) that cannot bind IMiDs was used.7 HEK293T cells were seeded in a 6ꢀwell
plate and after 24 h cells were transfected with 1 µg plasmid DNA using TransITꢀLT1 transfection
reagent (Mobitec).
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Immunoblotting. After designated treatment, cells were lysed in IP lysis buffer (Pierce) containing
HALT Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific). Protein content was
quantified with a BCA assay (Pierce) and equal amounts of proteins were separated by SDSꢀPAGE at
a constant voltage and transferred onto ImmobilonꢀP transfer membrane (Millipore). The following
antibodies were used in the study for western blot analyses: rabbit antiꢀCRBN (Sigma, HPA045910),
rabbit antiꢀIkaros (Cell Signaling, D6N9Y), rabbit antiꢀIKZF3 (Cell Signaling, D1C1E), goat antiꢀ
Casein kinase 1A1 (Santa Cruz, Cꢀ19), mouse antiꢀTubulin (Sigma, DM1A), rabbit antiꢀDYKDDDDK
(Cell Signaling), HRPꢀlinked antiꢀHA (Miltenyi, 130ꢀ091ꢀ972), FK2 (HRPꢀconjugate, Enzo
Lifescience), rabbit antiꢀCUL4A (Cell Signaling, #2699S). Western blots for drug treatments were
performed 2ꢀ5 times per experiment. Quantification was performed using ImageJ (Rasband, W.S.,
ImageJ, U.S. National Institutes of Health, Bethesda, Maryland, USA, https://imagej.nih.gov/ij/, 1997ꢀ
2016).
Co-immunoprecipitation. HEK239T cells were coꢀtransfected with an HAꢀCRBN and FLAGꢀCRBN
expressing vector. After 48 hours, cells were treated with DMSO or 100 nM 15a for 30 minutes. 10
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µM MLN4924 was added to block 15aꢀinduced degradation of CRBN. Cells was lysed in IP lysis
buffer (Pierce) and immunoprecipitation was performed with antiꢀFLAG sepharose beads (Sigma) for
2 hours in the presence of the drugs. After 3 washes with IP lysis buffer, FLAGꢀCRBN was eluted
with FLAG peptide and the samples were analyzed by western blot.
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Ubiquitination analysis. HEK293T cells were transfected with a FLAGꢀCRBN expressing plasmid.
48 hours after transfection cells were treated with DMSO or compound 15a for 3 hours. Cells were
then washed twice with iceꢀcold PBS and lysed under denaturing conditions using 2% SDSꢀ
containing lysis buffer and boiled for 10 minutes at 95 °C. The SDS was diluted with the addition of
10 × IP lysis buffer (Pierce) containing HALT Protease and Phosphatase Inhibitor Cocktail (Thermo
Scientific), MG132 (10 µM) and PR619 (50 nM) and incubated at 4 °C for 30 minutes.
Immunoprecipitation was performed using antiꢀFLAG M2 Affinity Gel (SigmaꢀAldrich) according to
the manufacturer’s protocol and protein was eluted with FLAG peptide (SigmaꢀAldrich). CRBN was
detected by a FLAGꢀspecific antibody and ubiquitinated CRBN by the FK2 antibody.
Cell viability assay. Cells were seeded in a 96ꢀ or 384 well plate and treated with the respective
concentrations of the test compounds. To determine the number of viable cells, CellTiterꢀGlo®
Luminescent Cell Viability Assay (Promega) was performed according to the manufacturer´s protocol.
Luminescence was detected on a PolarStar plate reader (BMG labtech).
CRISPR-mediated knockout of CRBN. For CRISPR gene editing, single guide RNAs were cloned
into the lentiviral SGL40C.EFS.dTomato vector (a gift from D. Heckl, Hannover).68 Guide RNA
sequences are listed in the Supporting Information. A luciferaseꢀspecific sgRNA was used as a
negative control and a sgRNA targeting the essential gene RNA polymerase II subunit A (PolR2A) as
a positive control. MM1S cells stably expressing Cas9 were seeded in a 96ꢀwell plate and infected
with sgRNAꢀexpressing lentiviral vectors. For drug treatment, cells were split 4 days post infection
1:1:1, seeded in 96 well plates and treated with 1 µM lenalidomide, 1 µM pomalidomide or DMSO as
a control. After 7, 11 and 14 days the percentage of infected cells was measured by flow cytometry.
Proteome analysis using liquid chromatography mass spectrometry. Cell lysis and peptide
digestion. Cell pellets were lysed for 30 minutes with ice cold urea lysis buffer containing 8 M urea,
75 mM NaCl, 50 mM TrisꢀHCl (pH 8.0), 1 mM EDTA, 2 µg/mL aprotinin (Sigma, A6103), 10 µg/mL
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leupeptin (Roche #11017101001), and 1 mM PMSF (Sigma, 78830). The samples were spun at 20,000
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lysate. Lysis buffer was used to bring the protein concentration of each lysate to 1 mg/mL. Protein
disulfide bonds were reduced with 5 mM dithiothreitol at 25 °C for 45 minutes. The proteins were
alkylated in the dark using 10 mM iodoacetamide at 25 °C for 45 minutes. Lysates were then diluted
1:4 using 50 mM Tris, pH 8.0, to lower the urea concentration to 2 M. LysC was added to each lysate
at a 1:50 enzymeꢀtoꢀsubstrate ratio and samples were digested at 25 °C for 2 hours. Afterwards,
trypsin was added at a 1:50 enzymeꢀtoꢀsubstrate ratio and the samples were digested at 25 °C
overnight. The digestion was quenched with 100% formic acid to reach a volumetric concentration of
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1% formic acid. Samples were spun at 5000
g for 5 minutes in order remove precipitated urea and
desalted using SepꢀPak C18 columns (Waters, 100 mg WAT023590). Columns were conditioned with
1 × 1 mL 100% acetonitrile, 1 × 1 mL 50% acetonitrile/0.1% formic acid, and 4 × 1 mL 0.1%
trifluoroacetic acid. Each sample was loaded onto a column and washed with 3 × 1 mL 0.1%
trifluoroacetic acid and 1 × 1 mL 1% formic acid. Peptides were eluted off the column with 2 × 0.6
mL 50% acetonitrile/0.1% formic acid and dried down.
TMT labeling and basic reverse phase fractionation. For TMT labeling, 60 µg of peptides from each
sample were labeled with a TMT10 reagent according to manufacturer instructions (Thermo
Scientific). The peptides were each reconstituted in 60 µL of 100 mM HEPES and labeled for 1 hour
at 25 °C with 0.48 mg TMT10 tag in 24.6 µL of 100% acetonitrile. Labeling efficiency was checked to
ensure proper and complete labeling. The labeling reaction was quenched with 4.8 µL of 5%
hydroxylamine for 15 minutes at 25 °C. Afterwards, all peptides were combined and desalted using a
SepꢀPak C18 column (Waters, 100 mg WAT036790) following a similar procedure as described
above. The sample reconstituted in 3% acetonitrile/0.1% formic acid and fractionated using basic
reverse phase fractionation as reported previously (ubi, ptm, cptac). A 4.6 mm Zorbax column
(Agilent) was used to fractionate the sample into 96 fractions that were concatenated in a
noncontiguous manner into 25 fractions. Each fraction was dried down and resuspended at 1 µg/µL
concentration in 3% acetonitrile/0.1% formic acid.
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LC-MS/MS and spectra analysis. All samples were analyzed using nanoflow HPLCꢀHCDꢀMS/MS
using an Orbitrap Fusion Lumos mass spectrometer and an EasyꢀnLC 1260 system. 1 µL of each
sample was injected at a flow rate of 500 nL/min onto a Picofrit column selfꢀpacked with 1.9 µm Cꢀ18
beads that was heated to 50 °C. The LCꢀMS/MS gradient and flow rate were used as previously
described.69 Spectra were acquired for 110 minutes. MS1 scans were acquired at 60 K resolution at a
scan range of 350ꢀ1800 m/z and a maximum injection time of 50 ms. Ions were fragmented with a
collision energy of 38%. MS2 scans were acquired at 50 K resolution with a maximum injection time
of 105 ms in a 0.7 isolation window. Spectra were searched using Spectrum Mill software (Agilent).
For peptide identification, MS/MS spectra were searched against the Uniprot human database with 150
contaminants.
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Statistical analysis. Statistical analysis was performed using Prism v5.0 (GraphPad Software). Data
are expressed as mean ± SEM. Statistical differences between two data sets were assessed using a twoꢀ
sided Student tꢀtest. P values less than 0.05 were considered statistically significant. P values greater
than 0.05 were indicated as not significant (n.s.) and the statistical significance is also reported in the
Figure Legends.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS publications website at DOI:
Supplementary Figures 1–9, Supplementary Table 1, as well as synthetic procedures (PDF)
AUTHOR INFORMATION
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Corresponding Authors
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*Address: Tel.: +49ꢀ228ꢀ732317. Fax: +49ꢀ228ꢀ732567. Eꢀmail: guetschow@uniꢀbonn.de (M.
Gütschow).
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*Address: Tel.: +49ꢀ731ꢀ50045718. Fax: +49ꢀ731ꢀ50045525. Eꢀmail: jan.kroenke@uniꢀulm.de (J.
Krönke).
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Author Contributions
#These authors contributed equally to this work. C.S. and M.G. designed homodimeric PROTACs.
C.S. synthesized the compounds. S.L., H.K., and S.K. performed drug treatments, western blot, and
cell viability analyses. N.D.U., D.M., and S.A.C. performed proteomic analyses. C.S., S.L., M.G., and
J.K. analyzed data and wrote the manuscript. M.G. and J.K. supervised the project.
Notes
The authors declare that they have no conflicts of interest with the contents of this article. This work
was supported by the Deutsche Forschungsgemeinschaft (EmmyꢀNoether Program Krꢀ3886/2ꢀ1 and
SFBꢀ1074 to J.K.; FOR2372 to M.G.).
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