A biphenyl inhibitor of eIF4E targeting an internal binding site enables the design of cell-permeable PROTAC-degraders

Article abstract of DOI:10.1016/j.ejmech.2021.113435

The eukaryotic translation initiation factor 4E (eIF4E) is the master regulator of cap-dependent protein synthesis. Overexpression of eIF4E is implicated in diseases such as cancer, where dysregulation of oncogenic protein translation is frequently observed. eIF4E has been an attractive target for cancer treatment. Here we report a high-resolution X-ray crystal structure of eIF4E in complex with a novel inhibitor (i4EG-BiP) that targets an internal binding site, in contrast to the previously described inhibitor, 4EGI-1, which binds to the surface. We demonstrate that i4EG-BiP is able to displace the scaffold protein eIF4G and inhibit the proliferation of cancer cells. We provide insights into how i4EG-BiP is able to inhibit cap-dependent translation by increasing the eIF4E-4E-BP1 interaction while diminishing the interaction of eIF4E with eIF4G. Leveraging structural details, we designed proteolysis targeted chimeras (PROTACs) derived from 4EGI-1 and i4EG-BiP and characterized these on biochemical and cellular levels. We were able to design PROTACs capable of binding eIF4E and successfully engaging Cereblon, which targets proteins for proteolysis. However, these initial PROTACs did not successfully stimulate degradation of eIF4E, possibly due to competitive effects from 4E-BP1 binding. Our results highlight challenges of targeted proteasomal degradation of eIF4E that must be addressed by future efforts.

Full text of DOI:10.1016/j.ejmech.2021.113435

European Journal of Medicinal Chemistry 219 (2021) 113435
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
A biphenyl inhibitor of eIF4E targeting an internal binding site enables
the design of cell-permeable PROTAC-degraders
,
b
,
c
,
,
e
,
1
,
, b
Patrick D. Fischer a
1, Evangelos Papadopoulos b
**, Jon M. Dempersmier a
,
Zi-Fu Wang a b, Radosław P. Nowak a b, Katherine A. Donovan a b, Joann Kalabathula a,
,
,
,
Christoph Gorgulla a b, Pierre P.M. Junghanns c, Eihab Kabha b, Nikolaos Dimitrakakis d,
,
Ognyan I. Petrov f, Constantine Mitsiades e, Christian Ducho c, Vladimir Gelev f,
Eric S. Fischer a b, Gerhard Wagner b, Haribabu Arthanari a
,
, b, *
a Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, 02215, USA
b Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, 02115, USA
c Department of Pharmacy, Pharmaceutical and Medicinal Chemistry, Saarland University, Saarbrücken, 66123, Germany
d Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, MA, 02115, USA
e Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, 02215, USA
f Faculty of Chemistry and Pharmacy, Sofia University, 1 James Bourchier Blvd., 1164, Sofia, Bulgaria
a r t i c l e i n f o
a b s t r a c t
Article history:
The eukaryotic translation initiation factor 4E (eIF4E) is the master regulator of cap-dependent protein
synthesis. Overexpression of eIF4E is implicated in diseases such as cancer, where dysregulation of
oncogenic protein translation is frequently observed. eIF4E has been an attractive target for cancer
treatment. Here we report a high-resolution X-ray crystal structure of eIF4E in complex with a novel
inhibitor (i4EG-BiP) that targets an internal binding site, in contrast to the previously described inhibitor,
4EGI-1, which binds to the surface. We demonstrate that i4EG-BiP is able to displace the scaffold protein
eIF4G and inhibit the proliferation of cancer cells. We provide insights into how i4EG-BiP is able to inhibit
cap-dependent translation by increasing the eIF4E-4E-BP1 interaction while diminishing the interaction
of eIF4E with eIF4G. Leveraging structural details, we designed proteolysis targeted chimeras (PROTACs)
derived from 4EGI-1 and i4EG-BiP and characterized these on biochemical and cellular levels. We were
able to design PROTACs capable of binding eIF4E and successfully engaging Cereblon, which targets
proteins for proteolysis. However, these initial PROTACs did not successfully stimulate degradation of
eIF4E, possibly due to competitive effects from 4E-BP1 binding. Our results highlight challenges of tar-
geted proteasomal degradation of eIF4E that must be addressed by future efforts.
Received 11 November 2020
Received in revised form
23 March 2021
Accepted 1 April 2021
Available online 8 April 2021
Keywords:
Translation inhibitor
eIF4E inhibitor
eIF4E-eIF4G inhibitor
Protein-protein interaction inhibitor
eIF4E PROTAC
Prodrug
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
the DEAD-box RNA helicase eIF4A, and the scaffold protein eIF4G
[1e7]. eIF4E’s interaction with eIF4G facilitates loading of the 40S
Cap-dependent translation in eukaryotes is initiated when eIF4E
binds to the m7GTP cap of mRNAs, a rate-limiting step that results
in the formation of the eIF4F complex, which is comprised of eIF4E,
small ribosomal unit onto the mRNA, triggering scanning for the
start codon. eIF4E binding proteins (4E-BPs) compete with eIF4G
for binding to eIF4E, and successful binding of 4E-BPs abolishes
cap-dependent translation, making 4E-BPs important regulators of
this process. This competition is regulated by the 4E-BP phos-
phorylation state. Upon phosphorylation by mTORC1, 4E-BP iso-
forms disassociate from eIF4E, freeing it to then engage with eIF4G
and form the eIF4F complex [8] (Fig. 1A). The critical role of eIF4E in
cancer was first identified when overexpression was observed to
cause tumorigenic transformation of fibroblasts [9]. eIF4E has since
been found to be overexpressed in a number of cancer types,
* Corresponding author. Department of Biological Chemistry and Molecular
Pharmacology, Harvard Medical School, Boston, MA, 02115, USA.
** Corresponding author. Medical Oncology, Dana-Farber Cancer Institute, Boston,
MA, 02215, USA.
E-mail addresses: Epapadopoulos@partners.org (E. Papadopoulos), hari@hms.
harvard.edu (H. Arthanari).
1
Joint Authors.
https://doi.org/10.1016/j.ejmech.2021.113435
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

P.D. Fischer, E. Papadopoulos, J.M. Dempersmier et al.
European Journal of Medicinal Chemistry 219 (2021) 113435
Fig. 1. A. Assembly of the translation initiation complex eIF4F, which consists of eIF4E, eIF4G and eIF4A. eIF4E is negatively regulated by hypo-phosphorylated 4E-BP, which in turn
is regulated by mTORC1. B. Structures of the small molecule inhibitors 4EGI-1 and i4EG-BiP. C. Fluorescence polarization assay for displacement of an eIF4G peptide from eIF4E. Both
4EGI-1 and i4EG-BiP are capable of displacing eIF4G with similar affinities.
2

P.D. Fischer, E. Papadopoulos, J.M. Dempersmier et al.
European Journal of Medicinal Chemistry 219 (2021) 113435
including breast [10], non-Hodgkin lymphoma [11] and head and
neck [12].
the active head-head dimer (4EGI-dim) of 4EGI-1, and 4EGI-dim
displayed a slightly better Kd than 4EGI-1 in the FP assay. Next, we
synthesized i4EG-BiP, containing a biphenyl instead of cyclohexyl-
phenyl “head"(here BiP denotes the biphenyl moiety in the scaf-
fold), as a control for a series of “head-to-head” dimer series such as
4EGI-dim (Supplementary Fig. 2). We serendipitously discovered
that i4EG-BiP binds to a new internal cavity on eIF4E, near the 4EGI-
1 binding site.
The structure of eIF4E resembles a hand with a palm consisting
of b-strands and dorsally positioned a
-helices [13,14]. The m7GTP
cap binds tightly to the palm region and is stabilized by interactions
with four tryptophan sidechains. Both eIF4G and 4E-BPs engage
eIF4E, in part, through conserved motifs with the consensus
sequence YX4LF, where F is a hydrophobic amino acid. Structural
studies have revealed that the conserved 4G/4E-BP binding motif
binds to the dorsal surface, opposite of the cap-binding surface
[15,16]. Studies have shown crosstalk between the cap-binding and
4G/4E-BP binding events with binding at one site affecting the af-
finity of the other [17]. All published structures of eIF4E in complex
with minimal binding epitopes from eIF4G or 4E-BPs are nearly
identical and only provided information about the eIF4G and 4E-BP
peptides engaging eIF4E. However, structures with larger frag-
ments of eIF4G or 4E-BP in complex with eIF4E have been recently
determined [15,16]. These structures show that eIF4G and 4E-BPs
use interfaces beyond their minimal consensus sequence to engage
eIF4E.
We were also interested in leveraging information from high-
resolution structures of 4EGI-1 and i4EG-BiP bound to eIF4E to
engineer PROteolysis-TArgeting Chimeras (PROTACs) in an effort to
trigger specific degradation of eIF4E as an alternative to traditional
small molecule inhibitors that target protein-protein interfaces
[30e35]. PROTACs are heterobifunctional molecules in which a
small molecule ligand is conjugated to an E3 ligase ligand with a
linker, frequently PEG- or carbon-based. PROTACs take advantage of
the host E3 ubiquitin ligase machinery to induce targeted protea-
somal degradation of the protein of interest. Studies with PROTACs
based on promiscuous kinase degraders have shown that ligands
with weak binding (KD > 10 mM) can still be turned into potent
Despite this structural data, the atomic detail of how 4E-BPs
outcompete eIF4G is still not fully known. Although the canonical
binding helix is conserved between eIF4G and 4E-BPs, the residues
beyond the consensus sequence are not, and in the case of 4E-BPs,
this non-consensus sequence harbors several phosphorylation sites
[15,16,18e20]. It has been reported that phosphorylation of T37 and
T46 of 4E-BP2 induces folding of the protein into a four b-strand
folded domain that sequesters the eIF4E binding motif [21]. More
recently, it was found that hyper-phosphorylation of the C-terminal
degraders [30,36]. Kaur et al. previously attempted to employ
PROTAC strategies against eIF4E using derivatized cap analogues
coupled to lenalidomide or von Hippel-Lindau (VHL) ligands [37].
Bn7GDP linked to lenalidomide was shown to be an effective eIF4E
binder in vitro with an affinity of 50 mM, but all tested compounds
failed to degrade eIF4E in cellular assays. This was discovered to be
due to insufficient cell permeability, a known problem for highly
negatively charged cap analogues. Our secondary goal in this study
was therefore to generate cell-permeable degraders of eIF4E based
on our inhibitors 4EGI-1 and i4EG-BiP. Here, we present the
structural and biochemical characterization of i4EG-BiP and our
PROTAC derivatives of 4EGI-1 and i4EG-BiP. Since there are many
intrinsically disordered phosphor-sites of 4E-BP2 stabilizes the b-
stranded folded domain, further reducing 4E-BP2 binding to eIF4E
[22]. Dislodging eIF4G by binding of either 4E-BPs or a small
molecule inhibitor would stop cap-dependent translation and this
latter approach could be used for the therapeutic treatment of
eIF4E-mediated pathogenic dysregulation of translation.
acronyms and abbreviations, we have included
a
table
(Supplementary Table 2) in the supplementary section to ease
readership.
4EGI-1 was the first identified small molecule inhibitor of the
eIF4E-eIF4G interaction, binding to eIF4E with low micromolar af-
2. Results
finity (IC50 ¼ 57
1 mM [23,24]). 4EGI-1 has been shown to be
effective in arresting proliferation in a number of cancer cell lines
[25e28]. The crystal structure of 4EGI-1 in complex with eIF4E
reported in 2014 (PDB: 4TPW) [24] showed that 4EGI-1 bound to
eIF4E at a site that is distinctly different from the primary binding
site of the eIF4G/4E-BP consensus sequence or the m7GTP cap
binding site. In the crystal structure, the binding of 4EGI-1 induces a
2.1. Discovery of i4EG-BiP
In an effort to increase the efficacy of 4EGI-1, we synthesized
and screened multiple analogues of 4EGI-1. The analogues syn-
thesized included variations of the di-chlorophenyl moiety, the
nitrophenyl moiety or the thiazole core. However, none of these
compound variations improved the binding affinity. Amongst the
synthesized compounds were also dimeric versions of 4EGI-1,
mirrored along the di-chlorophenyl moiety. One of the in-
termediates from this synthesis was the biphenyl compound i4EG-
BiP (Fig. 1B). This compound caught our attention due to its similar
eIF4G displacement properties when compared to 4EGI-1 (Fig. 1C);
i4EG-BiP displaces an eIF4G peptide from eIF4E with an IC50 value
conformational change in helix
a1 of eIF4E, thus leading to the
hypothesis that 4EGI-1 is an allosteric inhibitor. Another small
molecule inhibitor that displaces eIF4G from eIF4E is 4E1RCat,
which was discovered with a high-throughput screen using a time
resolved (TR)-FRET based assay [29]. There is no high-resolution
structures of 4E1RCat bound to eIF4E. Analogues of 4EGI-1 co-
crystallized by our group reveal binding at the same site as 4EGI-
1 [24]. This common binding site is proximal to a cavity on the
surface of eIF4E.
In an effort to increase the efficacy of 4EGI-1, we previously
synthesized and screened multiple analogues of this compound.
Systematic variation of the di-chlorophenyl “head”, the nitrophenyl
“tail” or the thiazole core did not produce analogues with signifi-
cantly increased binding affinity to eIF4E. Removal of the hydra-
zone linker consistently reduced binding affinity. In contrast,
certain significant changes in the aromatic “head” could be
accommodated without significant loss in binding affinity. For
example, an analogue (4EGI-1A, Supplementary Fig. 2) containing
an additional cyclohexyl ring showed activity similar to 4EGI-1 in
an FP assay measuring displacement of an eIF4G peptide from eIF4E
and was a part of the original publication [23]. We then synthesized
of 68
2 mM, which is similar to that of 4EGI-1. Since 4EGI-1 binds
on the surface of eIF4E, we were intrigued as to how the extra ar-
omatic ring would be stabilized, so to answer this question, we
solved the structure of i4EG-BiP in complex with eIF4E by X-ray
crystallography.
2.2. The structure of eIF4E bound to i4EG-BiP
The eIF4E structure in complex with i4EG-BiP was resolved by
molecular replacement to a resolution of 1.9 Å (Fig. 2A). The
structure can be accessed at the PDB databank with the PDB ID
7MEU. The refined model allowed us to unambiguously locate the
position of the ligand in the electron density map (Fig. 2B). i4EG-BiP
binds to a cavity on the surface of eIF4E that is near the 4EGI-1
3

P.D. Fischer, E. Papadopoulos, J.M. Dempersmier et al.
European Journal of Medicinal Chemistry 219 (2021) 113435
Fig. 2. A. Structure of eIF4E bound to i4EG-BiP. The asymmetric unit consists of two cap-bound eIF4E structures, of which one shows density for the small molecule. B. i4EG-BiP
density is unambiguously located between helix 1 and 2. C. Surface representation of the co-crystal structure shows the cavity into which i4EG-BiP binds.
a
a
binding site, opening up possibilities for the development of a new
class of eIF4E inhibitors. The structure of the protein itself has a
striking similarity to that of eIF4E bound to 4EGI-1 (PDB ID 4TPW).
The unit cell is comprised of a dimer with two copies of palm-like
eIF4E molecules arranged at 180ꢀ orientations relative to each other
around a pseudo-symmetry axis positioned between the two dor-
sal surfaces. m7GTP, which constitutes the cap structure of mRNA, is
bound to the palm of eIF4E. However, only one copy of the protein
(chain A) is occupied by the i4EG-BiP ligand, while the binding site
on the other copy (chain B) is empty. This is similar to the case of
4EGI-1, where we found the ligand bound to only one of the two
proteins in the unit cell. Furthermore, as observed in the case of
4EGI-1, the 3e10 helix between residues S82 and L85 has melted
Y76, Y91 and W130. Hydrogen bonds are formed between the nitro
group of i4EG-BiP and the amide side chain of Q80 as well as be-
tween the hydrazone NH of i4EG-BiP and the backbone carbonyl
oxygen of Q80. The carboxylic acid moiety of i4EG-BiP forms a
hydrogen bond with the NH backbone of L85 (Fig. 3C), instead of
the salt-bridge with K49, which represents the strongest interac-
tion found for 4EGI-1 (Fig. 3D). Upon binding of i4EG-BiP, the
helix is extended by the amino acids Q80, L81, S82 and S83. These
four residues constitute a loop that faces the 2 helix in other eIF4E
a1
a
structures without a small molecule inhibitor. This observed helix
extension is needed to create room for the thiazole, hydrazone and
nitrophenyl moieties of i4EG-BiP and the formation of hydrogen
bonds between Q80 and the ligand. It is unclear why the same helix
extension is observed with the binding of 4EGI-1, since in this case
the structural rearrangement neither enables new protein in-
teractions nor does it serve to avoid steric clashes. This helix
extension does, however, appear to be a crucial factor in eIF4G
displacement which is discussed in greater detail later.
into a loop while the a1 helix (residues H78-L85) is extended by
one turn upon engagement of i4EG-BiP. This conformational
change induced by i4EG-BiP is seen in chain A, but is obviously not
present in chain B, which lacks the small molecule.
i4EG-BiP fits well within its binding cavity on the surface of
eIF4E, burying 334 Å2 of the eIF4E surface area, yet leaving room to
expand the small molecule near the thiazole moiety, which is
proximal to a deep cavity as shown in Fig. 2C. There is an estimated
200 Å2 of additional surface area available that could be leveraged
to increase the binding affinity of i4EG-BiP. The i4EG-BiP binding
mode to eIF4E is distinct from any previously described eIF4E
2.3. i4EG-BiP is able to displace the eIF4G peptide from eIF4E
To assess the ability of inhibitors binding to eIF4E to disrupt the
eIF4E-eIF4G interaction, we used a previously reported fluores-
cence polarization (FP) assay [23]. This assay leverages the fact that
a free fluorescently labelled eIF4G peptide has a molecular corre-
lation time that is vastly different from when this peptide is bound
to eIF4E, thereby allowing displaced (free) peptide to be distin-
guished from bound peptide. In this displacement assay, increasing
concentrations of compounds were titrated against eIF4E bound to
binder. The compound engages the pocket between helix
a1 and
helix 2 (Fig. 3A). The biphenyl moiety is buried deeply, while the
a
carboxylic acid and the nitrophenyl functionalities are solvent
exposed. The compound engages eIF4E using hydrophobic in-
teractions with residues L45, L75, I79, L93 and L134. Edge-to-face
p
-p
interactions are formed between the biphenyl moiety and
a fluorescently labelled eIF4G peptide. Compounds that can
4

P.D. Fischer, E. Papadopoulos, J.M. Dempersmier et al.
European Journal of Medicinal Chemistry 219 (2021) 113435
Fig. 3. A. Interaction profile of i4EG-BiP. eIF4E is depicted in light blue, i4EG-BiP is depicted in salmon and the side chains of eIF4E involved in the interaction are depicted in blue. B.
Interaction profile of 4EGI-1. eIF4E is depicted in light blue, 4EGI-1 is depicted in salmon and the side chains of eIF4E involved in the interaction are depicted in blue. C. i4EG-BiP
makes hydrophobic interactions with most residues of helix a1 and a2. Hydrogen bonds are observed between the small molecule and the side chains of Gln80 and Leu85. D. 4EGI-1
makes primarily hydrophobic interactions with different residues than those interacting with i4EG-BiP. A salt-bridge and hydrogen bond between the small molecule and Lys49 is
also formed. E. Depiction of eIF4E residues within 4 Å of i4EG-BiP.
displace the eIF4G peptide led to a reduction in FP signals. Our
results show that i4EG-BiP was able to displace the eIF4G peptide
lines due to reported sensitivity to eIF4E ablation in the DepMap
database [39]. Cells were treated once with serial dilutions of either
4EGI-1 or i4EG-BiP and assayed at 24, 48 or 72 h (Fig. 4A). 24 h of
from eIF4E with an IC50 of 68
observed for 4EGI-1 (Fig. 1C).
2 mM, which is similar to that
treating MCF7 cells with 4EGI-1 resulted in an IC50 of 71
mM, which
decreased to 43 M at 48 h and 35 M at 72 h, thus resulting in a 2-
m
m
2.4. i4EG-BiP impairs viability in MCF7 and MM1S cells
fold decrease in viability as compared to 24 h. i4EG-BiP treatment
inhibited cell viability of MCF7 cells over a treatment period of 24 h
To test the activity of i4EG-BiP in cells, we performed time-
dependent CS-BLI cell viability assays [38] in two different cell
lines. MCF7 and MM1S cells were chosen as representative cell
with an IC50 value of 59
18 M when treatment is prolonged for 72 h. This corresponds to a
decrease in viability of more than 3-fold from 72 h treatment as
mM, which decreased to 27 mM at 48 h and
m
5

P.D. Fischer, E. Papadopoulos, J.M. Dempersmier et al.
European Journal of Medicinal Chemistry 219 (2021) 113435
Fig. 4. A. Cell viability assay of 4EGI-1 and i4EG-BiP in two different cell lines (MCF7 and MM1S) as a function of concentration over 24 h, 48 h and 72 h. Error bars represent
standard deviation and in all cases n ¼ 3. B. A dual luciferase assay for 4EGI-1 and i4EG-BiP shows a greater decrease in cap-dependent translation with increasing compound
concentration for i4EG-BiP. C. Western blot of an eIF4E pull down with m7-GDP functionalized agarose beads in the absence and presence of increasing concentrations of i4EG-BiP.
With increasing concentrations of i4EG-BiP, the eIF4E/eIF4G ratio increases, while the eIF4E/4E-BP ratio decreases.
compared to 24 h. In MM1S cells, we did not observe a significant
time-dependent effect with either compound. The IC50 values for
2.5. i4EG-BiP inhibits cap-dependent translation better than 4EGI-1
in cellular assays
4EGI-1 were 23
and 16 M for 72 h. i4EG-BiP was less active in MM1S cells, with
IC50 values of 67 M after treatment for 24 h, 62 M for 48 h and
56 M for 72 h. This data indicates that i4EG-BiP affects the viability
of MCF7 to a similar extent to 4EGI-1. In MM1S cells i4EG-BiP is less
active compared to 4EGI-1, while both compounds do not exert
significant time dependency in their cytotoxic effects.
mM for a treatment period of 24 h, 19 mM for 48 h
m
To test the inhibition of cap-dependent protein translation by
i4EG-BiP in cells, we transfected a plasmid into HEK293 cells that
produces a bicistronic transcript containing a 50-Firefly luciferase
followed by an IRES and a 30-Renilla luciferase. The Firefly luciferase
therefore reports on cap-dependent translation while the Renilla
luciferase reports on cap-independent translation. The day after
m
m
m
6

P.D. Fischer, E. Papadopoulos, J.M. Dempersmier et al.
European Journal of Medicinal Chemistry 219 (2021) 113435
transfection, cells were treated with increasing concentrations of
compounds and following a 3 h incubation, Firefly and Renilla
luciferase activity were measured by chemiluminescence. The
interaction between eIF4E and eIF4G is essential for cap-dependent
translation, but not IRES-dependent translation, and therefore only
the Firely luciferase activity should be affected by compound
treatment. Interestingly, we found a 65% reduction in the Firefly to
Renilla (L/R) luciferase activity ratio with i4EG-BiP treatment while
treatment with 4EGI-1 resulted in a considerably lower 15% L/R
ratio reduction (Fig. 4B). This data indicates that i4EG-BiP inhibits
cap-dependent translation to a greater extent than 4EGI-1.
As a starting point, we chose to synthesize the first PROTAC as a
4EGI-1 analogue linked to a 2-phenoxyacetamide derivative of
thalidomide, as described for dBET1, a BET bromodomain degrader
developed by Winter et al. [33]. To achieve this, 4-
Hydroxythalidomide 8 was synthesized by reacting 3-Hydroxy
pththalic anhydride and 3-Aminopiperidine-2,6-dione in glacial
acetic acid with 96% yield (Fig. 6A). The linker was prepared using a
benzyloxycarbonyl (CBz) protected diamine, which is more
economical than the previously used Boc protected variant [33].
Benzyl (4-aminobutyl)carbamate was reacted with 2-Chloroacetyl
chloride to produce the CBz-protected 2-Chloroamide 9 in 91%
yield, which was subsequently transformed into the more reactive 2-
Iodoamide10 viaFinkelsteinreactioninaquantitativemanner.10 was
then coupled to 4-Hydroxythalidomde 8 to yield the CBz-protected
Thalidomide linker, which was deprotected using hydrogen gas and
a Palladiumblack catalysttogivethefreeThalidomidelinker11 in41%
yield.
To selectively deprotect the acid moiety on the thiazole ring and
generate the final PROTAC d4E-1, methyl ester hydrolysis of the
tert-butyl protected 4EGI-1 building block 7 was performed using
lithium hydroxide at 4 ꢀC to create the ready-to-couple 4EGI-1
derivative 12 (Fig. 6B). The final product d4E-1 was then generated
with a total yield of 41% over two steps using HATU-mediated
coupling of the 4EGI-1 derivative 12with the Thalidomide linker
11 and subsequent tert-butyl deprotection using 12% trifluoroacetic
acid in dichloromethane.
2.6. i4EG-BiP disrupts the eIF4E-eIF4G and strengthens the eIF4E-
4E-BP1 interactions
We assessed the interaction between eIF4E and its binding
partners eIF4G and 4E-BP1 in pull-down experiments using a
simplified cap-analogue (m7GDP-agarose) resin [40]. HeLa cell ly-
sates were incubated with either DMSO as a negative control or
increasing concentrations of i4EG-BiP. After incubation of the
mixture with m7GDP agarose resin, beads were washed, followed
by elution of bound proteins and analysis by Western Blot. As can
be seen in Fig. 4C, the amount of eIF4G relative to eIF4E decreases
with increasing amounts of i4EG-BiP, while the amount of 4E-BP1
increases. These results suggest that the extension of the a1-helix
in eIF4E, caused by binding of i4EG-BiP, is detrimental for eIF4G
binding to eIF4E, but does not adversely impact 4E-BP1 binding.
2.8. d4E-1 interacts with eIF4E, but does not engage Cereblon in
cells
2.7. Design and synthesis of the 4EGI-1-based PROTAC d4E-1
Next, we decided to leverage the structural information we
had to design eIF4E-targeting degrader molecules (PROTACs). The
idea was to deal a double-blow to inhibit translation, one by
blocking the eIF4E-eIF4G interaction, and the other by degrading
eIF4E. We first assessed the structure of 4EGI-1 for functional
groups to which a flexible linker could be coupled for the
attachment of thalidomide. The carboxylic acid moiety appeared
to be ideal for linkage; however, it is also involved in a crucial
interaction with eIF4E (Fig. 3D). We therefore decided to attach
the linker to the free carbon of the thiazole moiety, which is
surface exposed in the co-crystal structure (PDB ID: 4TPW).
Based on the simulated modelling of eIF4E bound to 4EGI-1 in
complex with Cereblon (CRBN) bound to lenalidomide (PDB ID:
4CI2), we decided to synthesize the first test compound with a 4-
carbon linker (Fig. 5A).
After successful synthesis of the 4EGI-1 based PROTAC d4E-1, we
next wanted to analyse binding of the bifunctional molecule to its
targets: eIF4E and Cereblon.
2.8.1. Binding to eIF4E by displacement of the eIF4G peptide
To test whether d4E-1 can displace eIF4G in a similar manner
compared to 4EGI-1, the compound was tested for its ability to
displace a fluorescently tagged eIF4G peptide in an FP assay
(Fig. 8A). At the three measured concentrations (33, 100 and
300
mM), both d4E-1 and 4EGI-1 displaced the 4G peptide in a
similar manner. We therefore concluded that the binding affinity of
our PROTAC compound is comparable to that of the parent com-
pound, 4EGI-1.
The synthetic challenge was to introduce the acetic acid func-
tionalitytothethiazoleringandchooseaselectiveprotectionstrategy
for the resulting two carboxylic acids. Therefore, succinic anhydride
was reacted with 1,2-dichlorobenzene in a Friedel Crafts acylation
reaction to produce the phenyl-4-oxobutanoic acid 1 with 41% yield
(Fig. 5B). The carboxylic acid was protected using iodomethane as a
methylation agent to quantitively yield the methyl ester 2. Bromi-
nation of 2 in the alpha-keto position produced the phenyl-3-bromo-
4-oxobutanoate 3 with 96% yield. The hydrazine functionalized
thiazole 4 was obtained by reacting this product with thio-
semicarbazide. Due to the high reactivity of 4 it was used without
further purification. To finishthesynthesis isthe 4EGI-1 analogue, the
nitrophenyl-2-oxopropanoic acid 5 was created by hydrolysis of an
oxazol-5-one derivative, which was obtained from reacting 2-
Nitrobenzaldehyde with acetyl glycine. The overall yield of this 2-
step reaction was 70%. Then, the acid was protected using tert-butyl
acetate to form the tert-butyl ester 6 with 79% yield. The crude
hydrazineylthiazol4was then coupled to the tert-butyl ester6 toform
anE/Z-isomeric mixture of the protected 4EGI-1 buildingblock7with
63% yield.
2.8.2. Cellular assay to evaluate the ability of 4EGI-1 degraders to
engage Cereblon
Next, we wanted to assess the cellular CRBN engagement of d4E-
1. We used a previously described assay [41,42] in which com-
pounds are tested for their ability to rescue dBET6-induced (a
CRBN-dependent BET bromodomain degrader) degradation of
BRD4BD2 by competing for binding to CRBN. Flp293T cells stably
expressing a BRD4BD2-GFP fusion protein and an mCherry reporter
were co-treated with dBET6 at 100 nM and compounds in dose
response, with lenalidomide used as a positive control. The GFP/
RFP signal ratio was quantified using an Acumen laser scanning
cytometer (TTP Labtech). Active compounds were identified by an
increased GFP/mCherry ratio resulting from inhibition of BRD4BD2
-
GFP degradation by dBET6. To our surprise, d4E-1 did not prevent
degradation of BRD4BD2 in a dose dependent manner (results
shown later). We hypothesized that this could be due to the net
negative charge of d4E-1, which could hinder cellular uptake in a
similar manner to the cap-based degraders previously reported
[37]. Therefore, our next goal was to synthesize an optimized
prodrug version of d4E-1.
7

P.D. Fischer, E. Papadopoulos, J.M. Dempersmier et al.
European Journal of Medicinal Chemistry 219 (2021) 113435
Fig. 5. A. Docking of lenalidomide bound to Cereblon (PDB ID: 4CI2) and eIF4E bound to 4EGI-1 (PDB ID: 4TPW). Surface exposed parts of the small molecule can be arranged in
proximity without steric clashes. The target degrader molecule derived from this docking pose (d4E-1) is depicted on the right. B. Synthesis of the double protected 4EGI-1 building
block 7.
2.9. Design, synthesis and CRBN engagement of a 4EGI-1-based
prodrug PROTAC
scaffold, as this functionality reduces cellular uptake (results from
i4EG-BiP PROTAC analogues shown later).
The arylamine linked thalidomide derivative 15 was synthesized
as previously described by others [45]. To make the free acid of the
modified 4EGI-1 PROTAC, the hydrazine derivative 4 was reacted
with pyruvic acid and subsequently protected using tert-butylace-
tate (Fig. 7). HATU-mediated amide coupling with 15 and acidic
deprotection with 12% trifluoroacetic acid in dichloromethane
yielded the free acid of the 4EGI-1-prodrug PROTAC d4E-6. Despite
repeated efforts, the purity of the d4E-6 free acid could not be
improved to satisfying levels, hence the crude product was reacted
with pivaloyloxymethyl iodide to generate pure d4E-6 with an
overall yield of 31% over 5 steps.
To negate the hypothesized negative effects from the negative
charge on the carboxylic acid, a commonly applied prodrug approach
was employed. In this approach, the carboxylate is modified with a
protective groupthat can becleavedoff incells afteruptake. We chose
touseapivaloyloxymethylesterasaprodrugunit, amoietythatisalso
used in prodrug versions of ampicillin (pivampicillin) [43] or butyric
acid (AN-9) [44], for example. In addition to the prodrug strategy,
arylamine linkage to thalidomide was used as these CRBN ligands
reduce synthetic efforts significantly. To further improve cellular
uptake, the 2-nitrophenyl moiety was removed from the 4EGI-1
8

P.D. Fischer, E. Papadopoulos, J.M. Dempersmier et al.
European Journal of Medicinal Chemistry 219 (2021) 113435
Fig. 6. A. Synthesis of the 2-phenoxyacetamide thalidomide linker 11. B. Endgame of the synthesis of target structure d4E-1.
Using the same assay for cellular CRBN engagement, we evalu-
ated d4E-6 and its parent compound. While the free acid was not
capable of rescuing dBET6-induced degradation of BRD4 (data not
while beneficially eliminating the net negative charge of the
product. Furthermore, the structure suggests that the 2-
nitrophenyl moiety plays a minor role for interaction. On the ba-
sis of this observation, we decided to include structures that lack
this functional group. We tried to further improve CRBN binding by
switching from phthalimide ether to arylamine phthalimide. This
was driven by the fact that the field developed improvements in
linker design, which encouraged us to opt for arylamine phthali-
mides instead of the phthalimide ether used for d4E-1 [46].
Regarding chirality of the thalidomide analogues it is well estab-
lished that the stereocenter interconverts in vivo [47].
shown), the prodrug did with an IC50 value of ~20 mM (Fig. 8B).
These encouraging results supported our previous hypothesis that
the net negative charge in our 4EGI-1-based PROTAC d4E-1 was
root cause for its lack of activity.
2.10. Design and synthesis of the i4EG-BiP-based PROTACs (d4E-2
to d4E-5)
Starting from 1-([1,10-biphenyl]-4-yl)-2-bromoethan-1-one, the
crude reactive hydrazine derivative 13 was synthesized using thi-
osemicarbazide (Fig. 9A). i4EG-BiP and the i4EG-BiP derivative
lacking the 2-nitrophenyl moiety 14 were generated using 2-
nitrophenyl pyruvic acid and pyruvic acid with 63% and 57%
yield, respectively. The arylamine linked thalidomide derivatives
To optimize and expand the spectrum of possible eIF4E de-
graders, we switched to using i4EG-BiP as a scaffold for new PRO-
TACs. In this case, the carboxylic acid moiety is not involved in the
same crucial interaction found with 4EGI-1 (Fig. 3C and D).
Therefore, we hypothesized that direct linking of the acid to a
thalidomide linker should not result in a significant loss of affinity,
9

P.D. Fischer, E. Papadopoulos, J.M. Dempersmier et al.
European Journal of Medicinal Chemistry 219 (2021) 113435
Fig. 7. Synthesis of the 4EGI-1 prodrug PROTAC d4E-6 by POMylation of the free acid 4EGI-1 analog with iodomethyl pivalate.
Fig. 8. A fluorescence polarization assay shows that d4E-1 displaces the eIF4G peptide in a manner comparable to 4EGI-1. B. Cellular Cereblon engagement assay used to elucidate
intracellular activities of d4E-1 and d4E-6.
15, 16 and 17 were synthesized as previously described by others
[45] (Fig. 9B). As before, HATU-mediated amide coupling was used
to generate the PROTACs d4E-2, d4E-3, d4E-4 and d4E-5 with 43%,
39%, 40% and 43% yields.
2-nitrophenyl moiety on cellular uptake: d4E-2 (4-carbon linker,
with no nitrophenyl), and d4E-3 (4-carbon linker, nitrophenyl
included). The best molecule tested was d4E-2 with an IC50 value of
1.6
mM. In contrast, d4E-3 was approximately 8-fold less active
(with an IC50 value of 13
m
M), suggesting the nitrophenyl moiety
2.11. i4EG-BiP-based degraders engage Cereblon in cells
had a negative effect on cellular uptake (Fig. 9D). Based on these
findings, we tested three nitrophenyl-free compounds with varying
carbon linker lengths (d4E-2, d4E-4 and d4E-5) for cytotoxicity in
HeLa cells (Fig. 9C). The efficacy of the tested compounds decreased
To test whether the new PROTACs were able to engage CRBN in
cells, we tested two model compounds to evaluate the effects of the
10

P.D. Fischer, E. Papadopoulos, J.M. Dempersmier et al.
European Journal of Medicinal Chemistry 219 (2021) 113435
Fig. 9. A. Synthesis of i4EG-BiP and the i4EG-BiP deriative lacking the 2-nitrophenyl moiety (14). B. Endgame of the synthesis of target structures d4E-2, d4E-3, d4E-4 and d4E-5. C.
Cell viability data for PROTACs in HeLa cells (triplicates over 24 h). D. Cellular Cereblon engagement assay used to elucidate intracellular activities of d4E-2 and d4E-3, to compare
the influence of the 2-nitrophenyl moiety on cellular uptake. E. Representative Western Blot of eIF4E degradation by d4E-4 and d4E-6 at 10 mM compound concentration.
11

P.D. Fischer, E. Papadopoulos, J.M. Dempersmier et al.
European Journal of Medicinal Chemistry 219 (2021) 113435
with increasing linker length, but more strikingly all PROTACs
displayed strong cytotoxic effects, even at rather low concentra-
tions, with d4E-4 and d4E-5 killing over 50% of the cells at the
3. Discussion
The cap-dependent translation machinery represents a rela-
tively underexplored area of vulnerability for targeted therapeutics
in cancer, with many genes (eIF4A, eIF4E, eIF4G) listed as sensitive
in DepMap. Inhibiting the protein-protein interaction of eIF4E with
eIF4G and concomitantly disrupting the translation initiation
complex has been shown to be an attractive therapeutic option.
Inhibitors of the eIF4E-eIF4G interaction have been shown to
inhibit translation, and exhibit antiproliferative and putative anti-
tumor activities [51,52].
lowest concentration measured (10 mM).
After these promising initial biochemical and cellular results, we
proceeded to test our two best compounds (d4E-4 and d4E-6) for
their potential to induce eIF4E degradation in HEK293T cells. We
performed two experiments: 1) treatment of HEK293T cells with
PROTAC concentrations varying between 0.1 and 10
of 24 h, and 2) treatment of the same number of HEK293Tcells with
M PROTAC for varying lengths of time, ranging from 0.5 h to 24 h.
mM for a period
1
m
Unfortunately, neither experiment showed significant degradation
of eIF4E, as assessed by immunoblotting (representative Western
Blot shown in Fig. 9E). However, we did observe that eIF4E levels
varied at different time points for both compounds as well as DMSO
treated cells. This suggests that a cellular response may be coun-
teracting eIF4E degradation. To test whether eIF4E was being
ubiquitinated in the first place, HEK293T cells were incubated with
The previously described 4EGI-1 and i4EG-BiP, introduced here,
bind to different allosteric sites on eIF4E, but both result in a similar
conformational change (extension of the helix
a1 by one turn)
which in turn displaces eIF4G. i4EG-BiP binds to an internal cavity
of eIF4E, rather than on the surface as done by 4EGI-1. There is a
distinct possibility that an endogenous metabolite could also
engage this i4EG-BiP cavity, thus modulating translation, but such a
molecule has yet to be identified. We found that 4EGI-1 and i4EG-
BiP can displace eIF4G but not 4E-BP1 in cells, although both eIF4G
and 4E-BP1 share the same consensus binding motif. We posit that
extension of the helix resulting from engagement of the small
molecules hinder binding of eIF4G due to a steric clash. Specifically,
the side chain of Ser-82 in eIF4E will clash with a conserved Leu-
1 mM PROTACs in the presence of 100 nM Bortezomib, a known
inhibitor of the 26S proteasome [48]. We compared eIF4E ubiq-
uitination in these cells to those from cells treated with Bortezomib
and a DMSO control; however, we were not able to detect any
significant differences (data not shown), suggesting that our PRO-
TACs did not induce CRBN-mediated ubiquitination of eIF4E.
We performed quantitative proteomics experiments to measure
the global change in protein expression resulting from treatment
with d4E-2 relative to DMSO control. Proteomics experiments have
confirmed that d4E-2 is engaging CRBN in cells as we see down-
regulation of C2H2 zinc finger targets, SALL4, ZNF692 and ZNF827,
which are commonly degraded as a result of the IMiD CRBN-
binding handle of the degrader [58] (Supplementary Fig. 3). We
also observed that d4E-2 did not downregulate eIF4E.
641 in eIF4G that is
a part of the extended interface
(Supplemnentary Fig. 1). In the case of 4E-BP1, this steric clash
would not occur. Instead, we predict the complex with eIF4E would
be stabilized by a putative hydrogen bond between the sidechain of
Ser-82 in eIF4E and the backbone of Ser-83 from 4E-BP1 (Supple-
mentary movie M1). This model in which the small molecule would
inhibit the translation activator eIF4G and stabilize the inhibitor 4E-
BP1 presents a two-fold attack on translation initiation. Since the
inhibitory effect stems from a similar allosteric change, both 4EGI-1
and i4EG-BiP, although binding to different sites, have similar
ability to displace the eIF4G/4E-BP consensus peptide and exhibit
2.12. Solution NMR studies confirm binding modes of the i4EG-BiP-
based degraders to eIF4E are similar to that of the parental
compound
similar cellular activity. Exactly how the extension of the helix a1
displaces the eIF4G/4E-BP consensus peptide, which lacks the
extended binding region of the full-length proteins, still remains an
open question.
Supplementary video related to this article can be found at
https://doi.org/10.1016/j.ejmech.2021.113435
Due to these unexpected results, we wanted to confirm that the
derivatization of i4EG-BiP with thalidomide did not affect the
binding of the degraders to eIF4E. To do this, we used solution-state
nuclear magnetic resonance (NMR) spectroscopy as an additional
method to identify the binding site of i4EG-BiP on eIF4E and
compare it with the i4EG-BiP-based PROTAC d4E-4 (Fig. 10A and C).
Chemical shift perturbations (CSPs) were observed when 15N-
labelled GB1-eIF4E was titrated with i4EG-BiP or d4E-4. Significant
CSPs were observed in both cases at the binding interface identified
from the co-crystal structure (Fig. 10B and D). A molecular docking
simulation of d4E-4 was carried out with QuickVina 2 [49], which is
based on AutoDock Vina [50]. The obtained docking score
was ꢁ8.2 kcal/mol. CSPs plotted onto the structure of eIF4E bound to
i4EG-BiP as well as the docked structure of d4E-4 reveal significant
CSPs located in similar regions for both small molecules (Fig. 10E and
F). Although the overall CSP patterns look similar for i4EG-BiP and
d4E-4, indicating similar binding modes, some additional CSPs were
observed towards the N- and C-termini of the protein when d4E-4
was added. This could originate from non-specific binding events
due to the 3-fold excess of inhibitor that was used in these experi-
ments, in order to obtain significant CSPs. Alternatively, these
additional CSPs could be the result of conformational changes
induced by ligand binding. In particular, the C-terminal residues
constitute the cap-binding region and there have been previous re-
ports of crosstalk between eIF4G binding to the dorsal surface and
cap binding to the palm of eIF4E. This may be the result of dynamic
loops in the cap-binding region, which have been observed in
multiple conformations in the various published crystal structures.
To further improve the potency of the inhibitors 4EGI-1 and
i4EG-BiP, we designed PROTAC versions of the two molecules. Here,
we employed 4EGI-1 and the novel inhibitor i4EG-BiP as scaffolds
to create cell-permeable thalidomide conjugates that bind to eIF4E
and engage Cereblon. Engagement of Cereblon as well as VHL with
eIF4E ligands has been previously attempted by Kaur et al. using
cap analogues. Even though they observed binding to eIF4E by their
PROTACs, target degradation was not achieved. The authors
attributed this failure to the net negative charge of the m7GDP-
based compounds [37]. Here we show that the design of a neutral
PROTAC based on 4EGI-1 using a prodrug approach and neutral
PROTACs based on i4EG-BiP using the carboxylic acid moiety as a
linking point seems to alleviate this hurdle on efficacy. Using
cellular Cereblon engagement assays, we were able to show a
concentration-dependent cell-based effect of our compounds.
However, we were not able to detect any targeted cellular
degradation of eIF4E. This could, in part, be due to the relatively low
binding affinity of the parent compounds, 4EGI-1 and i4EG-BiP.
Using medicinal chemistry approaches to improve the binding af-
finity of i4EG-BiP might help to improve the PROTAC strategy for
our i4EG-BiP-based molecules. This is particularly possible as there
is unoccupied space on eIF4E in the vicinity of the i4EG-BiP binding
site which can be leveraged to improve molecule affinity. The failed
degradation of eIF4E could also be explained by increased binding
12

P.D. Fischer, E. Papadopoulos, J.M. Dempersmier et al.
European Journal of Medicinal Chemistry 219 (2021) 113435
Fig. 10. A. 15Ne1H-HSQC overlay of GB1-eIF4E in the absence (blue) and presence (red) of i4EG-BiP. B. Chemical shift perturbation (CSP) plot of changes induced by i4EG-BiP as a
function of residue number. The solid line represents the mean value of all CSPs, the dashed line represents the mean value plus one standard deviation of all CSPs. C. 15Ne1H-HSQC
overlay of GB1-eIF4E in the absence (blue) and presence (orange) of d4E-4. D. CSP plot of changes induced by d4E-4 as a function of residue number. The solid line represents the
mean value of all CSPs, the dashed line represents the mean value plus one standard deviation of all CSPs. E. CSPs plotted onto the eIF4E-i4EG-BiP structure: Highlighted in orange
are residues with CSPs > mean þ one standard deviation, highlighted in red are residues with CSPs > mean þ two standard deviations. F. CSPs plotted onto the docked eIF4E-d4E-4
structure. Highlighted in orange are residues with CSPs > mean þ one standard deviation, highlighted in red are residues with CSPs > mean þ two standard deviations.
13

P.D. Fischer, E. Papadopoulos, J.M. Dempersmier et al.
European Journal of Medicinal Chemistry 219 (2021) 113435
of 4E-BP1 to eIF4E as a result of eIF4E binding 4EGI-1 or i4EG-BiP.
Increased binding of 4E-BP1 to eIF4E could result from conforma-
tional changes in eIF4E upon small molecule binding, which could
increase the affinity for 4E-BP1, and/or the fact that the competing
binding protein, eIF4G, is no longer able to engage eIF4E. It has been
previously shown that ubiquitination of eIF4E at K159 is orches-
trated by the E3 ubiquitin ligase CHIP, which leads to subsequent
proteasomal degradation, and furthermore that this ubiquitination
can be blocked by overexpression of 4E-BP1 [53]. Therefore,
increased 4E-BP1 binding as a result of 4EGI-1 or i4EG-BiP binding
could protect eIF4E from ubiquitination and stabilize the protein. It
should however be noted that in our current study the ubiquiti-
nation is performed in a Cereblon-dependent manner, which might
not have the same inhibition response to 4E-BP1 as CHIP. Future
studies will be needed to address this possible obstructive role of
4E-BP1 on targeted degradation of eIF4E. If indeed the association
of 4E-BP1 prevents the ubiquitination of eIF4E, an interesting
approach would be to use 4E1RCat as a scaffold to create eIF4E
targeting PROTACs, since this inhibitor has been shown to displace
both eIF4G and 4E-BP1 binding to eIF4E [29]. Utilizing the syn-
thetical method we provide in this manuscript to link the carbox-
ylic acid moiety of 4E1RCat to lenalidomide could circumvent both
cell-permeability hurdles as well as evasion of eIF4E ubiquitination
by enhanced 4E-BP1 binding.
Following concentration, up to 4 mL of eluate was subjected to size-
exclusion chromatography using a Superdex75 16/10 preparative
column (GE Healthcare) equilibrated with 10 mM HEPES, pH 7.5,
125 mM NaCl, and 1 mM TCEP buffer. The collected pure protein
fractions were concentrated to a final concentration (by ultrafil-
tration) to 1 mg/mL as evaluated by NanoDrop™ at 280 nm. The
total yield of the aforementioned process was approximately
3e5 mg of pure protein from 1 L of culture.
4.2. Protein crystallization
Crystallization was performed using the sitting drop method
and all crystals were grown in drops containing 1
mL of protein-
ligand solution and 1 L of crystallization solution containing
m
10e25% (vol/vol) 3.3-kDa PEG, 100 mM MES, pH 6.0, 10% (vol/vol)
isopropanol. The protein-ligand solution used for crystallization
was prepared by mixing eIF4E (9 mg/mL) and the small molecule
(i4EG-BiP or analogues) solution (12.5 mM in DMSO) at an
approximately 1:1 stoichiometry. The resulting proteinesmall
molecule mixture was then serially diluted from 9 to 1 mg/mL
and used to set up sitting drop crystal trials. Crystals began to form
in 2 days and grew to full size at day 4. They were inspected and
based on their size and morphology, the optimal conditions of
protein concentration and PEG were determined. Crystals were
harvested and quickly transferred to a cryoprotectant solution of
crystallization buffer plus 10% (v/v) glycerol before flash freezing in
liquid nitrogen.
Blocking cap-dependent translation provides an attractive op-
portunity for future efforts to target cancer cells and eIF4E is the
master regulator of this process. Disrupting the crucial interaction
of eIF4E with its scaffold protein eIF4G or degrading eIF4E alto-
gether are two independent routes to inhibit eIF4E function. Here
we attempted to synergistically apply both these approaches.
Although we did not achieve successful degradation of eIF4E, our
successful targeting of eIF4E in this study with a newly identified
small molecule inhibitor, i4EG-BiP, paves way for future efforts.
4.3. X-ray diffraction data collection
X-ray diffraction data were collected at the APS X-Ray Syn-
chrotron Source at the Argonne National Laboratory from single
protein ligand complex crystals. The data were integrated using
XDS. The results indicated monoclinic space group P21 crystals
with diffraction up to 1.9 Å resolution. Phases were calculated by
molecular replacement using PHASER and an initial model of eIF4E
(PDB ID 4TPW). Subsequently, models were manually inspected
and refined in Coot followed by further rounds of phase calcula-
tions by molecular replacement in PHENIX and model structure
refinement until a minimum R-free was reached. The position of
the i4EG-BiP ligand was clearly seen in the Fo-Fc map before the
ligand was included in the model.
4. Material and methods
4.1. Expression and purification of eIF4E
A construct of human eIF4E was expressed in transformed
Escherichia coli BL21(DE3). A D26-eIF4E or GB1-eIF4E construct in a
pET-28(þ) backbone was used for crystallography and NMR studies,
respectively. Bacteria were grown in Luria Broth (LB) at 37 ꢀC.
Protein expression was induced by the addition of 0.1 mM isopro-
pyl-
b
-
D
-thiogalactopyranoside at OD600 ¼ 0.6 followed by incuba-
4.4. Nuclear magnetic resonance experiments
tion overnight at 23 ꢀC. Cells were harvested with a yield of 3 g/L
wet pellet and stored at ꢁ30 ꢀC. Bacterial pellets were resuspended
by slow pipetting in lysis buffer constituting of 50 mM Tris,HCl, pH
7.5, 100 mM NaCl, 1% Triton-X, 5 mM tris(2-carboxyethyl)phos-
phine (TCEP), 1 cOmplete™ Protease Inhibitor Cocktail tablet,
lysozyme, RNase, and DNase.
15Ne1H-TROSY-HSQC spectra were recorded on a Bruker Avance
III 800 MHz spectrometer with a TXO-style cryogenically cooled
probe. 15N-labelled GB1-eIF4E was concentrated to a final concen-
tration of 100 mM in a buffer composed of 50 mM sodium phos-
phate, pH 6.5, 50 mM potassium chloride, 2 mM DTT and 5% D2O. A
reference spectrum was recorded at 298 K by addition of DMSO to a
final concentration of 1.5%. NMR titration was performed by
recording 15Ne1H-TROSY-HSQC spectra in the presence of either
Cells were subsequently homogenized in a cell microfluidizer
and the lysates were centrifuged at 38,000ꢂg for 1.5 h. After
centrifugation the clarified lysate supernatant was first passed
through a 0.45
m
m cellulose acetate syringe-filter and then passed
i4EG-BiP or d4E-4 at 50, 100 and 300 mM concentrations (diluted
over diethylaminoethylcellolose (DEAE) column previously
a
from 20 mM stock solutions in DMSO). NMR experiments were
processed with nmrPipe and analyzed using the ccpNMR software
(version 2.4.1) [54].
equilibrated with the same lysis buffer. The DEAE flow-through was
loaded on adipiceagarose-m7GDP column and after 0.5 h of bind-
ing, the column was washed with 50 mL of wash buffer (10 mM
HEPES, pH 7.5, 125 mM NaCl, and 1 mM TCEP) five times followed
by elution four times with 10 mL elution buffer (10 mM HEPES, pH
4.5. Docking of d4E-4 to eIF4E
7.5, 125 mM NaCl, 100
m
M m7GTP plus 10 mM TCEP). The eluted
The protein structure used for docking was the structure of
i4EG-BiP bound to eIF4E, with i4EG-BiP removed. The receptor
structure was prepared in PDBQT format with AutoDock Tools,
which is part of MGLTools [55], by assigning AutoDock atom types
and merging the nonpolar hydrogen atoms. The ligand was
protein concentration was assessed by Bradford assay (Bio-Rad,
Hercules, CA, USA) according to the manufacturer’s instructions
and then concentrated to a final volume of 3 mL by ultrafiltration
through
a 15 mL, 10-kDa-cutoff Millipore centrifugal filter.
14

P.D. Fischer, E. Papadopoulos, J.M. Dempersmier et al.
European Journal of Medicinal Chemistry 219 (2021) 113435
prepared in PDBQT format with Open Babel [56], which included
computation of the 3-dimensional structure. The docking box was
18 Å ꢂ 30 Å x 18 Å, and the docking exhaustiveness was set to 4. The
receptor was held rigid during the docking procedure, and 10
replicates of the docking procedure were executed.
format. The resulting images were analyzed using CellProfiler [57].
A series of image analysis steps (an ‘image analysis pipeline’) was
constructed. First, the red and green channels were aligned and
cropped to target the middle of each well (to avoid analysis of the
heavily clumped cells at the edges). A background illumination
function was calculated for both red and green channels of each
well individually and subtracted to correct for illumination varia-
tions across the 384-well plate from various sources of error. An
additional step was then applied to the green channel to suppress
the analysis of large auto fluorescent artifacts and enhance the
analysis of cell specific fluorescence by way of selecting for objects
under a given size (30 A U.) and with a given shape (speckles).
mCherry-positive cells were then identified in the red channel by
filtering for objects 8e60 pixels in diameter and by using intensity
to distinguish between clumped objects. The green channel was
then segmented into GFP positive and negative areas and objects
were labelled as GFP positive if at least 40% of it overlapped with a
GFP positive area. The fraction of GFP-positive cells/mCherry-pos-
itive cells in each well was then calculated, and the green and red
images were rescaled for visualization. The values for the concen-
trations that led to a 50% increase in BRD4BD2-eGFP accumulation
(EC50) were calculated using the nonlinear fit variable slope model
(GraphPad Software).
4.6. Fluorescence polarization experiments
For the fluorescence polarization assays, a GST-eIF4E construct
was used. It was expressed in E. coli and purified using the same
procedure described above for
D26-eIF4E without the DEAE col-
umn step. The assay was used to test the activity of various com-
pounds against eIF4E/eIF4Gepeptide complex formation. For the
fluorescent probe we used a purified eIF4G-peptide conjugated
with fluorescein derived by peptide synthesis with the sequence
KKQYDREFLLDFQFK-FITCH. Assay mixtures consisted of 150 nM
eIF4G-peptide plus 0.3 mM GST-eIF4E in 100 mM Na-phosphate, pH
7.5. A 384-well black plate was used to prepare serial dilutions of
i4EG-BiP and other compounds from a 12.5 mM stock solution in
DMSO at a starting ligand concentration of 500 mM. The fluores-
cence polarization signal was recorded using an EnVison™ plate
reader.
4.7. Cellespecific bioluminescence imaging (CS-BLI) cell viability
assay
4.10. Proteomics methods
CS-BLI was performed according to a previously published high-
throughput cell viability assay [38]. Briefly, Luciferase-expressing
MM1S cells and MCF7 were cultured in T75 flasks. RPMI or
DMEM media (as appropriate), supplemented with 10% FBS, and
penicillin and streptomycin were used accordingly. 104 cells were
seeded in 50 mL of media in each well of a 96-well, tissue-culture-
treated, white flat bottom plate and left to adhere overnight in a
cell-culture incubator. The next day we supplemented the well
4.10.1. Global quantitative proteomics sample preparation
Kelly cells were treated with DMSO (biological triplicate) or d4E-
2 at 1 m
M for 5 h and cells were harvested by centrifugation at 4 ꢀC.
Cell lysis was performed by resuspension of the cell pellet in
denaturing Urea buffer (8 M Urea, 50 mM NaCl, 50 mM 4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid (EPPS) pH 8.5,
Protease and Phosphatase inhibitors), followed by manual ho-
mogenization by 20 passes through a 21-gauge needle. Cell lysate
was clarified by centrifugation and global protein quantified using a
volume with 50
mL of media with serially diluted compounds. After
24 h we added 5
m
L of 1 mg/mL stock of beetle -luciferin, let it
D
Bradford assay (Bio-Rad). 100 mg of protein from each treatment
equilibrate at 37 ꢀC for 30 min, and top-read the chem-
iluminescence plate using a BioTek Synergy HTX plate reader. The
reads were repeated at 48 and 72 h. Collected data were normal-
ized, processed, and plotted with Scilab and GraphPad-Prism.
was reduced, alkylated, digested and TMT labelled for LC-MS
analysis as previously described [58]. The TMT labelled sample
was offline fractionated into 96 fractions by high pH reverse phase
HPLC (Agilent LC1260) through an aeris peptide xb-c18 column
(phenomenex) with mobile phase A containing 5% acetonitrile and
10 mM NH4HCO3 in LC-MS grade H2O, and mobile phase B con-
taining 90% acetonitrile and 5 mM NH4HCO3 in LC-MS grade H2O
(both pH 8.0). The resulting 96 fractions were recombined in a non-
contiguous manner into 24 fractions and desalted using C18 solid
phase extraction plates (SOLA, Thermo Fisher Scientific) followed
by subsequent mass spectrometry analysis.
4.8. Cell culture
HeLa cells were grown in 4.5 g/L glucose DMEM supplemented
with 10% fetal bovine serum (Gibco 16000-49) and 1% penicillin/
streptomycin. Cells were seeded into opaque white 96-well assay
plates at a density of 5x103 cells per well. 24 h post-seeding, the
medium was replaced with medium containing the drug at the
specified concentrations in equal volumes of DMSO. Cells were
assayed for viability 48 h later using CellTiter-Glo (Promega) and
plates were read on a CLARIOstar plus (BMG Labtech) plate reader.
Values were normalized to the DMSO control. A minimum of two
experiments was performed with representative data shown.
4.10.2. LC-MS data collection and analysis
Data were collected using an Orbitrap Eclipse Tribrid mass
spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) and
coupled with an UltiMate 3000 RSLCnano System. Peptides were
separated on an EasySpray ES803a.rev2 75
mm inner diameter
microcapillary column (Thermo Fisher Scientific). Peptides were
separated over a 190 min gradient of 9e32% acetonitrile in 1.0%
formic acid with a flow rate of 300 nL/min. Quantification was
performed using a MS3-based TMT method as described previously
(McAlister et al., 2014), with the addition of Real-Time Search MS3
acquisition implemented between MS2 and MS3 scans. The data
were acquired using a mass range of m/z 340e1350, 120,000 res-
olution, standard AGC and maximum injection time of 50 ms for the
peptide measurements in the Orbitrap. Data dependent MS2
spectra were acquired in the ion trap with a normalized collision
energy (NCE) set at 34%, custom AGC target and a maximum in-
jection time of 35 ms. Real-Time Search was performed with a
4.9. Cereblon engagement assay
Cells stably expressing the BRD4BD2-GFP with mCherry reporter
[41] were seeded at 30e50% confluency in 384-well plates with
50 mL per well of FluoroBrite DMEM media (Thermo Fisher Scien-
tific A18967) supplemented with 10% FBS a day before compound
treatment. Compounds and 100 nM dBET6 were dispensed using a
D300e Digital Dispenser (HP), normalized to 0.5% DMSO, and
incubated with the cells for 5 h. The assay plate was imaged
immediately using an Acumen High Content Imager (TTP Labtech)
with 488 nm and 561 nm lasers in a 2
mm ꢂ 1
mm grid per well
15

P.D. Fischer, E. Papadopoulos, J.M. Dempersmier et al.
European Journal of Medicinal Chemistry 219 (2021) 113435
Swissprot human database (December 2019), searching for tryptic
peptides with maximum of 1 missed cleavage, static alkylation of
cysteines (57.0215 Da), static TMT labelling of lysine residues and
peptide N-termini (304.2071 Da) and variable oxidation of methi-
onine (15.9949 Da).
Data availability
Atomic coordinates and structure factors for the reported crystal
structures have been deposited with the Protein Data Bank under
accession number 7MEU.
MS3 scans were acquired in the Orbitrap with HCD collision
energy set to 45%, custom AGC target, maximum injection time of
86 ms, resolution at 50,000 and with a maximum synchronous
precursor selection (SPS) precursors set to 10.
Declaration of competing interest
The authors declare the following financial interests/personal
relationships which may be considered as potential competing
interests: E. S. F. is a founder, science advisory board member and
equity holder in Civetta, Jengu (board member) and Neomorph, an
equity holder in C4, and a consultant to Astellas, Novartis, Deerfield,
RA Capital and EcoR1. The Fischer lab receives or has received
research funding from Novartis, Astellas, Ajax and Deerfield. G.W. is
co-founder of PIC Therapeutics, Cellmig biolabs, Skinap therapeu-
tics and Virtual Discovery. H.A. is an equity holder in PIC Thera-
peutics. The research described here is scientifically and financially
independent of the efforts in any of the above-mentioned
companies.
Proteome Discoverer 2.4 (Thermo Fisher Scientific) was used
for.RAW file processing and controlling peptide and protein level
false discovery rates, assembling proteins from peptides, and pro-
tein quantification from peptides. The MS/MS spectra were
searched against a Swissprot human database (December 2019)
containing both the forward and reverse sequences. Searches were
performed using a 20 ppm precursor mass tolerance, 0.6 Da frag-
ment ion mass tolerance, tryptic peptides containing a maximum of
two missed cleavages, static alkylation of cysteine (57.0215 Da),
static TMT labelling of lysine residues and N-termini of peptides
(304.2071 Da), and variable oxidation of methionine (15.9949 Da).
TMT reporter ion intensities were measured using a 0.003 Da
window around the theoretical m/z for each reporter ion in the MS3
scan. The peptide spectral matches with poor quality MS3 spectra
were excluded from quantitation (summed signal-to-noise across
channels < 100 and precursor isolation specificity < 0.5), and the
resulting data was filtered to only include proteins with a minimum
of 2 unique peptides quantified. Reporter ion intensities were
normalized and scaled using in-house scripts in the R framework (R
Development Core Team, 2014). Statistical analysis was carried out
using the limma package within the R framework [59].
Acknowledgement
PDF acknowledges the Chleck Foundation. HA acknowledges
funding from the Claudia Adams Barr Program for Innovative
Cancer Research and from NIH (GM136859). G.W. acknowledges
support from NIH grant GM132079 and CA200913. We thank Dr.
Kendra Leigh for her critical comments on the manuscript.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113435.
4.11. eIF4E pull down and Western Blot
HEK293 and HeLa cells were grown for 24 h, harvested by
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