Cover Picture: Identification and Structure of Small-Molecule Stabilizers of 14–3–3 Protein–Protein Interactions (Angew. Chem. Int. Ed. 24/2010)

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Protein–Protein Interactions
DOI: 10.1002/anie.200907203
Identification and Structure of Small-Molecule Stabilizers of 14–3–3
Protein–Protein Interactions**
Rolf Rose, Silke Erdmann, Stefanie Bovens, Alexander Wolf, Micheline Rose, Sven Hennig,
Herbert Waldmann, and Christian Ottmann*
Inhibition of protein–protein interactions (PPIs) with small
molecules has gained substantial interest in pharmaceutical
research and provided novel opportunities for the treatment
of disease.
[1–5]
However, the alternative development of small
molecules that stabilize protein–protein interactions has been
achieved in only very few cases,
[6,7]
typically including
structurally complex natural products that are usually pro-
duced by fermentation and isolation from natural sources.
[8–11]
These compounds are however widely employed in basic
research and even in clinical use, thereby demonstrating the
value of this approach. Therefore, the identification and
development of small-molecule PPI stabilizers that are
tractable by organic synthesis and can be optimized in terms
of potency and specificity is of major interest.
A particularly relevant case is given by the 14–3–3
proteins and their interaction with target proteins. The 14–
3–3 proteins are a highly conserved class of adapter proteins
that are involved in the regulation of several hundred
proteins, among them important pharmaceutical targets
such as Raf, p53, Cdc25, Cdk2, and histone deacetylases
(HDACs).
[12]
Binding of 14–3–3 proteins can either be
inhibitory, such as in the case of the Cdc25 phosphatases,
[13]
or it can be stimulatory as in the case of the tumor suppressor
protein p53.
[14]
Depending on the physiological context, for
example in different cancers, the stabilization of a regulatory
14–3–3 protein–protein complex might be of therapeutic
value. However, small molecule stabilizers of these thera-
peutically interesting interactions are not known. An example
of a natural 14–3–3 PPI-stabilizing small molecule is the
specific 14–3–3 protein–protein interaction with the plant
proton pump PMA2, which can be stabilized by the fungal
toxin fusicoccin.
[15]
This natural compound binds to a site
preformed by the two proteins and mediates its stabilizing
function by simultaneously contacting both partner pro-
teins.
[16,17]
Herein we report on the identification of two small
molecules that selectively stabilize the 14–3–3/PMA2 pro-
tein–protein interaction by binding to two adjacent sites in the
interface, and which are active in vivo. To identify potential
stabilizing molecules, we screened a 37 000-member com-
pound library in a surface-based format monitoring the
binding of green fluorescent protein (GFP) fused with 14–3–3
to surface-immobilized gluthatione S-transferase (GST) fused
with PMA2-CT52 (the C-terminal 52 amino acids of PMA2;
see the Supporting Information). The initial screen success-
fully identified two structurally unrelated compounds with the
desired activity (Figure 1a). For these compounds, the
kinetics of the stabilizing activity were determined by
means of surface plasmon resonance (SPR; Figure 1b). To
this end, the 14–3–3 interaction domain of PMA2, that is, the
52 C-terminal amino acids (CT52)
[17]
was immobilized on the
dextran matrix of a BiaCore chip, and binding of the 14–3–3
protein was measured in the presence of the protein-protein
interaction-stabilizing small molecule. The trisubstituted
pyrrolinone hit compound, pyrrolidone1, showed association
kinetics similar to fusicoccin (Figure 1b) but with a more
rapid dissociation, resulting in a K
D
of 80 mm. The dipeptide
epibestatin displayed a different kinetic behavior in stabiliz-
ing the 14–3–3/PMA2 complex, with a slower association than
fusicoccin and pyrrolidone1 (Figure 2b). However, the dis-
sociation kinetics of epibestatin resembled that of fusicoccin;
that is, the protein complex was very stable once it was
constituted and dissociation was very slow. As a result, the
calculated K
D
of the 14–3–3/PMA2 complex in the presence
of epibestatin was 1.8 mm. In the absence of any stabilizing
compound no binding of 14–3–3 to immobilized PMA2 could
be measured (Figure 1b, DMSO control).
To unravel the mechanistic basis of the obviously different
binding modes of pyrrolidone1 and epibestatin, the crystal
structures the ternary complexes with 14–3–3 and the C-
terminal 30 amino acids of PMA2 (PMA2-CT30) were solved.
We obtained co-crystals by mixing 14–3–3 protein and PMA2-
CT30 (1:1.5 ratio) in the presence of either 2 mm pyrrolidone1
or 2 mm epibestatin. Both protein complexes crystallized in
[*] Dr. R. Rose, Dr. S. Erdmann, Dr. S. Bovens, Dr. A. Wolf, M. Rose,
Dr. S. Hennig, Dr. C. Ottmann
Chemical Genomics Centre of the Max Planck Society
Otto-Hahn-Strasse 15, 44227 Dortmund (Germany)
Fax : (+ 49)231-133-2499
E-mail: christian.ottmann@cgc.mpg.de
Homepage: http://www.cgc.mpg.de
Prof. Dr. H. Waldmann
Max-Planck-Institut fr molekulare Physiologie
Abteilung Chemische Biologie
Otto-Hahn-Strasse 11, 44227 Dortmund (Germany)
and
Technische Universitt Dortmund, Fakultt Chemie
Lehrbereich Chemische Biologie
Otto-Hahn-Strasse 6, 44227 Dortmund (Germany)
[**] This work was supported by the Max Planck Society and BMBF grant
GO-Bio 0313873 (to R.R., S.B., S.E., A.W., M.R., and S.H.). We
thank Alfred Wittinghofer for helpful discussions and the staff at the
Swiss Light Source, beamline X10SA, for support during crystallo-
graphic data collection. The atomic coordinates and structure
factors of the T14-3e/CT30/pyrrolidone1 and the T14-3e/CT30/
epibestatin complex have been deposited in the Protein Data Bank
(PDB) under the ID codes 3M51 and 3M50.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200907203.
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the same space group, but whilst the epibestatin crystals
diffracted reproducibly to 2.6 , the crystals of the pyrroli-
done1 complex did not diffract beyond 3.2 . Nevertheless,
we were able to determine the binding and orientation at the
interface of the two protein partners
for both molecules (Figure 2e,f;
Supporting Information, Figur-
e S1,S2).
The different association/disso-
ciation kinetics of pyrrolidone1 and
epibestatin are reflected in their
binding modes. Pyrrolidone1 shares
most of its protein contact surface
with 14–3–3 (288.2
2
from a total of
349.7
2
; 83 %) and binds to a site
that is highly accessible from the
solvent space, which might contrib-
ute to its fast association kinetics
(Figure 1b,c). The contacts to
PMA2 are rather limited (Figure 1 f,
Figure 2a), a fact that together with
the general exposed character of its
binding pocket might account for
the high dissociation rate of the
complex. In contrast to pyrroli-
done1, epibestatin is more deeply
buried in the protein complex and is
literally trapped between 14–3–3
and PMA2 (Figure 1 d), and shares
a roughly equal contact surface with
14–3–3 (164.4
2
, 55%) and PMA2
(135.5
2
, 45%; Figure 1g). These
characteristics are in accordance
with the observed slow association
and dissociation kinetics.
Pyrrolidone1 shares part of its
binding site with fusicoccin (Fig-
ure 2a) but is coordinated in quite
a different way. Fusicoccin almost
completely fills the cavity of the
binding groove of 14–3–3, whereas
pyrrolidone1 only partially fills this
region (Figure 2a–c). This binding
mode indicates that appropriate var-
iation of the structure of this inhib-
itor type may yield compounds that
completely fill the gap in the inter-
face of the two proteins. Epibestatin,
like fusicoccin, fills its binding site, a
crevice between the two proteins, to
a considerably higher extent than
observed for pyrrolidone1 (Fig-
ure 2d). Therefore, the binding
mode of epibestatin suggests a
strong shape-driven component
that is comparable to the binding
mode of fusicoccin. The more inti-
mate contact to both protein part-
ners probably accounts for the stronger potency of epibestatin
in stabilizing the 14–3–3/PMA2 complex. Details of the
protein contacts of both compounds are shown in Figure 2 e
and f.
Figure 1. Pyrrolidone1 and epibestatin stabilize the 14–3–3/PMA2 complex in different ways to each
other and to fusicoccin. a) Structures of fusicoccin, pyrrolidone1, and epibestatin. b) The associa-
tion/dissociation of 14–3–3 to the binding domain of PMA2 (CT52) was measured with surface
plasmon resonance (BiaCore) in the presence of fusicoccin (5 mm, gray) pyrrolidone1 (50 mm,
yellow), and epibestatin (50 mm, orange) or in DMSO as control (open). The turning point from the
association to the dissociation phase of the experiment is indicated. c) Crystal structure of
pyrrolidone1 (yellow) bound to the binary complex of 14–3–3 (green) and PMA2-CT30 (blue surface).
d) Epibestatin (orange) bound to the 14–3–3/PMA2-CT30 complex. e) Superimposition of fusicoccin
(gray) bound to the 14–3–3/PMA2-CT30 (surface representation, color coding as in (c) and derived
from our previously published 14–3–3/PMA2-CT52 complex structure (PDB code: 2O98).
[17]
f) Bind-
ing site of pyrrolidone1 (yellow) in the 14–3–3/PMA2-CT30 complex. g) Binding site of epibestatin
(orange) in the 14–3–3/PMA2-CT30 complex.
Communications
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The stabilization of the 14–3–3/PMA2 complex by fusi-
coccin leads to activation of the proton pump, opening of the
stomata, which are the gas-exchanging pores on the leaf
surface, and subsequent wilting of the plant.
[18,19]
To deter-
mine whether the two compounds identified in our biochem-
ical screen also display in vivo activity, we employed a
stomata-opening test in which isolated epidermis of leaves
from the Asiatic dayflower (Commelina communis) is incu-
bated with the test compounds. After three hours, the opening
width of the stomatal pores was measured by light microscopy
(Figure 3a). In the presence of 50 mm pyrrolidone1 or
epibestatin, stomatal pores opened 3 mm and 3.2 mmon
average, respectively. This data clearly indicate in vivo
potency of pyrrolidone1 and epibestatin as activators of the
plant proton pump.
The fact that pyrrolidone1 and epibestatin are chemically
unrelated to each other and furthermore show no structural
resemblance to fusicoccin is of particular note for the strategy
to employ small molecule 14–3–3 protein–protein stabilizers.
The initial discovery of different chemotypes from a limited
primary screening for a certain 14–3–3 protein–protein
interaction indicates that further compound classes may be
found. As the actual binding sites of protein–protein stabiliz-
ing molecules are constituted by both protein partners, the
intrinsic specificity of these molecules may be very high. To
investigate this possibility, we tested epibestatin and pyrroli-
done1 in regard to their potential ability to stabilize other
14–3–3 protein–protein interactions, including Raf1, p53,
Cdc25C, RNF11, Mlf1, AICD, Cby, and YAP (Supporting
Information, Figure S3,S4). Both compounds showed no
activity in stabilizing the binding of 14–3–3 to any of these
targets. Although this investigation is not comprehensive, our
data indicate that it might be possible to identify specific small
molecules that stabilize distinct 14–3–3 protein–protein
Figure 2. Comparison of the binding modes of fusicoccin, pyrroli-
done1, and epibestatin. a) Superimposition of fusicoccin (gray) and
pyrrolidone1 (yellow) bound to the 14–3–3/PMA2-CT30 complex
(green and blue surface, respectively). b) Fusicoccin (gray) fills the
profile of the 14–3–3 binding grove (green) almost completely.
c) Pyrrolidone1 (yellow), in contrast to fusicoccin, occupies only about
one third of the 14–3–3 binding grove profile. d) Binding of epibestatin
(yellow) to a crevice-like site between 14–3–3 (green) and PMA2-CT30
(blue). e) Residues from 14–3–3 (green) and PMA2 (blue) implicated
in the coordination of pyrrolidone1 (yellow). Polar contacts are
indicated as yellow lines, the 2F
obs
F
calc
electron density of pyrroli-
done1 (contoured at 1s) and two water molecules (red spheres) is
shown in white. f) Residues of the 14–3–3/PMA2 complex (color
coding as in (e)) implicated in binding of epibestatin (orange) with the
the 2F
obs
F
calc
electron density of epibestatin (contoured at 1s) shown
in white.
Figure 3. Pyrrolidone1 and epibestatin show fusicoccin-specific activity
in plant cells. a) Opening of stomatal pores in the presence of
fusicoccin (5 mm), pyrrolidone1 (50 mm), and epibestatin (50 mm). The
epidermis of leaves from the Asiatic dayflower (Commelina communis)
were incubated for 3 h in buffered solutions containing fusicoccin,
pyrrolidone1, epibestatin, or DMSO (control) and the opening width
were analyzed by light microscopy. b) Median opening width of
stomata after 3 h treatments of epidermal preparations from Comme-
lina communis.
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interactions with high selectivity. Such specific molecules
would be valuable tools in investigating the biology of 14–3–3
protein–protein interactions with a plethora of target proteins
and might be promising starting points for the development of
medicinally active agents.
Received: December 21, 2009
Revised: March 1, 2010
Published online: April 30, 2010
.
Keywords: 14–3–3 proteins · crystal structures · drug discovery ·
protein–protein interactions · surface plasmon resonance
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