An Exploration of Chemical Properties Required for Cooperative Stabilization of the 14-3-3 Interaction with NF-κB—Utilizing a Reversible Covalent Tethering Approach

Article abstract of DOI:10.1021/acs.jmedchem.1c00401

Protein-protein modulation has emerged as a proven approach to drug discovery. While significant progress has been gained in developing protein-protein interaction (PPI) inhibitors, the orthogonal approach of PPI stabilization lacks established methodologies for drug design. Here, we report the systematic ″bottom-up″ development of a reversible covalent PPI stabilizer. An imine bond was employed to anchor the stabilizer at the interface of the 14-3-3/p65 complex, leading to a molecular glue that elicited an 81-fold increase in complex stabilization. Utilizing protein crystallography and biophysical assays, we deconvoluted how chemical properties of a stabilizer translate to structural changes in the ternary 14-3-3/p65/molecular glue complex. Furthermore, we explore how this leads to high cooperativity and increased stability of the complex.

Full text of DOI:10.1021/acs.jmedchem.1c00401

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Article
An Exploration of Chemical Properties Required for Cooperative
Stabilization of the 14-3‑3 Interaction with NF-κBUtilizing a
Reversible Covalent Tethering Approach
Madita Wolter,§ Dario Valenti,§ Peter J. Cossar,§ Stanimira Hristeva, Laura M. Levy, Thorsten Genski,
Torsten Hoffmann, Luc Brunsveld,* Dimitrios Tzalis,* and Christian Ottmann*
Cite This: J. Med. Chem. 2021, 64, 8423−8436
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ABSTRACT: Protein−protein modulation has emerged as a proven approach to
drug discovery. While significant progress has been gained in developing protein−
protein interaction (PPI) inhibitors, the orthogonal approach of PPI stabilization
lacks established methodologies for drug design. Here, we report the systematic
″bottom-up″ development of a reversible covalent PPI stabilizer. An imine bond was
employed to anchor the stabilizer at the interface of the 14-3-3/p65 complex, leading
to a molecular glue that elicited an 81-fold increase in complex stabilization. Utilizing
protein crystallography and biophysical assays, we deconvoluted how chemical
properties of a stabilizer translate to structural changes in the ternary 14-3-3/p65/
molecular glue complex. Furthermore, we explore how this leads to high cooperativity and increased stability of the complex.
identify hit compounds for the development of PPI stabilizers.
Fragment-based drug discovery has emerged as a promising
approach leading to several clinically approved drugs.15,16
Since the clinical approval of ibrutinib, the stigma relating to
covalent drugs and fragments has dissipated,17 resulting in a
rise in the application of covalent tethering, particularly for
challenging drug targets.18,19 The emergence of fragment
tethering approaches using covalent chemistry has simplified
the identification of hit compounds as binding fragments are
easily detectable, and instrumentation is not reliant on
detecting transient fragments/complex interactions to identify
hits.20,21 Covalent fragment screening uses a covalent bond
that is formed between a natural or engineered amino acid
inside the targeted binding pocket and a reactive handle
installed on the fragments.22 Recent fragment screening
approaches utilizing disulfide tethering have proven successful
in identifying hit fragments for PPI stabilization. Such site-
directed fragment identification has recently been reported by
us for stabilization of the protein 14-3-3 in complex with
estrogen receptor α (ERα)23 and estrogen related receptor
gamma (ERRγ)24 utilizing a disulfide tethering approach. We
have also reported a new aldimine-based covalent tethering
approach for the 14-3-3/p65 subunit of NF-κB PPI.25 This
1. INTRODUCTION
Protein−protein interaction (PPI) modulation has fundamen-
tally changed drug discovery, expanding the druggable
proteome.1−4 This shift in perspective has rapidly expanded
the number of entry points for novel therapeutic intervention
for many diseases.2,5 The development of PPI inhibitors has
progressed steadily, leading to the emergence of clinically
approved PPI inhibitors such as Venetoclax for chronic
lymphocytic leukemia or small lymphocytic lymphoma.5 In
contrast, PPI stabilization has emerged slowly, with the
retrospective elucidation of the mode of action of clinically
approved immunosuppressants, rapamycin,6,7 and the cyto-
static paclitaxel.8,9 Successes of immunomodulators, lenalido-
mide, and cooperative PROTACs like AT1 provide valuable
″proofs-of-concepts″ for modulating cell homeostasis using
synthetically derived PPI stabilizers.10−12 However, a system-
atic understanding of how to design small molecules that
stabilize PPIs is urgently needed. The disconnection between
these orthogonal PPI modulation approaches is primarily
driven by a poor understanding of drug design rules for PPI
stabilizers due to the complicated kinetics of dynamically
interacting components that form the complex,13,14 shallow
surface pockets, and limited or no structural information about
the essential interactions that drive complex formation. A
major obstacle for the development of PPI stabilizers is
identifying chemical starting points, as the transient nature of
complexes makes detecting ligand binding challenging.
Further, affinity of a ligand to a complex is not necessarily
correlated to the ligand’s stabilizing activity.
Received: March 5, 2021
Published: June 2, 2021
There is a significant need to develop novel drug discovery
methodologies and alternative types of chemical matter to
© 2021 The Authors. Published by
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Figure 1. Exploring the p65/14-3-3 interface with imine forming fragments. (A) Overview of the composite binding pocket of 14-3-3 and the
p65_45 peptide (PDB: 6QHL).38 (B) Crystal structure of the NF-κB subunit p65 (PDB: 1IKN,39 IκBα and p50 hidden for clarity) with known
phosphorylated serine residues Ser281 and Ser45 labeled. (C) Interface of the ternary complex of 1/p65_45/14-3-3 (PDB: 6YOW).25 Indicated
are three subpockets (the roof of 14-3-3, the FC-binding pocket, and the deep binding pocket), which can be exploited by fragment extension.
Shown are 14-3-3 (white sticks, surface and cartoon), p65_45 peptide (red sticks), and fragment 1 (cyan sticks). (D) Fragment hits based on X-
crystallography screening with benzaldehyde binding to Lys122 of 14-3-3.
approach employs a dynamic imine-based tether to anchor the
fragment at the interface of the composite pocket. Lysine
tethering is a particularly attractive approach as the large
percentage of the proteinogenic amino acids makes it
amendable to a wide array of chemical probe development
projects.26−29
Here, we extend upon our initial communication reporting
on an imine-based site-directed fragment approach to develop
a 14-3-3/p65 molecular glue.25 Critical to the development of
molecular glues is a robust understanding of molecular
interactions and structural changes in the protein−protein−
ligand complex that result in cooperative behavior. To
understand the chemical properties that produce cooperative
ligands, we employed a fragment extension design process
using structural information gathered from X-ray crystallog-
raphy soaking experiments and fluorescence anisotropy (FA)
measurements. This enabled us to design initial fragments into
molecular glues that showed stabilizing activity for the 14-3-3/
p65 complex, culminating with the discovery of compound 24j
that elicits an 81-fold stabilizing effect on the 14-3-3/p65
complex.
Targeting the p65 subunit of NF-κB is of specific interest
since NF-κB is a homo- and/or heterodimeric transcription
factor involved in the regulation of immune responses, cell
proliferation, and inflammation and therefore is connected to
cancer and autoimmune diseases, among others.30−32 Attempts
to directly inhibit the transcriptional activity of NF-κB have
typically failed due to the inability to identify NF-κB-targeting
matter.33,34 Interestingly, increased transcriptional activity of
p65 has been correlated with downregulation of 14-3-3 in
studies on ischemia−reperfusion and breast cancer.35,36 Also,
upregulation of 14-3-3 has been shown to favor cytosolic
localization of p65,37 subsequently preventing transcriptional
activity. Stabilization of the 14-3-3/p65 complex could
therefore furnish a novel entry point for targeting NF-κB and
enabling a controlled therapeutic intervention.
Point mutational studies on p65 revealed three potential 14-
3-3 binding sites surrounding the phosphorylation sites S45,
S281, and S340.37 Binding affinities and structural information
for two of these sites, pS45 and pS281, were reported, showing
the direct physical interaction between both proteins.38
Benzaldehyde-based molecular fragments were shown to bind
specifically to Lys122 of 14-3-3 via imine bond formation,
thereby stabilizing the interaction with the p65 motif around
phosphorylation site pS45 via hydrophobic contacts with
p65.25
2. RESULTS AND DISCUSSION
We have previously shown that aldimine bond formation is
highly selective for Lys122 of 14-3-3 that lies at the interface
between 14-3-3 and p65 in the composite binding pocket
(Figure 1A). The enhanced selectivity for Lys122 is the result
of a combination of the local hydrophobic character of the
composite pocket, a lowered pKa of the lysine side chain, and
the templating effects of p65 binding.25 Given the intrinsically
disordered nature of large parts of the p65 subunit of NF-κB
(Figure 1B), we utilized a 13-mer phosphopeptide representing
(EGRSAG pSer45 IPGRRS) the recognition sequence of 14-3-
3 to expedite chemical matter elucidation. Our initial
investigation used X-ray crystal soaking experiments and a
fragment library of commercially available aldehydes (34
fragments) to identify four key chemotypes that induced imine
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Figure 2. Nitrobenzaldehyde structural analogues. (A) Initial fragment hit 3 covalently bound to the 14-3-3/p65_45 complex. The fragment can be
extended from its meta-position (black arrow shows proposed vector for extension) (PDB: 6YOY).25 (B) Ternary structure of 9a (salmon sticks;
PDB: 7NMH), p65_45 (red sticks), and 14-3-3 (white cartoon/sticks). Water molecules: red spheres; hydrogen bonds: black dashes. (C) Ternary
structure of 10a/p65_45/14-3-3 (PDB: 7BJF). Details as in (B). (D) Ternary structure of 13e (left; PDB: 7BJL), 13f (middle; PDB: 7BJW), or
13l (right; PDB: 7BKH) binding to the p65_45/14-3-3 complex. Compounds are shown as yellow sticks; the rest are as described before. The 2Fo-
Fc electron density map is contoured at 1σ for all.
Scheme 1. Synthesis of Focused Fragment Libraries 1 to 3
bond formation with Lys122 within the 14-3-3/p65 composite
pocket (Figure 1C,D): methylsulfonyl (1 and 2), 1-nitro,3-
hydroxybenzene (3), methyl acetamide (4), and five-
membered N-heterocycles (5, 6, and 7). Biochemical assess-
ment of fragments 1−7 used a fluorescence anisotropy (FA)
assay. To assess the fragments’ capacity to induce a ternary
complex, the fragments were titrated to a solution of 100 nM
fluorescently labeled p65_45 peptide (FITC-βAla-EGRSAG
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Figure 3. Imidazole-benzaldehydes are stabilizing the p65_45/14-3-3 complex. (A) The imidazole moiety of 5 (teal sticks) offers three vectors for
fragment growth (arrows) (PDB: 6YP3).25 (B) Fragment induced p65_45/14-3-3γ ternary complex formation, measured with compound titrations
in FA (r/mAU). The compounds were titrated to 50 μM 14-3-3γ and 100 nM p65_45. (C) Ternary structure of 16d (light blue sticks; PDB:
7NM9) binding to the p65_45/14-3-3σΔC complex. (D) Mixture of structural isomers of 16j and 16k. (E, F) Crystal structure of 16k binding to
the p65_45/14-3-3σΔC complex (PDB: 7NR7). The electron density map indicates two binding poses (E). Based on the position of the methoxy
substitution, the 2-chloro substitution points either toward the peptide (16k-5) or to the FC pocket (16k-6). Both binding poses engage in
hydrophobic contacts with 14-3-3 and p65 (sphere representation, F). For all: 14-3-3: white cartoon, surface, sticks; p65_45: red sticks; 2Fo-Fc
electron density map (contoured at 1σ): gray mesh.
pSer45 IPGRRS) and 50 μM of 14-3-3γ. The subsequent
outputs are herein termed ″half maximal complex concen-
tration (CC50)″. Results from the assay showed that these
fragments did not increase complex formation. However, the
lack of increase in complexation at relevant fragment
concentrations was in accordance with crystal data, which
indicated little to no direct contacts between the fragments and
the p65 peptide. Fragments that are detectable in crystallog-
raphy experiments but do not induce a detectable effect on
complex formation are termed ″silent-binders″. We sought to
develop a structure−activity relationship (SAR) based upon
these four chemotypes with the specific goal of developing
molecular glues that orthosterically engage with p65 and
stabilize the 14-3-3/p65 complex.
To facilitate rapid optimization of the initial hit compounds
into molecular glues, six focused libraries were designed based
on the four chemotypes from the initial screen. Key to the
development of molecular glues is understanding which
specific interactions (hotspots) between the fragments and
the 14-3-3/p65 complex lead to cooperative binding. Within
our lab, we have developed a highly robust crystallography
screening system to rapidly access X-ray crystal structures of
soaked fragments. Utilizing this system, we gain unparalleled
structural information that guides hit optimization.
2.1. Focused Library Development around the 3-
Hydroxy,4-nitrobenzaldehyde Scaffold. Initial focused
library development concentrated on extension of 3 based
on crystal data, which showed complete coverage of the
fragment by the electron density map, indicating a high
occupancy for 3 within the composite pocket. Analysis of the
X-ray crystal structure of 3 indicated that a relatively large
solvent exposed pocket was present in front of the fragment
formed by p65 (Figure 2A). We sought to explore this
chemical space via extension of 3 from the 3-hydroxy position.
To expedite exploration, focused libraries 1 and 2 were
obtained from a commercial supplier and screened using X-ray
crystallography (Scheme 1).
Analysis of focused libraries 1 and 2 using X-ray crystal
soaking experiments showed three sulfonate fragments (9a−c)
and four ester fragments (10a, d, j, and m) bound to Lys122 in
the crystal structures (Figure 2B,C; Figure S1A,B; Table S1).
The various substituents elicited poor to moderate coverage by
the electron density map, except for the sulfonates 9a and 9b.
However, neither 9a nor 9b were active in functional FA assays
(Figure S1A). For these smaller substitutes (9a and 9b), no
favorable contacts with the peptide could be observed, whereas
the larger substitutions resulted in a loss of electron density,
indicative of a high conformational freedom of the ester side
chain (9c, 10a, and 10j; Figure S1A,B).
An additional collection of fragments (focused library 3) was
synthesized based on tertiary amines at the meta-position. The
replacement of the phenol oxygen of 3 with a ternary amine
increased the number of vectors for fragment extension,
reduced conformational freedom, and enabled the exploration
of a different chemical space. A one-step nucleophilic aromatic
substitution using the key benzaldehyde intermediate 11 and
the corresponding amine (12a−u) was employed to afford 21
analogues (13a−u) with yields ranging from 20 to 68%
(Scheme 1). Focused fragment library 3 was then soaked into
p65_45/14-3-3 crystals and tested in the FA assay. Of this
collection, four fragments bound in crystallography experi-
ments, with 13e, f, l, and q showing significant electron density
coverage (Figure 2D, Figure S1C, Table S1). Notably, all four
fragments contained six-membered saturated N-heterocyclic
rings with polar functional groups that formed either direct
hydrogen bonds with the backbone of p65 or water-mediated
hydrogen bonds. The binding poses of these compounds were
well defined, whereby the saturated N-heterocyclic ring
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Scheme 2. Synthesis of Focused Fragment Libraries 4 and 5
Figure 4. Acetamide-benzaldehydes in complex with 14-3-3/p65_45. (A) 3D (beige sticks) and 2D structure of 4-formyl-benzamide. The C−C−
C−N torsion angles (orange lines) cluster at τ ≈ 0 and 180°.40 (B) Initial fragment hit 4 can be extended from its para-position (black arrows;
PDB: 6YOX).25 (C) Ternary structure of 19e (green sticks) in complex with p65_45 (red sticks) and 14-3-3σΔC (white cartoon and sticks)
(PDB: 7BIY). Hydrogen bonds are indicated with green dashes; the 2Fo-Fc electron density map is shown as gray mesh, contoured at 1σ.
extends toward the C-terminus of the p65 peptide. These
results suggest that these fragments are shielded by the
amphiphilic amino acids Pro47, Gly48, Arg49, and Arg50 of
p65, which facilitate covalent tethering. Biophysical assessment
of 13e, f, l, and q using FA compound titration assays showed
that the fragments did not elicit significant ternary complex
formation at biochemically relevant concentrations (Figure
S1C). Surprisingly, 13f and 13l showed no increased complex
formation, considering that both fragments engage in hydrogen
bonding with p65. Given the lack of 14-3-3/p65 increased
complex formation in FA compound titration assays at
concentrations practical for hit optimization, we shifted focus
to the five-membered N-heterocycles chemotype.
2.2. Focused Library Development around the 4-(1H-
Imidazol-1-yl)benzaldehyde Scaffold. Fragment 5 was
selected for fragment library development as the para-
substitution proved to be more solvent exposed compared
with 7 and the 1,3-substituted imidazole provided the
possibility for fragment extension from three vectors of the
N-heterocycle (Figure 3A). A focused library of 13 fragments
(focused library 4) was synthesized using a nucleophilic
aromatic substitution reaction with cesium fluoride, triethyl-
amine, 4-fluoro-nicotinaldehyde (14), and an array of
substituted imidazoles (15a−e) or benzimidazoles (15f−k)
(Scheme 2). The starting reactant 4-fluoro-nicotinaldehyde
(14) was used to improve the solubility of the fragments and
to provide a further point for a polar interaction compared
with 5−7. The resulting library was then subjected to X-ray
crystal soaking experiments and FA assay. Notably, fragments
16b, c, f, and g−k were tested as mixtures of regioisomers.
Analysis of the fluorescence anisotropy assay identified that
fragments 16d, e, j, and k induced an increase in anisotropy
(Figure 3B); however, only 16d, j, and k were detected in the
electron density map of soaking experiments. Notably, all three
fragments showed a mixture of conformational poses (Figure
3C−F, Figure S2, Table S2). Both structural isomers of 16j (2-
methyl-5-methoxy-benzimidazole) and 16k (2-chloro-5-me-
thoxy-benzimidazole) were observed to bind within the
composite binding pocket as result of the soaking experiment
using mixtures of the structural isomers (Figure 3D−F). For
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Figure 5. Sulfonamide-benzaldehydes are active in FA compound titrations. (A) 3D and 2D chemical structure of 4-formyl-benzenesulfonamide
with a C−C−S−N torsion angle τ as indicated.40 (B) The benzenesulfonamides are extended toward the FC pocket or toward the p65 peptide
(red; 14-3-3: white; both with sticks and transparent surface) (PDB: 6YOW).25 (C) FA compound titrations in the presence of 50 μM 14-3-3γ and
100 nM p65_45. The compounds are divided into weak (left), medium (middle), and high (right) activity. The legends are sorted by activity.
instance, the chlorine atom in 16k points toward the p65
peptide, while the methoxy-substitution can be detected in the
5-position (Figure 3E). For the other binding pose, the
chlorine atom in 16k points to the FC pocket, whereas the
methoxy-substitution located in the 6-position is positioned
above Ile46 of p65. Both structural isomers of 16k engage in
hydrophobic contacts with the roof of 14-3-3 and Ill46 of
p65_45 (Figure 3F). Despite their similar binding poses, 16k
(CC50 = 260 μM) elicited a significantly higher increase in
anisotropy compared to 16j, which induced a negligible ternary
complex formation in the FA assay (Figure 3B). The
replacement of a chloro-moiety for a methyl group in 16k
(16j) on the imidazole ring has a significant impact on
complex formation, resulting in diminished anisotropy.
2.3. Focused Library Development around the 4-
Formylbenzamide Scaffold. We next turned our attention
to fragment extension based upon fragment 4. Analysis of the
co-crystal structure showed that the N-acetyl group of 4 probes
up and toward the p65 peptide. To expediate the rapid parallel
synthesis of focused library 5, the N-(4-formylphenyl)-
acetamide (4) was inverted to a N-substituted 4-formyl-
benzamide (19a−t), improving the nucleophilicity of the
amine in the corresponding amide coupling reactions as well as
providing greater chemical diversity of the fragment extension
(Scheme 2). Further, a previously published cluster analysis of
occurring torsion (τ) angles of benzamide report a τ ≈ 30 and
150° between the benzene ring and the acetamide head group
(Figure 4A).40 We hypothesized that extension of the fragment
from this vector provides the opportunity to engage with the
p65 peptide and enhance fragment binding (Figure 4B).
Focused library 5 was synthesized using standard amide
coupling conditions, 1-formylbenzoic acid (17), and amines
18a−t. In total, 13 fragments (19a−t) with amide substitution
in the para-position of the aldehyde functionality were
synthesized, soaked in p65_45/14-3-3σΔC crystals, and tested
in FA compound titrations. All fragments from library 5 were
shown to bind within the composite binding pocket using X-
ray crystallography (Figure S3, Table S2). Subsequent
biophysical analysis identified that all compounds were silent
binders, not inducing detectable formation of a ternary
complex at assay relevant concentrations. X-ray co-crystal-
lization experiments and an assessment of the C−C−C−N
torsion angles provided an explanation for the lack of activity
for this series of fragments. The electron density map of the
aldehydes showed highly resolved electron density for the
benzaldehyde ring and the amide of the benzamide. The
carbonyl of the benzamide engages in polar contacts with the
water shell of 14-3-3, thereby stabilizing this orientation of the
fragments (Figure 4C). The binding poses of the different R-
substitutions are poorly resolved by the electron density map,
suggesting a high level of conformational freedom and a high-
level entropy of the substitutes, unfavorable for binding.
Assessment of this library of fragments showed the R-
substitution pointing toward the solution above the p65
peptide or Asp215 of 14-3-3 with limited possibilities to
engage in favorable contacts. For a few fragments, the R-
substitutes engage in polar contacts with the 14-3-3 water shell,
exemplified by 19e (Figure 4C). However, the additional polar
contacts do not translate to significant increases in ternary
complex formation potentially due to entropic penalties or lack
of contacts with the p65 peptide. Given the lack of cooperative
ternary complex formation of this library at biochemically
relevant concentration, we shifted focus to the 4-formyl
benzenesulfonamide chemotype.
2.4. Focused Library Development around 4-Formyl-
benzenesulfonamides. Having investigated focused libraries
1−5, we shifted our attention to development of a focused
library based on fragment 1. Analysis of the crystal structure of
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b
Table 1. Exploration of 4-Formyl-benzenesulfonamides
a
b
Fit did not converge. CC50: values of compound titrations with 50 μM 14-3-3γ. KD,app: value of protein titrations in the presence of 1 mM of the
fragment. SF: the fold-change of apparent KD in comparison to a DMSO control. ND: not determined. All fragments bound to the p65_45/14-3-
3σΔC complex. For additional data, see Figure S4 and Table S3.
fragment 1, specifically the torsion angle of τ = 90
30°
compound titrations. From the focused library 6, nine
fragments elicited CC50 values ranging from 57 to 430 μM.
An additional assessment of stabilization using the FA protein
titration assay showed for 19 fragments a significant shift of
apparent KD values in the presence of 1 mM of the fragments.
Fragments 23e, f, k, t, w, and z showed KD values of <30 μM
compared to the binary complex of full-length 14-3-3 and the
p65_45 peptide that gave a KD = 300 100 μM. Analysis of
this subset of fragments showed that these shifts in KD’s
(stabilization factor, SFs range from ∼12- to 65-fold).
Fragment 23z, possessing a N-substituted 1,2,3,4-tetrahydro-
quinoline, showed the greatest SF of ∼65-fold.
Comparison of X-ray crystal soaks provided an explanation
for the FA assay results. Structural data showed that 2-methyl
pyrrolidine (23e) and piperidine (23f) were tolerated within
the composite binding pocket. Ring expansion to the N-methyl
diazepane (23 g) proved to be detrimental to stabilization
(CC50 = 640 μM). Notably, polar functionalities within six-
membered N-heterocycles were not well tolerated; specifically,
hydrogen bond accepting and donating groups (23h, n−u)
elicited high CC50 values and modest SFs (6−16-fold). This
can be exemplified by fragment 23f containing piperidine (app
between the benzene ring and the mesyl group, proved
interesting (Figure 5A).40 Extension of the fragment from the
methyl group provided a potential point of reaching over the
p65 peptide, trapping its binding to 14-3-3 (Figure 5B).
Alternatively, we postulated that fragment extension could also
result in a change in the conformation of the fragment leading
to the occupation of the FC pocket and increasing 14-3-3-
based affinity. To expedite fragment development, the methyl
group was replaced for N-substituted amines to facilitate rapid
access to a library of 25 structural analogues of 4-formyl-
benzenosulfonamides. This library of sulfonamides was
synthesized by conversion of sodium 2-formylbenzene-1-
sulfonate (20) to 4-formyl-benzenesulfonyl chloride (21)
using thionyl chloride in DMF. Subsequent coupling of 21
with N-substituted amines (22a−z) afforded fragments 23a−z.
In contrast to focused libraries 1−5, most of the tested 4-
formyl-benzenesulfonamide fragments showed significant
stabilization in FA assays, allowing a differentiated SAR
analysis (Table 1, Figure 5C). The activity ranged from a
weak affinity with a small, not quantifiable increase in
anisotropy to a two-digit micromolar CC50 potency in
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Figure 6. Sulfonamide-benzaldehydes inducing complex stabilization. (A) Overlay of 23h (green sticks; PDB: 7NLE), 23k (orange sticks; PDB:
7NK5), 23f (cyan sticks; PDB: 7NJB), and 23u (yellow sticks; PDB: 7BJB) binding to p65_45 (red sticks) and 14-3-3 (white cartoon and sticks).
Hydrogen bonds are shown as black dashes, and hydrophobic contacts are indicated with transparent spheres. A conserved water molecule (small
sphere) is shown colored based on the related fragment color. (B) Fragments 23h, k, f, and u show complex stabilization in FA compound
titrations (left; [14-3-3γ]: 50 μM, [p65_45]: 100 nM) and protein titrations (right; [fragment]: 1 mM, [p65_45]: 100 nM). (C) Overlay of 23n
(brown sticks; PDB: 7NM3) and 24m (light blue sticks; PDB: 7NM1) in complex with 14-3-3σΔC/p65_45. (D) Crystal structures of 23w (blue
sticks; PDB: 7NLA) binding to the composite binding pocket of p65 (red sticks) and 14-3-3 (white cartoon and sticks). The 2Fo-Fc electron
density map (1σ) is displayed as gray mesh, and hydrogen bonds are shown as black dashes. (E) Overlay of 23y (green sticks; PDB: 7NK3) and
23z (yellow; PDB: 7NJ9) binding to the p65_45/14-3-3 complex. Upon binding of 23y, Pro47 changes its conformation (red sticks, as reference:
Pro47 of the 23z/p65_45/14-3-3σΔC complex shown as yellow sticks). (F) Ternary structure of 23z (yellow sticks; PDB: 7NJ9) binding to the
p65_45/14-3-3 complex. Details as in (D). (G) Ternary complex formation of 23x, y, and z was measured with FA compound titrations (left; [14-
3-3γ]: 50 μM, [p65_45]: 100 nM) and the stabilizing activity of 23w, y, and z with protein titrations (right; [fragment]: 1 mM, [p65_45]: 100
nM).
KD = 15 μM, SF =22) compared with N-methylpiperazine 23h
(app KD = 100 μM, SF = 3.3) and morpholine 23u (app KD =
52 μM, SF = 6.3) (Figure 6A,B). Analysis of the X-ray
structures showed that introduction of a polar functionality
typically resulted in reorientation of the R-substitute of the
fragment toward the FC-binding pocket. Introduction of a
hydrophobic functionality such as 23k (app KD = 27 μM, SF =
12.2) and 23v (app KD = 430 μM, SF = 10.6)25 recovered
stabilizing activity, with the fragments re-establishing hydro-
phobic contacts with the p65 peptide. Notably, large
amphiphilic modifications were also not well tolerated, such
as 23m (app KD = 74 μM, SF = 4.5) and 23n (app KD = 94
μM, SF = 3.5) (Figure 6C, Table 1), with both 23m and 23n
occupying solvent exposed space within the composite pocket
but not engaging in contacts with p65. Interestingly, fragment
23w (app KD = 26 μM, SF = 12.7) showed a significant drop in
stabilization with respect to 23f. Crystal soaking experiments
showed that fragment 23w engages in hydrophobic inter-
actions with Ile46, Pro47, and Gly48 of p65 (Figure 6D).
However, unlike other fragments, 23w induces a conforma-
tional change in the C-terminus of the peptide shifting toward
the pS45. Analysis of soaking experiment with fragment 23y
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Figure 7. Optimization of 23z. (A) Ternary structure of 23z (yellow spheres) in complex with 14-3-3σΔC (white surface) and p65_45 (red
cartoon, transparent spheres) (PDB: 7NJ9). Carbons of the bicyclic head group are numbered. (B) Ternary structure of 24b (orange sticks) in
complex with p65_45/14-3-3σΔC (PDB: 7BIQ). The distance between the 2-methyl and water (red spheres) is indicated with black dashes. Polar
contacts are shown as black dashed lines. (C) Structure of 24e (cyan sticks) binding to the p65_45/14-3-3σΔC complex (as in A; PDB: 7BI3).
Hydrophobic contacts are indicated with transparent spheres. (D) FA compound titrations with 50 μM 14-3-3γ and 100 nM p65_45. (E) FA
protein titrations with 1 mM fragment and 100 nM p65_45. (F) Structure of 24j binding to the p65_45/14-3-3σΔC complex (as in A; PDB:
7BIW). A beneficial hydrogen bond is formed between the backbone carbonyl of p65s’ Pro47 and 24j (black dash).
showed that the tetrahydroisoquinoline ring engages in
hydrophobic contacts with the p65 peptide; however, unlike
23e and 23f, the bicyclic rings repulse Pro47 of p65 (Figure
6E). This provides an explanation for the poor stabilizing
activity of 23y. In contrast to 2-tetrahydroquinoline (23y), 1-
tetrahydroquinoline (23z) was highly tolerated, eliciting CC50
= 57 μM and app KD = 5.1 μM; this translated to ∼65-fold
stabilization. X-ray crystallography analysis of fragment 23z
provided valuable explanation for the high stabilization
observed in FA assays. Specifically, the bicyclic substructure
in 23z engages the p65 peptide and is positioned upwards
occupying the hydrophobic roof of 14-3-3σΔC shaped by
residues Ile219, Leu218, and Leu222 (Figure 6F). Further, 23z
also engages in direct hydrophobic contacts with Ile46 and
Pro47 of p65. Most notably, the 14-3-3/p65/23z ternary
complex formation results in a reorientation of the p65 peptide
leading to additional 14-3-3/p65 contacts. Fragment 23z
appears to function as a template for the additional binding of
p65, increasing cooperative behavior of the ternary complex.
Fragment 23z facilitates additional 14-3-3/p65 contacts by
enabling the C-terminus of p65 to wrap over 23z and engage in
electrostatic contacts with 14-3-3. Specifically, salt bridges are
observed between Arg49/Glu14 and Arg50/Asp215 of
p65_45/14-3-3, respectively. These structural features translate
to high activity in FA assays, with a CC50 of 57 μM and an SF
of 65 (Figure 6G). The additional interactions between 14-3-3
and p65 induced by the binding of 23z lead to increased
cooperativity of the ternary complex. This improved
cooperative behavior directly translates into improved
stabilization of the 14-3-3/p65 complex.
fragment 23z. Due to the rearrangement of the p65 peptide in
the presence of 23z forming a narrow and enclosed binding
pocket, this limited the sites for fragment modifications to the
5-, 6-, or 7-position of the tetrahydroquinoline ring (Figure
7A). As a result, we focused on small modifications to build
additional interactions with the hydrophobic patch in the roof
of 14-3-3 or extension of the fragments over Ile46 of p65.
Additionally, we investigated the addition of heteroatoms in
the 4-position of the tetrahydroquinoline ring to engage with
the backbone carbonyl or nitrogen of Pro47 or Gly48 to
establish additional polar contacts. A small library of 10
analogues was synthesized using our previously mentioned
sulfonamide coupling (Table 2).
Structural analysis of this library showed that this series of
analogues retained a similar cooperative behavior as 23z,
inducing additional PPI contacts. Biophysical analysis using an
FA assay provided valuable insight into the SAR around
fragment 23z. Analysis of the CC50 concentration indicated
that 23z showed the highest ternary complex formation, with
all other analogues in this series eliciting a drop in
complexation (CC50 values ranging from 110 to 310 μM).
However, significant insight into complex stabilization was
gained from the analysis of app KD values. Substitution of 23z
for an indoline (24a) or racemic 2-methyl-tetrahydroquinoline
(24b) was tolerated; however, a 2-fold reduction was observed
in stabilization, with app KD values of 11 and 9.6 μM,
respectively. Structural analysis of 24a showed additional space
around the five-membered heterocycle potentially providing
additional sites for functionalization in the future (Figure S5).
Fragment 24b showed excellent electron density across the
entire fragment within the X-ray crystal structure, enabling
unambiguous assignment of stereochemistry. From the
structure, it was identified that the R-enantiomer exclusively
2.5. Optimization of Fragment 23z. Encouraged by the
high stabilizing effect of covalent fragment 23z, we looked to
develop a library of 10 sulfonylamides based on tricyclic
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b
24j eliciting an app KD of 4.3 μM, translating into the highest
stabilizing effect with an 81-fold stabilization (Figure 7D,E).
Structural analysis showed that the introduction of a nitrogen
atom in the saturated ring installs an additional hydrogen bond
with the backbone carbonyl of Pro47 of p65 (Figure 7F).
To assess the selective ternary complex formation of 24j
with 14-3-3/p65, the fragment was further screened in a
compound titration FA assay against three well-established 14-
3-3 interactions (p53, ERα, and TAZ; Figure S6). Gratifyingly,
no ternary complex formation was detected for p53, ERα, or
TAZ complexes, proving that 24j is not a pan-stabilizer of 14-
3-3 interactions and indicating an initial selectivity of 24j
toward the p65/14-3-3 interaction.
Table 2. Exploration of structural analogs of 23z
3. CONCLUSIONS
Summarizing, here we build upon our initial report of a novel
covalent fragment screening approach using an aldehyde
fragment library. These covalent fragments form a covalent
tether with Lys122 within the 14-3-3/p65 composite binding
pocket. Specifically, we describe the optimization of initial hit
covalent fragments into a p65/14-3-3 molecular glue that
elicited an 81-fold stabilization of the 14-3-3/p65 complex.
Critical to this success was the use of X-ray crystallography and
FA measurements to develop a robust understanding of the
structural activity relationship. Direct hydrophobic engagement
with p65 was the driving interaction that established complex
stabilization. Hydrophobic contacts between 23z or 24j and
p65 resulted in a conformational change in the 14-3-3/p65
interface with extended interactions, in turn increasing the
cooperativity of the ternary complex. Fragment 23z or 24j
might serve as a valuable starting point for optimization for the
development of a chemical probe to study the effects of 14-3-3-
mediated regulation of p65 in a cellular context. Particularly,
the aldehyde functionality requires additional investigations to
validate its activity and oxidative stability in a cellular context
such as with the aldehyde-containing drug voxelotor.41
Alternatively, the aldehyde could be replaced with a more
benign chemical handle that is less susceptible to oxidation, or
the application of a prodrug approach to protect the aldehyde
may need to be considered when moving toward cellular
studies.
Considering the lack of rational methodologies to develop
PPI stabilizers, here we describe a systematic approach to
developing initial hit fragments into molecular glues.
Observations from this research indicate that the direct
engagement of molecular glues with the partner peptide is
significant for stabilization. Specifically, hydrophobic contacts
are effective to enhance ternary complex formation and
support a cooperative binding mode. Analysis of SAR results
indicates that fragments that facilitate templating of the p65
peptide and promotion of additional contacts between 14-3-3
and p65 resulted in increased complex stabilization. This
research has direct applications to further development of PPI
stabilizers as well as the field of PROTACs. There is a growing
body of evidence showing that cooperative PROTACs, which
stabilize direct PPIs, lead to improved efficacy, reducing
problems associated with the hook-effect and the necessity for
highly potent molecules.42−45 Additionally, research from the
field of PROTACs and our findings appear to be in agreement
that the molecular glue/cooperative PROTAC potency of a
ligand for a complex is not the driving force for increased
complex stabilization; rather, the cooperativity behavior of the
a
b
Tested as an enantiomeric mixture. CC50: values of compound
titrations with 50 μM 14-3-3γ. KD,app: values of protein titrations in
the presence of 1 mM of the fragment. SF: the fold-change of
apparent KD in comparison to a DMSO control. ND: not determined.
All fragments bound to the p65_45/14-3-3σΔC complex. For
additional data, see Figure S5 and Table S4.
bound in the crystal, suggesting that the pure R-enantiomer
may have significantly underestimated CC50 and app KD
(Figure 7B). This site may provide an excellent point for
fragment extension toward the p65 peptide. Addition of a
halogen or methoxy in the 6-position resulted in a reduction in
stabilization, with 6-fluoro (24c), 6-chloro (24d), or 6-
methoxy (24e) substituted tetrahydroquinoline fragments
affording app KD values of 6.2, 9.9, and 10 μM, respectively.
Analysis of soaking experiments shows that modifications in
this position resulted in unfavorable contacts with the roof of
14-3-3 (Figure 7C). Addition of an oxygen to the saturated six-
membered ring (24f) proved detrimental to stabilization (app
KD = 15 μM). Further addition of halogens or methoxy groups
in the 6 or 7 positions did not re-establish lost stabilization,
such as 6-chloro (24h), 6-methoxy (24i), or 7-fluoro (24g).
Substitution of the benzomorpholine (24f) for tetrahydroqui-
noxaline (24j) resulted in an improvement in stabilization with
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3γ and 10 nM peptide. The protein concentration was adjusted to
one-third of the KD of the binary protein/peptide complex (final 14-3-
3γ concentration for the measurement with TAZ: 0.1 μM; with p53:
0.3 μM and with ERα: 0.1 μM). For a better comparison, the baseline
of each measurement was subtracted (Δr/mAU).
ternary complex appears to be the driving force for PPI
stabilization.11,42,45,46
4. EXPERIMENTAL SECTION
All measurements were performed as single measurements. Data
analysis was performed with Origin 2019 (V9.6.0.172, OriginLab
Corporation) with the inbuilt ″Hill1″ function for data fitting. For
data sets without an upper plateau, the Hill coefficient n was set to n =
1.
4.1. Protein Expression and Purification. The 14-3-3 proteins
were expressed and purified using standard protocols. In short,
pPROEX HTb vectors encoding the 14-3-3σΔC (truncated C-
terminus ΔC17) and 14-3-3γ isoform were transformed into
BL21(DE3) cells. Protein expression was initiated with 0.4 mM
IPTG at a cell density OD600 = 0.8−1. The expression took place
overnight at 18 °C. The cells were harvested by centrifugation
(10,000g, 15 min) and resuspended in a lysis buffer (50 mM Tris/
HCl pH 8, 300 mM NaCl, 12.5 mM imidazole, and 2 mM β-
mercaptoethanol). The cells were lysed with a homogenizer, and the
lysate was cleared via centrifugation (40,000g, 30 min). Ni-NTA
columns were used to isolate the protein, which was washed with 10
CV lysis buffer and eluted with 250 mM imidazole (50 mM Tris/HCl
pH 8, 300 mM NaCl, 250 mM imidazole, and 2 mM β-
mercaptoethanol). The full-length 14-3-3γ was dialyzed against 25
mM HEPES pH 7.5, 100 mM NaCl, 10 mM MgCl2, and 0.5 mM
Tris(2-carboxyethyl)phosphine) and stored at −80 °C. For the 14-3-
3σΔC, the His6-tag was removed by the TEV protease; the TEV was
removed with Ni-NTA columns. The rest of the imidazole of the 14-
3-3σΔC solution was removed by size exclusion chromatography (20
mM HEPES pH 7.5, 150 mM NaCl, and 2 mM β-mercaptoethanol)
and stored at −80 °C.
4.2. X-ray Crystallography. Binary crystals with p65_45 peptide
(sequence: EGRSAG pS45 IPGRRS, C-terminus: amidation; N-
terminus: acetylation)47 and 14-3-3σΔC were grown at a 14-3-
3σΔC concentration of 12 mg/mL in a 1:2 ratio with the acetylated
peptide in 20 mM HEPES pH 7.5, 2 mM MgCl2, and 2 mM β-
mercaptoethanol. This complexation mixture was incubated over-
night. In a hanging drop setup, the complexation mixture was mixed
in 1:2 ratio with the precipitation buffer (95 mM HEPES pH 7.5, 27−
28% PEG400, 190 mM CaCl2, and 5% glycerol). For data acquisition,
crystals were directly flash-frozen in liquid nitrogen.
Fragment soaks were performed by adding compounds in DMSO
stock solutions direct to fully grown crystals with a final compound
concentration of 10 mM (≤1% DMSO). The soaks were incubated
for 7 days prior to data acquisition. Diffraction data were measured
either at the P11 beamline of PetraIII (DESY campus, Hamburg,
Germany) or the i-03/i-24 beamline of the diamond light source
(Oxford, UK) or in-house. The diffraction data were integrated with
the xia2/DIALS pipeline48 followed by molecular replacement with
MolRep.49,50 The binary p65_45/14-3-3σΔC structure was used as a
search model (PDB ID: 6QHL). Model refinement took place in
iterative cycles with Coot,51 Refmac5,52 and phenix.refine.53 3D
structures of ligands were prepared using the fragment SMILES and
elbow of the phenix suite.53 Figures were generated with PyMOL
(V2.0.6, Schrodinger LLC).
4.3. Fluorescence Anisotropy Assays. Complex stabilization
was measured using a fluorescently labeled p65_45 peptide (FITC-
βAla-EGRSAG pS45 IPGRRS) at a concentration of 100 nM
throughout all assays. During compound titrations, the 14-3-3γ
concentration was constant at 50 μM and the compound was titrated
in a 1:1 dilution series. In protein titrations, 14-3-3γ was titrated in a
1:1 dilution series in the presence of 1 mM compound. The plates
(Corning 384 well plates, black, round bottom, low binding) were
incubated for 3 h at RT prior to fluorescence anisotropy (FA)
measurements with the Tecan Infinite 500 plate reader (FITC dye:
excitation 485 nm and emission 535 nm). Dilution series were
prepared in an FA buffer (10 mM HEPES pH 7.4, 150 mM NaCl, and
0.1% Tween20).
4.4. Chemistry: General Information. All commercial chemicals
were used as received. Reagents were used without further purification
unless otherwise noted. TLC analysis was performed on TLC
aluminum sheets, silica gel layer, ALUGRAM SIL G UV254, 20 × 20
cm by MACHEREY-NAGEL. TLC plates were analyzed by UV
fluorescence (254 nm). UHPLC−MS analysis was performed using
the UPLC Agilent Technologies 1290 Infinity coupled with the
Agilent Technologies 6120 Quadrupole LC/MS DAD detector.
Column: ACQUITY UHPLC BEH C18 (1.7 μm) 2.1 mm × 50 mm.
Temperature: 40 °C. Detection: DAD + MS/6120 Quadrupole.
Injected volume: 1 μL. Flow: 1.2 mL/min. Solvent A: water + 0.1%
formic acid. Solvent B: acetonitrile + 0.1% formic acid. Gradient: 0
min 2% B, 0.2 min 2% B, 2.0 min 98% B, 2.2 min 98% B, 2.21 min 2%
B, and 2.5 min 2% B. Preparative HPLC was performed using the
UPLC Agilent Technologies 1260 Infinity coupled with the Agilent
Technologies 6120 Quadrupole LC/MS. Column: Waters XBridge
Prep C18 5 μm OBD 19 × 150 mm. Detection: DAD + MS/6120
Quadrupole. Flow: 32 mL/min. Solvent A: water + 0.1% formic acid.
Solvent B: acetonitrile + 0.1% formic acid. Gradient: 0 min 77% A/
23% B, 1 min 77% A/23% B, 9 min 16% A/84% B, 9.01 min 2% A/
98% B, and 11 min 2% A/98% B. The purity of the synthesized
1
compounds is ≥95%. H NMR and 13C NMR spectra were recorded
on a Bruker 300 MHz spectrometer at ambient temperature. The
chemical shifts are listed in ppm on the δ = scale, and coupling
constants were recorded in hertz (Hz). Chemical shifts are calibrated
relative to the signals corresponding of the nondeuterated solvent
(CHCl3: δ = 7.26 ppm for 1H and 77.16 ppm for 13C; DMSO: δ =
2.50 ppm for 1H and 39.52 ppm for 13C). Abbreviations are used in
the description of NMR data as follows: chemical shift (δ = ppm),
multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet, bs =
broad singlet, dd = doublet of doublets, td = triplet of doublets), and
coupling constant (J = Hz).
For general procedures, chemical characterization, NMR spectra,
and LC−MS traces, see the Supporting Information.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00401.
Additional results and X-ray crystallography tables,
chemical experimental, and chemical spectral data
(PDF)
Molecular formula strings (CSV)
Accession Codes
Coordinates and structure factors have been deposited in the
Protein Data Bank under the following accession codes:
7NMH, 7BI3, 7BIQ, 7BIW, 7BIY, 7BJB, 7BJF, 7BJL, 7BJW,
7BKH, 7NJ9, 7NJB, 7NK3, 7NK5, 7NLA, 7NLE, 7NM1,
7NM3, 7NM9, 7NR7, 7NV4, 7NVI, 7NWS, 7NXS, 7NXT,
7NXW, 7NXY, 7NY4, 7NYE, 7NYF, 7NYG, 7NZ6, 7NZG,
7NZK, 7NZV, 7O34, 7O3A, 7O3F, 7O3P, 7O3Q, 7O3R,
7O3S, 7O57, 7O59, 7O5A, 7O5C, 7O5D, 7O5F, 7O5G,
7O5O, 7O5P, 7O5S, 7O5U, 7O5X, 7O6F, 7O6G, 7O6I, 7O6J,
7O6K, 7O6M, and 7O6O. Authors will release the atomic
coordinates and experimental data upon article publication.
The activity of the hit compound 24j was measured for the TAZ-
peptide23 (FITC-βAla-RSH pS89 SPASLQ), p53-peptide54
(TAMRA-Ahx-SRAHSSHLKSKKGQSTSRHKKLMFK pT387
EGPDSD-COOH), or ERα-peptide13 (FITC-O1Pen-AEGFPA
pT594 V-COOH) and 14-3-3γ. Therefore, the compound was
titrated in a 1:1 dilution series to a constant concentration of 14-3-
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The authors declare the following competing financial
interest(s): LB and CO are co-founders of Ambagon
Therapeutics.
AUTHOR INFORMATION
■
Corresponding Authors
Luc Brunsveld − Department of Biomedical Engineering,
Laboratory of Chemical Biology and Institute for Complex
Molecular Systems, Eindhoven University of Technology,
5600 MB Eindhoven, The Netherlands; orcid.org/0000-
0001-5675-511X; Phone: +31 40−247-2870;
Email: l.brunsveld@tue.nl
Dimitrios Tzalis − Medicinal Chemistry, Taros Chemicals
GmbH & Co. KG, 44227 Dortmund, Germany;
Email: dtzalis@taros.de
Christian Ottmann − Department of Biomedical Engineering,
Laboratory of Chemical Biology and Institute for Complex
Molecular Systems, Eindhoven University of Technology,
5600 MB Eindhoven, The Netherlands; orcid.org/0000-
0001-7315-0315; Phone: +31 40−247-2835;
Email: c.ottmann@tue.nl
ABBREVIATIONS
■
app. KD, apparent KD; CC50, half-maximal complex concen-
tration; FA, fluorescence anisotropy; PPI, protein−protein
interaction; PROTAC, proteolysis targeting chimera; SAR,
structure−activity relationship
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Authors
Madita Wolter − Department of Biomedical Engineering,
Laboratory of Chemical Biology and Institute for Complex
Molecular Systems, Eindhoven University of Technology,
5600 MB Eindhoven, The Netherlands; orcid.org/0000-
0003-1430-9589
Dario Valenti − Department of Biomedical Engineering,
Laboratory of Chemical Biology and Institute for Complex
Molecular Systems, Eindhoven University of Technology,
5600 MB Eindhoven, The Netherlands; Medicinal Chemistry,
Taros Chemicals GmbH & Co. KG, 44227 Dortmund,
Germany
Peter J. Cossar − Department of Biomedical Engineering,
Laboratory of Chemical Biology and Institute for Complex
Molecular Systems, Eindhoven University of Technology,
5600 MB Eindhoven, The Netherlands
Stanimira Hristeva − Medicinal Chemistry, Taros Chemicals
GmbH & Co. KG, 44227 Dortmund, Germany
Laura M. Levy − Medicinal Chemistry, Taros Chemicals
GmbH & Co. KG, 44227 Dortmund, Germany
Thorsten Genski − Medicinal Chemistry, Taros Chemicals
GmbH & Co. KG, 44227 Dortmund, Germany
Torsten Hoffmann − Medicinal Chemistry, Taros Chemicals
GmbH & Co. KG, 44227 Dortmund, Germany
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00401
Author Contributions
§M.W., D.V., and P.J.C. contributed equally. The manuscript
was written through contributions of all authors. All of the
authors approved the final version of the manuscript.
Funding
The research was supported by funding from the European
Union through the TASPPI project (H2020-MSCA-ITN-
2015, grant number 675179), through the Eurotech Post-
doctoral Fellow program (Marie Skłodowska-Curie Co-funded,
grant number 754462), and through The Netherlands
Organization for Scientific Research (NWO) via VICI grant
016.150.366 and via Gravity Program 024.001.035.
Notes
L.B. and C.O. are scientific co-founders of Ambagon
Therapeutics.
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