Exploration of a 14-3-3 PPI Pocket by Covalent Fragments as Stabilizers

Article abstract of DOI:10.1021/acsmedchemlett.1c00088

The systematic discovery of functional fragments binding to the composite interface of protein complexes is a first critical step for the development of orthosteric stabilizers of protein-protein interactions (PPIs). We have previously shown that disulfide trapping successfully yielded covalent stabilizers for the PPI of 14-3-3 with the estrogen receptor ERα. Here we provide an assessment of the composite PPI target pocket and the molecular characteristics of various fragments binding to a specific subpocket. Evaluating structure-activity relationships highlights the basic principles for PPI stabilization by these covalent fragments that engage a relatively large and exposed binding pocket at the protein/peptide interface with a "molecular glue"mode of action.

Full text of DOI:10.1021/acsmedchemlett.1c00088

pubs.acs.org/acsmedchemlett
Letter
Exploration of a 14-3‑3 PPI Pocket by Covalent Fragments as
Stabilizers
Eline Sijbesma, Kenneth K. Hallenbeck, Sebastian A. Andrei, Reanne R. Rust, Joris M. C. Adriaans,
Luc Brunsveld,* Michelle R. Arkin,* and Christian Ottmann*
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ABSTRACT: The systematic discovery of functional fragments binding to the
composite interface of protein complexes is a first critical step for the development
of orthosteric stabilizers of protein−protein interactions (PPIs). We have
previously shown that disulfide trapping successfully yielded covalent stabilizers
for the PPI of 14-3-3 with the estrogen receptor ERα. Here we provide an
assessment of the composite PPI target pocket and the molecular characteristics of
various fragments binding to a specific subpocket. Evaluating structure−activity
relationships highlights the basic principles for PPI stabilization by these covalent
fragments that engage a relatively large and exposed binding pocket at the protein/
peptide interface with a “molecular glue” mode of action.
KEYWORDS: Protein−protein interactions, PPI stabilization, fragment-based drug discovery, covalent binder
he modulation of protein−protein interactions (PPIs) by
successfully selected fragments that enhanced the affinity
between 14-3-3 and ERα.25 We incubated the cysteine-
containing 14-3-3 protein with a disulfide library in the
presence or absence of ERα-derived phosphopeptide (ERα-
pp) and analyzed conjugate formation by intact-protein mass
spectrometry. The most effective covalent stabilizers increased
the PPI complex affinity by 40-fold. The molecular mechanism
was elucidated by X-ray cocrystal structures which revealed a
“molecular glue” mode of action, whereby the fragment
efficiently engaged a subpocket at the composite PPI interface,
interacting with both partners.
T
small molecule ligands is a highly sought-after strategy for
chemical biology and drug discovery.1−4 PPI stabilization and
inhibition are related approaches but based on profoundly
different underlying principles.5,6 The stabilization of protein
complexes by cooperatively binding ligands could have
tremendous benefit in terms of client selectivity since
druggable pockets at binding interfaces are constituted of
residues of both protein partners and thus only exist in the
context of the complex.2,7,8 In contrast, PPI inhibitors typically
affect multiple interactions of the target protein, an important
consideration when aiming to therapeutically target inter-
actions of “hub” proteins that have a large number of binding
partners.9−11 However, strategies to systematically screen for
small-molecule stabilizers are scarce. Since our interest focuses
on the interactions of the hub protein 14-3-3, we set out to
develop innovative strategies to discover new cooperative
ligands as stabilizers for 14-3-3 PPIs. The 14-3-3 proteins
regulate a number of important disease-related proteins; in
many cases, stabilization of the 14-3-3/client complex is
predicted to have a therapeutic effect.12−24 For instance,
binding of 14-3-3 to the C-terminus of estrogen receptor
(ERα) phosphorylated at T594 inhibits ERα activity;
stabilization of the interaction by the natural product
fusicoccin-A (FC-A) blocks growth of ERα-dependent
cells.22 Hence, small molecule 14-3-3 PPI stabilizers could be
useful chemical probes or starting points for drug discovery.
We have previously presented a site-directed fragment-based
screening approach (disulfide trapping), by which we
Here, we present the results of a study into the properties of
disulfide-tethered ligands and analyze both the affinity of
fragments at the 14-3-3 PPI pocket and the cooperativity
observed for fragments engaging a specific subpocket. The
position of the cysteine residue used for screening by disulfide
trapping was found to be crucial. Comparing covalent
fragments tethered to different engineered cysteine residues
along the rim of the pocket provided insight into pocket
ligandability by fragments. Residue C42 was suitable for
finding stabilizing fragments, whereas fragments bound to a
Received: February 9, 2021
Accepted: May 4, 2021
Published: May 10, 2021
© 2021 The Authors. Published by
American Chemical Society
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Figure 1. Disulfide trapping identify ligands for 14-3-3σ. (a) Target pocket for the site-directed disulfide-trapping approach, highlighting two
cysteine mutations (C42, C45; red surface areas) in the 14-3-3σ (white surface)/ERα-pp (green spheres) pocket. (b) Chemical structures of
previously described 14-3-3σ/ERα-pp stabilizers 1 and 2.25 (c−e) Chemical structures and disulfide trapping screening results for C45 hits. Mass
spectra for 14-3-3σ(C45) conjugated to fragment 3 (c), 4 (d), and 5 (e) in the absence (left) or presence (right) of ERα-pp. The adduct shift
between apo protein [expected mass 26 536 Da, 2-mercaptoethanol (βME)-capped mass 26 612 Da] and protein-disulfide conjugate mass is
indicated with arrows. Conditions for mass spectrometry: 100 nM 14-3-3σ, 200 nM ERα-pp, 100 μM fragment, 100 μM βME.
greater extent, but not cooperatively, to a cysteine at position
45 (C45). Both sites yielded several X-ray cocrystal structures
that provided hypotheses for binding affinity and cooperativity.
Even though these cysteine positions are non-native to 14-3-3
isoforms and optimization of fragments toward noncovalent
stabilizers represents a next challenge, initial structure−activity
relationships (SARs) were explored for the main cooperative
hit tethered to 14-3-3σ(C42), aiding our understanding of the
rules for 14-3-3/client stabilization by covalent fragments.
Covalent Fragments Tethered to 14-3-3σ C45.
Covalent fragments that strongly stabilized the interaction
between 14-3-3 and ERα were described previously.25 Briefly,
in a site-directed screening approach, we varied the position of
a cysteine residue serving as a reactive handle for thiol−
disulfide exchange with a library of ∼1600 disulfide-containing
fragments. We introduced cysteines at residues 42 and 45 on
14-3-3σ at the base of the target pocket adjacent to the C-
terminus of the ERα-pp in the protein/peptide complex
(Figure 1a). Fragments 1 and 2, tethered to 14-3-3σ(C42),
showed the best stabilization of the 14-3-3/ERα-pp complex
(Figure 1b; for which intact mass spectrometry (MS),
fluorescence polarization (FP), and X-ray crystallography
data was presented previously25); however, we also identified
fragments tethered to 14-3-3σ(C45). MS data of three hits are
presented here (Figure 1c−e). Of these, fragment 3 was ∼50%
bound to 14-3-3σ(C45) in the absence of ERα-pp and ∼90%
bound to 14-3-3σ(C45) in the presence of ERα-pp, based on
intact mass spectrometry (MS; Figure 1c). Additional
fragments displayed high % bound, as observed from the
protein-conjugate peaks for 4 and 5 (Figure 1d,e). Here, no
difference was observed between 14 and 3−3σ(C45) apo or
ERα-pp bound, indicating a strong affinity of these fragments
to 14-3-3 alone and no additional influence from the ERα
peptide on fragment binding. There is thus no initial indication
of PPI stabilizing or inhibiting activity.
Soaking cocrystals of 14-3-3σ(C45)/ERα-pp enabled the
observation of electron density for the three fragments 3−5,
with the most convincing, continuous density for 3 (Figure
2a). 4 only differs from 3 by the addition of a longer alkyl chain
(C3 versus C2 in 3), resulting in a less optimal binding pose in
the PPI complex (Figure 2b). Fragment 5 features a
chlorophenyl moiety, as is seen in fragments 1 and 2 (Figure
2c);25 interestingly, while the Cl moiety is positioned
identically, the phenyl ring of 5 is slightly tilted relative to 2
(Figure 2d). To determine whether fragments 3−5 stabilized
the 14-3-3σ(C45)/ERα-pp complex, we measured the binding
of fluorescein-labeled ERα-pp to 14-3-3σ(C45) by fluores-
cence anisotropy. Notably, fragments 3−5 did not induce 14-3-
3σ(C45)/ERα-pp complex formation, implying no stabiliza-
tion of the protein/peptide complex (Figure S1). This
observation was particularly striking for 3, given the close
proximity between the fragment and ERα-pp in the X-ray
cocrystal structure (Figure 2a), and its apparent increased
binding to 14-3-3σ(C45) in the presence of ERα-pp observed
by mass spectrometry (Figure 1c).
The lack of cooperativity for fragments 3−5 was confirmed
in MS titration experiments, performed under stronger
reducing conditions (1 mM βME) emphasizing the non-
covalent contribution of the fragment in stabilizing the PPI.
Here, the conjugation peak for 14-3-3σ(C45)-3 indicated
>80% tethering for all concentrations of fragment 3 (100 nM
to 1 mM), which was not influenced by the presence of ERα-
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only reached >80% tethering at high concentrations. The
cooperative difference for 3 tethered to C42 versus C45 was
further confirmed by fluorescence anisotropy, where fluo-
rescein-labeled ERα-pp binding was enhanced upon titration of
14-3-3σ(C42) with 3 (EC50 0.9 0.11 μM, Figure 3c). These
results indicated that, in addition to the appropriate chemo-
phore, the covalent tethering position was also important to
elicit stabilization. Together, these data suggested that even
though the C45-tethered fragments bound tightly to the 14-3-3
pocket, they lacked significant stabilizing activity toward the
motif, suggesting that the disulfide bond formation between 3
and 14-3-3 C45 contributed a degree of cooperativity observed
in the primary screen. Since they also did not show any
inhibition toward the PPI under study, these fragments, as
neutral binders, were compatible with the binary complex but
did not engage the composite interface enough to drive
orthosteric cooperativity. Thus, cooperativity in PPI binding is
finely tuned and depends on an optimal positioning of all
molecular elements.26,27
These results illustrate that strong binding of a fragment to
the PPI complex does not necessarily result in PPI stabilizing
activity. Indeed, when looking in more detail at data for C42
hits, strong tethering by itself or clear density in a cocrystal
structure are not per se good predictors of stabilizing activity
toward 14-3-3/ERα-pp, whereas differential dose−response
behavior of tethered fragments in absence or presence of ERα-
pp by MS is nicely correlated with stabilization in fluorescence
anisotropy. Fragment 1 displayed high % tethering to 14-3-
3σ(C42) only in the presence of ERα-pp, where >80%
tethering is observed for all concentrations of fragment 1
(Figure 3d), which is reflected by efficient stabilization of 14-3-
3/ERα-pp by 1.25 The X-ray cocrystal structures display
similarly clear density for fragments 1 and 3, further confirming
that a good binder (even to a composite pocket) is not
sufficient nor necessarily predictive of PPI stabilization
Figure 2. Close-up views of the binding pocket for cocrystal
structures of 14-3-3σ(C45)-tethered fragment 3−5 (a−c, mint sticks).
2Fo-Fc electron density maps are contoured at 1σ, 14-3-3σ is shown as
white surface, and ERα-pp as dark green sticks. (d) Crystallographic
overlay for C42-bound fragment 2 (blue) and C45-bound 5 (mint).
pp under these conditions (Figure 3a). Interestingly, a dose−
response effect was observed for titration of 3 to 14-3-
3σ(C42), where the presence of ERα-pp slightly increased
%-tethering at all concentrations (Figure 3b). In contrast,
fragment 1 showed cooperative tethering to both cysteine
positions, with the larger effect observed for C42 (Figure 3c,d).
Whereas 3 for C45 tethered similarly over the entire
concentration range, fragment 1 showed a steep S-curve and
Figure 3. Analysis of cooperative binding to 14-3-3σ of tethered fragments with ERα-pp. Dose−response data of protein−fragment conjugate
formation for 14-3-3σ C45 (a and c) or C42 (b and d), titrated with fragment 3 or 1, respectively, and analyzed by intact-protein mass
spectrometry (MS). 100 nM 14-3-3σ, 200 nM ERα-pp, 100 μM fragment, 1 mM βME. (e) Fluorescence anisotropy dose−response curves for 14-
3-3σ(C45 or C42) and fluorescein-labeled ERα-pp titrated with fragment 3. Data for time point 0 (t0) and at end point (after overnight incubation,
t
o/n) are displayed. (f) Schematic illustration of binding equilibria for ternary complex formation.
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Figure 4. Derivatives of 14-3-3σ(C42)/ERα-pp stabilizer 2. Chemical structures and X-ray cocrystal structures of compounds 6−13 in the binding
pocket tethered to C42 (yellow surface). 2Fo-Fc electron density maps are contoured at 1σ. Water molecules are depicted as red spheres.
potential. Furthermore, the data suggest that the C45 position
of 14-3-3 does not allow the fragments in our library to achieve
the proper orientation to stabilize the 14-3-3σ/ERα-pp
complex.
Derivatives of 14-3-3 C42-Tethered Stabilizers. A
small library of derivatives of fragment 2 was then synthesized
to assess the main contributing factors to the 14-3-3/ERα-pp
stabilizing activity. X-ray cocrystal structures were obtained for
eight disulfide fragments tethered to 14-3-3σ(C42) bound by
ERα-pp (Figure 4). The most resolved electron density was
observed for variants with a single para- or double meta-
halogen substituent on the phenyl (6−9, Figure 4a). An
unsubstituted phenyl (10) and a p-methylphenyl (11) showed
weaker electron density (Figure 4b). An m-methoxy in
addition to a p-bromo substituent resulted in well-resolved
electron density for 12, whereas a combination of o-chloro and
p-nitro substituents was less beneficial, resulting in electron
density mainly for the phenyl group and only part of the linker
for 13 (Figure 4c). Compared to the rest of the series, the
phenyl ring of 13 was also rotated by 90°, directed by the o-
chloro and resulting in the subsequent relocation of the linker.
Crystallographic overlays of compounds 6−12 with 2 (PDB
Figure 5. Crystallographic overlays for 6−12. Fragments tethered to
14-3-3σ(C42) bound by ERα-pp, overlaid on the X-ray cocrystal
entry 6HMT)25 reveals the apparent strict positioning of the
halogen in the pocket, specifically when comparing single
substituents on the para-position to double meta-substituents
(Figure 5a,b). For the double meta-substituted compounds,
the molecules are reorientated so that one of the halogens (in
8 and 9) overlays with the para-chloro position of 2. The
unsubstituted or p-fluorophenyl are less directing (Figure 5c),
structure of the original stabilizer 2 (PDB entry 6HMT).
while a p-methyl or the p-bromo/m-methoxy combination
perfectly overlays with the position of 2 (Figure 5d). While all
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Figure 6. Stabilization activity for a focused library of fragment 2 structure variations. Dose−response curves of 14-3-3σ(C42) and fluorescein-
labeled ERα-pp titrated with FC-A (orange), 2, and 6−13. Fluorescence anisotropy data and corresponding EC50 values, at time point 0 (t0) and at
end point (after overnight incubation, to/n) are displayed.
compounds occupy this same subpocket, 13 shows the most
divergent binding pose (Figure S2a).
highly beneficial for orientation into the identified subpocket,
as the strongest stabilization and most resolved electron
density are observed for both p-chloro (2) and p-bromo (6)
and doubly substituted m,m-fluoro (8) and m,m-chloro- (9)
phenyls. Finally, the constraining effect of the dimethyl moiety
on the linker appears important for achieving a specific
orientation. The surface complementarity of the fragments’
binding site, constituted jointly by the protein/peptide, further
explains the differences between C42/C45-binding fragments.
The binding pocket of 14-3-3/ERα is defined by several
molecular recognition elements, most optimally engaged by
the potent stabilizer and tool-compound FC-A (Figure S3a).
The part of the pocket toward the peptide C-terminus is
defined by a hydrophobic patch shaped by the “roof-of-the-
groove” 14-3-3 residues L218, I219, and L222 and V595 of
ERα. The C42-tethered fragments show optimal orientation of
a dimethyl moiety and of the phenyl-halogen facing toward
V595, thereby nestling in that hydrophobic patch in a shape-
complementary fashion, resulting in a stabilizing effect toward
the peptide binding (Figure S3b). In comparison, C45 binders
are tethered to the protein in too close proximity to the
peptide. Even though strong binding is observed to the protein
by itself, the majority of the noncovalent contributions are
directed toward the 14-3-3 protein pocket pointing away from
the peptide. Additionally, two 14-3-3 lysine residues at the base
of the groove close to the peptide C-terminus, K122 and K49,
hydrogen-bond to FC-A. Finally, an important feature of FC-A
stabilization of the 14-3-3/ERα complex is the hydrogen
bonding network between two FC-A hydroxyl groups and 14-
3-3′s D215, which is pulled toward the compound, thereby
A potential stabilization activity of 6−13 was analyzed in
fluorescence anisotropy experiments by titrating 14-3-3σ(C42)
and ERα-pp with the fragments (Figure 6). Data were
collected directly after preparing the samples (t0) and after
reaching equilibrium (at end point, after overnight incubation,
t
o/n). All derivatives were found to be stabilizers of the 14-3-
3σ/ERα-pp complex as indicated by enhanced ERα-pp binding
upon fragment titrations, displaying EC50 values (173−911
nM) in the same range as 2 (EC50 299 14 nM). Whereas the
equilibrium was near-instantaneous for stabilization by the
natural product FC-A, PPI stabilization induced by tethered
fragments binding upon thiol−disulfide exchange logically
displayed slower kinetics, perhaps due to the absence of βME
in these experiments. The to/n curve was shifted to the left,
resulting in roughly 10-fold improved EC50 values compared to
t0. Additionally, upper plateaus for 6, 8, and 9 reached
anisotropy values similar to FC-A and 2, while for 7, 10, and
11−13 the maximum anisotropy was lower, possibly caused by
a reduced stabilization of the distal region of ERα-pp. This was
reflected in the X-ray cocrystal structures, where the side chain
of the phenylalanine at the −2 position (F591) was flexible,
revealing different orientations and in two cocrystal structures;
additional density was also observed for G590 (Figure S2b).
This small set of derivatives is not sufficient for the
establishment of a complete SAR, yet it does provide several
valuable insights. First, it is interesting to find that all variations
are tolerated and only influence stabilization activity within a 3-
fold range of EC50 values. Second, a halogen on the phenyl is
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dragging helix 9 down toward the binding pocket (Figure S3a).
We have reported previously that this interaction is crucial for
a potent stabilizing allosteric effect.28 Whereas this residue is
engaged by neither C42/C45 binders reported here, it
constitutes an interesting opportunity for future optimization
of tethered fragments toward potent (noncovalent) 14-3-3 PPI
stabilizers.
Complex Molecular Systems (ICMS), Eindhoven University
of Technology, 5600 MB Eindhoven, The Netherlands
Kenneth K. Hallenbeck − Department of Pharmaceutical
Chemistry and Small Molecule Discovery Center (SMDC),
University of California, San Francisco 94134, United States
Sebastian A. Andrei − Laboratory of Chemical Biology,
Department of Biomedical Engineering and Institute for
Complex Molecular Systems (ICMS), Eindhoven University
of Technology, 5600 MB Eindhoven, The Netherlands
Reanne R. Rust − Laboratory of Chemical Biology,
Department of Biomedical Engineering and Institute for
Complex Molecular Systems (ICMS), Eindhoven University
of Technology, 5600 MB Eindhoven, The Netherlands
Joris M. C. Adriaans − Laboratory of Chemical Biology,
Department of Biomedical Engineering and Institute for
Complex Molecular Systems (ICMS), Eindhoven University
of Technology, 5600 MB Eindhoven, The Netherlands
In this work, we described covalent fragments that bound to
two engineered cysteine residues near the pocket formed by
the 14-3-3σ/ERα-pp complex. These fragments were identified
via disulfide trapping (“tethering”) screens that we proposed as
a systematic strategy for the discovery of PPI stabilizers.
Cooperative stabilization was achieved via tethering to C42,
whereas tethering to C45 resulted in neutral binders based on
similar chemophores. X-ray cocrystal structures combined with
biochemical binding studies revealed that tight binding alone
did not necessarily guarantee effective PPI stabilization. C42
appeared to be ideally located for identifying optimal stabilizers
for 14-3-3/ERα from this disulfide library. Some fragments,
particularly tethered to C45, strongly bound to 14-3-3 without
influencing ERα binding. Coupled with an understanding of
the features that lead to PPI stabilization, these tightly bound
compounds could perhaps be chemical optimized into effective
stabilizers. The ability to optimize screening for local
differences in target pockets is an important benefit of a
reversible covalent-fragment screening strategy and further
illustrates the suitability of the tethering approach to identify
stabilizers for adaptive interfaces and composite PPI pockets.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.1c00088
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Funding
This research was supported by The Netherlands Organization
for Scientific Research (through Gravity Program 024.001.035,
VICI grant 016.150.366 and ECHO grant 711.018.003).
Notes
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.1c00088.
■
The authors declare the following competing financial
interest(s): E.S. is full-time employee of Ambagon Therapeu-
tics, L.B., M.R.A., and C.O. are founders and shareholders of
Ambagon Therapeutics.
sı
Experimental procedures; fluorescence anisotropy dose−
response curves; crystallographic overlays; binding
modes of FC-A and C42- and C45-tethered fragments;
XRD data collection and refinement statistics; 13C NMR
Crystallographic structure data were deposited in the Protein
Data Bank (PDB) and obtained IDs: 7B9M, 7B9R, 7B9T,
7BA3, 7BA5, 7BA6, 7BA7, 7BA8, 7BA9, 7BAA, and 7BAB.
1
and H NMR spectra (PDF)
ACKNOWLEDGMENTS
■
We acknowledge the Renslo laboratory for synthesis of the
SMDC disulfide library and thank J. Schill for assistance with
synthesis and characterization.
AUTHOR INFORMATION
Corresponding Authors
■
Luc Brunsveld − Laboratory of Chemical Biology, Department
of Biomedical Engineering and Institute for Complex
Molecular Systems (ICMS), Eindhoven University of
Technology, 5600 MB Eindhoven, The Netherlands;
orcid.org/0000-0001-5675-511X; Email: l.brunsveld@
tue.nl
ABBREVIATIONS
■
PPI, protein−protein interaction; SAR, structure−activity
relationship; ERα, estrogen receptor alpha; FC-A, fusicoccin-
A; pp, phosphopeptide; MS, mass spectrometry; βME, 2-
mercaptoethanol; FA, fluorescence anisotropy.
Michelle R. Arkin − Department of Pharmaceutical Chemistry
and Small Molecule Discovery Center (SMDC), University of
California, San Francisco 94134, United States;
orcid.org/0000-0002-9366-6770;
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Eline Sijbesma − Laboratory of Chemical Biology,
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