Discovery and structural characterization of a small molecule 14-3-3 protein-protein interaction inhibitor

Department of Pharmacology, Emory UniversitySchool of Medicine, Atlanta, GA 30322, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 09/2011; 108(39):16212-6. DOI: 10.1073/pnas.1100012108
Source: PubMed
ABSTRACT
The 14-3-3 family of phosphoserine/threonine-recognition proteins engage multiple nodes in signaling networks that control diverse physiological and pathophysiological functions and have emerged as promising therapeutic targets for such diseases as cancer and neurodegenerative disorders. Thus, small molecule modulators of 14-3-3 are much needed agents for chemical biology investigations and therapeutic development. To analyze 14-3-3 function and modulate its activity, we conducted a chemical screen and identified 4-[(2Z)-2-[4-formyl-6-methyl-5-oxo-3-(phosphonatooxymethyl)pyridin-2-ylidene]hydrazinyl]benzoate as a 14-3-3 inhibitor, which we termed FOBISIN (FOurteen-three-three BInding Small molecule INhibitor) 101. FOBISIN101 effectively blocked the binding of 14-3-3 with Raf-1 and proline-rich AKT substrate, 40 kD(a) and neutralized the ability of 14-3-3 to activate exoenzyme S ADP-ribosyltransferase. To provide a mechanistic basis for 14-3-3 inhibition, the crystal structure of 14-3-3ζ in complex with FOBISIN101 was solved. Unexpectedly, the double bond linking the pyridoxal-phosphate and benzoate moieties was reduced by X-rays to create a covalent linkage of the pyridoxal-phosphate moiety to lysine 120 in the binding groove of 14-3-3, leading to persistent 14-3-3 inactivation. We suggest that FOBISIN101-like molecules could be developed as an entirely unique class of 14-3-3 inhibitors, which may serve as radiation-triggered therapeutic agents for the treatment of 14-3-3-mediated diseases, such as cancer.

Full-text

Available from: Fadlo R Khuri
Discovery and structural characterization of a small
molecule 14-3-3 protein-protein interaction inhibitor
Jing Zhao
a,b,1
, Yuhong Du
a,c,1
, John R. Horton
d,1
, Anup K. Upadhyay
d,1
, Bin Lou
a
, Yan Bai
a
, Xing Zhang
d
, Lupei Du
e
,
Minyong Li
e
, Binghe Wang
e
, Lixin Zhang
a,f
, Joseph T. Barbieri
g
, Fadlo R. Khuri
h
, Xiaodong Cheng
d,2
, and Haian Fu
a,h,c,2
a
Departments of Pharmacology;
d
Biochemistry; and
h
Hematology and Medical Oncology;
c
Emory Chemical Biology Discovery Center, Emory University
School of Medicine, Atlanta, GA 30322;
b
Department of Biochemistry and Molecular Biology, the Fourth Military Medical University, 17 Changle West
Road, Xian 710032, China;
e
Department of Chemistry, Georgia State University, Atlanta, GA 30302;
f
Institute of Microbiology, Chinese Academy of
Sciences, Beijing 100101, China; and
g
Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, WI 53226
Edited by Tony Pawson, Samuel Lunenfeld Research Institute, Toronto, Canada, and approved July 27, 2011 (received for review January 1, 2011)
The 14-3-3 family of phosphoserine/threonine-recognition proteins
engage multiple nodes in signaling networks that control diverse
physiological and pathophysiological functions and have emerged
as promising therapeutic targets for such diseases as cancer and
neurodegenerative disorders. Thus, small molecule modulators of
14-3-3 are much needed agents for chemical biology investigations
and therapeutic development. To analyze 14-3-3 function and mod-
ulate its activity, we conducted a chemical screen and identified
4-[(2Z)-2-[4-formyl-6-methyl-5-oxo-3-(phosphonatooxymethyl)pyri-
din-2-ylidene]hydrazinyl]benzoate as a 14-3-3 inhibitor, which we
termed FOBISIN (FOurteen-three-three BInding Small molecule IN-
hibitor) 101. FOBISIN101 effectively blocked the binding of 14-3-3
with Raf-1 and proline-rich AKT substrate, 40 kD
a
and neutralized
the ability of 14-3-3 to activate exoenzyme S ADP-ribosyltransfer-
ase. To provide a mechanistic basis for 14-3-3 inhibition, the crystal
structure of 14-3-3ζ in complex with FOBISIN101 was solved.
Unexpectedly, the double bond linking the pyridoxal-phosphate
and benzoate moieties was reduced by X-rays to create a covalent
linkage of the pyridoxal-phosphate moiety to lysine 120 in the
binding groove of 14-3-3, leading to persistent 14-3-3 inactivation.
We suggest that FOBISIN101-like molecules could be developed as
an entirely unique class of 14-3-3 inhibitors, which may serve as
radiation-triggered therapeutic agents for the treatment of 14-3-
3-mediated diseases, such as cancer.
small molecule 14-3-3 modulator
I
nitially discovered as a protein abundant in the brain, the 14-3-3
family of proteins consists of seven defined isoforms (β, ϵ, γ, η, σ,
τ, and ζ) in mammals and is widely expressed in all tissues and
organs examined (13). 14-3-3 acts as an adaptor protein that
controls the function of its target proteins through highly regu-
lated proteinprotein interactions. Studies on the interaction of
14-3-3 with phosphorylated Raf-1 led to the discovery of 14-3-3 as
the founding member of the class of phosphoserine/threonine-
binding protein modules (48). Reversible phosphorylation of
target proteins at a defined motif dictates the 14-3-3 association
in response to dynamic actions of cellular kinases and phospha-
tases. These 14-3-3 recognition motifs include the prototype
sequence, RSxpS/TxP (mode 1), and RxxxpS/TxP (mode 2), pS/TX-
COOH (mode 3), where x stands for any amino acid (811). The
availability of well characterized 14-3-3 recognition motifs coupled
with advanced genomics, proteomics, and functional biology ap-
proaches has revealed an entirely new landscape in which 14-3-3
binds a variety of signaling molecules, controlling their function
in response to environmental signals (8, 9, 1214). More than 200
ligand proteins have been identified for 14-3-3 (12). Depending on
the nature of its target proteins, 14-3-3 binding impacts multiple
signaling pathways that determine cell fate and organ development.
For example, 14-3-3 association controls Raf signaling fidelity,
neutralizes Bad-mediated apoptosis, and couples histone H3 with
H4 to create a histone code for transcriptional elongation (2, 3, 15).
Through these highly regulated interactions, 14-3-3 proteins govern
diverse physiological processes as well as a wide range of pathophy-
siological events. For example, dysregulated 14-3-3 signaling con-
tributes to the development of a number of human diseases, such
as cancer and neurodegenerative diseases (13). Thus, 14-3-3 pro-
teins are promising molecular targets for probe discovery and
therapeutic development.
In an effort to discover 14-3-3 proteinprotein interaction
modulators, we have previously reported the development and
structural characterization of peptide 14-3-3 antagonists, R18
and difopein (16 18), which have been widely used in the field
to manipulate 14-3-3/client protein interactions for functional
studies. It is expected that small molecule 14-3-3 modulator dis-
covery would provide added advantages to rapidly advance the
14-3-3 field, which impacts a wide range of biomedical areas.
Here we report our experimental chemical screening effort, the
identification and analysis of FOBISIN101 as a phosphoSer/Thr-
mimetic agent, and the structural details of the FOBISIN101
14-3-3ζ interaction. This study revealed an unexpected covalent
modification of 14-3-3ζ by a FOBISIN 101 derivative at a critical
ligand binding site, Lys120, explaining its potent 14-3-3 inhibitory
effect.
Results and Discussion
Using a fluorescence polarization-based 14-3-3 binding assay
(19), we screened the LOPAC library for compounds that disrupt
the interaction of 14-3-3γ with the pS259-Raf-1 peptide and
identified FOBISIN101 (F1 in Fig. 1A) as a potential 14-3-3 in-
hibitor (Fig. S1) (19, 20). F1 consists of a pyridoxal-phosphate
moiety linked to p-amino-benzoate via an N ¼ N bond. Because
the screening assay utilized a 14-3-3-binding peptide, it is essen-
tial to demonstrate that FOBISIN101 is capable of disrupting
the interaction of 14-3-3 with its full-length binding proteins.
We employed three complementary biochemical and functional
assays for this purpose. A Glutathione S-transferase (GST) fusion
14-3-3 affinity chromatography assay was used to examine the
ability of F1 to disrupt the 14-3-3 association with two well estab-
lished partners, Raf-1 and proline-rich AKT substrate, 40 kD
a
(PRAS40). The addition of increasing concentrations of F1 to
the cell lysates led to a dose-dependent release of PRAS40 and
Raf-1, supporting an effective inhibitory role of F1. This inhibi-
Author contributions: Y.D., F.R.K., X.C., and H.F. designed research; J.Z., Y.D., J.R.H., A.K.U.,
B.L., Y.B., and X.Z. performed research; B.L., L.D., M.L., B.W., L.Z., and J.T.B. contributed
new reagents/analytic tools; J.Z., Y.D., J.R.H., A.K.U., Y.B., X.Z., L.Z., F.R.K., X.C., and H.F.
analyzed data; and J.Z., Y.D., J.R.H., A.K.U., X.C., and H.F. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The atomic coordinates have been deposited in the Protein Data Bank,
www.pdb.org (PDB ID code 3RDH).
1
J.Z., Y.D., J.R.H., A.K.U. contributed equally to this work.
2
To whom correspondence may be addressed. E-mail: hfu@emory.edu or xcheng@
emory.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/
doi:10.1073/pnas.1100012108/-/DCSupplemental.
1621216216 PNAS September 27, 2011 vol. 108 no. 39 www.pnas.org/cgi/doi/10.1073/pnas.1100012108
Page 1
tion occurred in a manner similar to the action of a defined
14-3-3 antagonist peptide, R18 (17) (Fig. 1B). F1 appears to be
a pan-14-3-3 inhibitor as it decreased the binding of Raf-1 to all
seven 14-3-3 isoforms in a dose-dependent manner (Fig. S2). A
quantitative enzyme-linked immunosorbent assay established
a half-maximal inhibitory concentration (IC
50
) for F1, which was
9.3 or 16.4 μM, respectively, for the binding of 14-3-3ζ or 14-3-3γ
to PRAS40 (Fig. 1C). Furthermore, F1 effectively blocked the
ability of all seven 14-3-3 isoforms to stimulate exoenzyme S
(ExoS) ADP-ribosyltransferase (IC
50
¼ 619 μM) in a functional
assay (21) (Fig. 1D). Because ExoS is a nonphosphorylated client
protein, F1 is capable of interfering with the binding of both
phosphorylated and nonphosphorylated client proteins to 14-3-3.
Together, these data suggest a direct action of F1 on 14-3-3
proteins.
To provide a structural explanation for the inhibitory effect
of F1 on 14-3-3 proteins, compound F1 was soaked into pre-
formed crystals of 14-3-3ζ. These cocrystals were bright orange
in color (Fig. 2A). During the X-ray data collection, immediately
after exposure to synchrotron radiation, the cocrystals turned
brownish, then yellow, and eventually became colorless. We de-
termined the complex structure to a resolution of 2.39 Å
(Table S1). The crystal contains four monomers (two dimers)
within the asymmetric unit (Fig. 2B), with a root-mean-square-
deviation of <0.5 Å of 230 pairs of Cα atoms between the mono-
mers. Each monomer consists of nine α helices that form an a
mphipathic groove where a client protein is located (9, 2224).
F1 is bound to the basic surface of the peptide-binding groove
of each monomer. However, only the pyridoxal-phosphate moiety
of F1 was found in this groove (Fig. 2C). The F1 exocyclic nitro-
gen atom formed a covalent bond with the side chain terminal
nitrogen of Lys120, forming a diazene adduct with a N-N distance
of approximately 1.2 Å. The F1 phosphate group interacts with
the side chain of Lys49 and Asn173, while one face of the pyri-
doxal ring makes van der Walls contact with the side chain of
Ile217 (Fig. 2D, Fig. S3). These interactions position the F1 de-
rivative in a defined conformation. In addition, a solvent mole-
cule bridges Arg56 and Arg127.
We superimposed the F1-bound structure of 14-3-3ζ to that of
14-3-3 bound to either the pS259-Raf-1 (PDB 3CU8) or pS10-
histone H3 (25) (PDB 2C1N) peptide. In order to interact with
phosphorylated ligands, 14-3-3ζ engages a cluster of basic or
polar residues, including (i
) Arg56, Arg127, and Tyr128, which
coordinates the binding of the phosphate group (Fig. 2E) and
(ii) Asn173, whose side chain oxygen atom forms a hydrogen
bond with the main chain amide nitrogen of the residue C-term-
inal to the phosphoserine and involves an intramolecular inter-
action network forming a hydrogen bond with Asp114, which
in turn forms a salt bridge with Lys120 (Fig. 2F). The conforma-
tion of peptide residues at þ 2 and beyond differs when the pep-
tide exits from the 14-3-3 ligand binding groove, with Raf-1 to the
left and histone H3 to the right of Lys49, as shown in Fig. 2E.
It appears as though the phosphate group of covalently linked
F1 shifted approximately 4 Å away from the phosphoserine bind-
ing site, towards Lys120 (Fig. 2G). Mutagenesis of 14-3-3ζ
coupled with direct binding studies using isothermal titration
calorimetry indicated the importance of R56 and R60 in the bind-
ing of native uncleaved F1 (Fig. S4, Table S2), which supports
the proposed model in Fig. 2. We reasoned that the phosphate
moiety of F1 might be critical for its inhibitory activity by mimick-
ing the phosphorylated peptide motif for 14-3-3 binding. We thus
generated the compound F2, which lacks the phosphate group,
and observed that this compound had a drastically reduced effect
in blocking 14-3-3 binding to Raf-1 or PRAS40 (Fig. 1 B and C,
Fig. S2) and in inhibiting 14-3-3-mediated activation of ExoS
(Fig. 1E). Moreover, changes in the phenyl ring structure also
showed some effect on the 14-3-3/Raf-1 interaction (Fig. S5),
demonstrating the involvement of the azophenyl substructure
in 14-3-3 interaction. These data support F1 as a 14-3-3 inhibitor
and highlight its phosphate moiety as a primary functional com-
ponent. We modeled intact F1 by superimposing the F1 phos-
phate group onto that of phosphoserine and rotating the torsion
angles to reach maximum overlap with the bound peptide
(Fig. 3A). The model suggests that the side of the pyridoxal ring
with the phosphate group superimposes well with peptide back-
bones before and after phosphoserine, while the benzoate ring
Fig. 1. Discovery of FOBISIN (A) Chemical structures of FOBISIN 101 (F1), F2 (dephosphorylated F1), and F3 (pyridoxal-phosphate). (B) F1 blocks the binding of
14-3-3 to full-length Raf-1 and PRAS40 in cell lysates. GST-14-3-3γ was mixed with COS-7 cell lysate in the presence of test compounds. The 14-3-3γ complex was
isolated by GST-pull-down . The presence of Raf-1 and PRAS40 in the 14-3-3γ complex was revealed by Western blot analysis. (C) Inhibition of the interaction
between PRAS40 and 14-3-3γ or 14-3-3ζ by F1 in an ELISA assay. Interaction of PRAS40 with GST-14-3-3γ or 14-3-3ζ immobilized on an anti-GST antibody-coated
plate gave rise to robust ELISA signals as detected by anti-PRAS40 antibod y. (D) F1 inhibits ExoS activation by all seven isoforms of 14-3-3. ExoS and its substrates
were incubated with 14-3-3 proteins in the presence or absence of F1. 14-3-3-dependent ExoS enzymatic activity was quantified by the amount of
32
P-ADP-
ribose incorporated into SBTI, % control relative to samples without F1 was calculated. (E) Dephosphorylated F1 (F2) does not affect 14-3-3-mediated ExoS
activity in an ExoS assay as described in (D). The results in c e were presented as means SD (n ¼ 3) of one representative experiment. The experiments were
repeated at least three times with similar results.
Zhao et al. PNAS September 27, 2011 vol. 108 no. 39 16213
BIOCHEMISTRY
Page 2
could point to the peptide exit pathway similar to that of Raf-1
(Fig. 3A, left box).
To explore the possible cause of the covalent modification of
14-3-3 by F1, we hypothesized that radiation exposure cleaves the
N ¼ N diazene bond thereby releasing the paraaminobenzoic
acid moiety into the solvent, while the hydrogen binding inter-
action holds the pyridoxal-phosphate moiety in place within the
14-3-3 binding site (Fig. 3B). In this model, the reactive nitrogen
of the pyridoxal-phosphate group is approximately 67 Å from
either Lys120 or Lys49, respectively (Fig. 3C). However, the side
chain Nϵ of Lys120 is roughly parallel, while that of Lys49 is
roughly perpendicular, to the plane of the pyridoxal ring. We sug-
gest that bond-breaking and bond-making processes proceed
through specific attack trajectories. The preferred attack trajec-
tory might be the one which lies parallel to the plane of the ring
and facilitates the formation of a new nitrogen bond of the
cleaved compound with the side chain of Lys120, leading to cova-
lent modification and inactivation of 14-3-3 function. Indeed, mu-
tating Lys120 to Glu alone is sufficient to inactivate 14-3-3ζ
(Fig. S6).
The N ¼ N bond in diazene compounds is generally sensitive
to radiation and is known to undergo photolysis to generate re-
active organic radicals (26). We note that another plausible
source of the covalent adduct is through imine (Schiff base) for-
mation between the ϵ-amino group of a lysine and the aldehyde
group (with the loss of water) on the pyridoxal-phosphate portion
of the inhibitor. However, three lines of evidence argue against
this possibility. First, the electron density is of sufficient quality
to identify the phosphate group, its associated pyridine ring, and
the aldehyde (with a two bond length -C ¼ O away from a ring
atom). If the site of covalent attachment is via imine formation,
irradiation-induced cleavage of the N ¼ N bond would be re-
duced to NH
2
(in this case, one bond length away from a ring
atom). Second, the covalent adduct is only formed after exposure
Fig. 2. Structure of the 14-3-3ζF1 fragment covalent complex (A) Example color change of the 14-3-3ζF1 crystal after exposure to X-rays. (B) Two dimers of
14-3-3ζ in the asymmetric unit. (C) Covalently modified Lys120 in the peptide-binding groove of 14-3-3ζ. Omit electron densities, Fo-Fc (red mesh) and 2Fo-Fc
(green mesh), contoured at 4σ and 1.2σ above the mean, respectively, are shown for F1-modified Lys120. (D) Details of 14-3-3ζ and F1 fragment interactions.
The letter w indicates a solvent [either water in molecule (A-C) or a partially occupied phosphate in molecule (D)]. (E) Superimposition of 14-3-3ζ in complex
with a Raf-1 peptide (blue) (PDB 3CU8) and a histone H3 peptide (gray) (PDB 2C1N). For clarity, H3 residues 1214 (which point toward the viewer) were
removed. (F) Lys120 is involved in intramolecular interactions. (G) Superimposition of a covalently bound F1 fragment (yellow) with that of Raf-1 (blue)
and H3 (gray ) phosphoserine peptides.
Fig. 3. Model of intact F1 bound to 14 -3-3ζ (A) Superimposition of the pyr-
idoxal-phosphate moiety and the phosphoserine of a Raf-1 peptide (left) or
histone H3 peptide (right). For clarity, H3 residues 13-14 (which point toward
the viewer) were removed. (B) Proposed bond-breaking and bond-making
process. (C) Lys120 lies in a position for a preferred attack trajectory. (D) Mass
spectrum profiles (in the reflection mode of acquisition of the spectrum)
showing a 14-3-3ζ fragment containing Lys120 digested from crystals with
(bottom) and without (top) exposure to X-rays. The addition of 182 Da cor-
responds to modification of Lys120 (262 Da) with the loss of a phospha te
group (HPO
3
) from the peptide (by a reduction of 80 Da in the mass), pre-
sumably due to laser (337 nm) induced metastable decomposition during the
MALDI ionization process (3538).
16214 www.pnas.org/cgi/doi/10.1073/pnas.1100012108 Zhao et al.
Page 3
to X-rays. It is not known whether imine formation with pyridox-
al-phosphate requires photolysis. Gel digestion and mass spectro-
metric analysis of digested peptides obtained from F1-soaked
crystals that were not exposed to X-rays showed no adduct peak
(Fig. 3D, top box). Importantly, only the X-ray-treated samples
showed a mass addition corresponding to a peptide fragment of
residues 114-131 with modified Lys120 (Fig. 3D, bottom box).
Therefore, the fragmentation and covalent adduct formation
of F1 observed in the 14-3-3ζ crystal structure very well could
be induced by X-ray radiation during data collection, as evident
by the change of color (Fig. 2A). Third, assuming the site of cova-
lent attachment is via imine formation and the pyridine ring takes
on the same conformation (by flipping the ring 180° horizontally
as shown in Fig. 2C), the paraaminobenzoic acid moiety would
point to the solvent without any specific contact to the protein.
However, the pyridoxal-phosphate (F3 in Fig. 1A) has much re-
duced potency (Fig. 1E), indicating that the phenyl ring structure
still contributes to the inhibitory activity of F1, which is supported
by data in Fig. S5.
In conclusion, we have identified and experimentally con-
firmed a series of small molecule phospho-binding site inhibitor
and revealed structural details of one such molecule with 14-3-3ζ.
Because of the specificity of the mode of binding as revealed by
the cocrystal structural studies and the potent effect on both
phosphorylated and nonphosphorylated client protein binding
to 14-3-3 proteins, this pyridoxal-phosphate class of compounds
are expected to define a unique class of 14-3-3 inhibitors for phy-
siological and therapeutic investigations. It is important to note
that the F1 class of compounds have been investigated as iono-
tropic P2X receptor antagonists (20). Rich medicinal chemistry
information around this structural scaffold will greatly facilitate
their development as 14-3-3 modulators. Importantly, we also
offer a prodrug concept for 14-3-3-mediated diseases. For exam-
ple, F1-like molecules could be developed as radiation-triggered
therapeutic agents for the treatment of cancer. It is envisioned
that such 14-3-3 inhibitors alone may show negligible toxicity
to the host; however, radiation therapy targeted to a particular
tumor area may specifically cleave such designed 14-3-3 inhibitors
and lead to their covalent modification and potent inactivation of
14-3-3 proteins in tumors.
Materials and Methods
Molecular and Cell Biology Reagents. The expression vectors for GST-14-3-3
and His-14-3-3 isoforms were constructed as previously described (27).
Glutathione agarose beads and nickel-affinity columns were purchased from
GE Healthcare. Anti-Raf-1 and anti-GST antibodies were from Santa Cruz and
the anti-PRAS40 antibody was from Biosource. 14-3-3ζ was expressed in
Escherichia coli BL21(DE3) harboring pET-15b-derived plasmids and purified
using Ni
2þ
chelating chromatography essentially as described (21). Hexahis-
tidine tags were removed by thrombin digestion. The 14-3-3 protein used
for crystallization was further purified by gel filtration chromatography
(Superdex 200 in a Pharmacia FPLC system). ExoS was purified as previously
described (28). The LOPAC library was purchased from Sigma-Aldrich. COS-7
cells were grown in DMEM supplemented with 10% FBS.
Florescence Polarization Assay and Chemical Screening. The 14-3-3 FP assay was
carried out in black 384-well microplates in a total volume of 50 μL (19). Assay
reaction buffer (49 μL: 1 μM GST-14-3-3γ and 2 nM TMR-pS259-Raf peptide in
Hepes buffer) was dispensed to each well. Test compound (1 μL of 2 mM stock
in DMSO) was added to the reaction buffer using a Sciclone liquid handler
(Caliper LifeSciences). Plates were incubated at room temperature and the FP
value in millipolarization (mP) units was recorded with an Analyst HT reader
(Molecular Devices). An excitation filter at 545 nm and an emission filter at
610 to 675 nm were used with a dichroic mirror at 565 nm. Data analysis
was conducted using CambridgeSoft software. Compounds with recorded
mP values less than three standard deviation from the negative controls were
considered positive hits.
Enzyme-Linked Immunosorbent Assay. The 14-3-3 ELISA assay was developed
in 96-well microplates coated with either anti-GST antibody or glutathione
(Pierce Biotechnology). This assay monitors the interaction of recombinant
GST-tagged 14-3-3 proteins with endogenous client proteins, such as PRAS40,
in COS-7 cell lysate. Briefly, GST-14-3-3 protein (1 μM) immobilized on an
anti-GST plate was incubated with a test compo und before adding COS-7 cell
lysates in 1% NP-40 lysis buffer (16). After incubation and washing, antibo-
dies specific to PRAS40 along with peroxidase-labeled anti-rabbit IgG (50 μL;
11;000 dilution) were added. After washing, 100 μL of tetramethylbenzidine
was added. The reaction was stopped with sulfuric acid (0.1 N) and recorded
at 450 nm on an Envision reader (Perkin Elmer). IC
50
values were calculated
using GraphPad software.
GST Pull-Down Assay and Western Blotting. For binding assays, GST-14-3-3 pro-
teins (1 μg) were preincubated with various concentrations of test compound
before COS-7 cell lysates were added. Client proteins associated with GST-14-
3-3 were captured by glutathione Sepharose beads while unbound proteins
were removed by washing (1% NP-40 buffer). The fraction that was bound
to the beads was analyzed by SDS-PAGE followed by immunoblotting with
antibodies specific to Raf-1, PRAS40, and GST.
ExoS Activation Assay. To examine the functional effect of test compounds on
14-3-3 proteins, we utilized the 14-3-3-dependent ExoS ADP-ribosyltransfer-
ase assay (21). This assay is used as a functional readout for 14-3-3 inhibitors.
Briefly, 14-3-3 protein was preincubated with test compounds, followed by
incubation with ExoS in the presence of substrates (SBTI, NAD, and 0.35 μCi of
½adenylate-
32
PNAD
þ
as a reaction tracer). The reaction was terminated by
spotting assay mixture onto P81 phosphocellulose paper (Whatman). After
washing, radioactivity incorporated into SBTI by ExoS was determined by
liquid scintillation counting. Enzyme activities were expressed as picomoles
of ADP-ribose incorporated per min per microgram of ExoS. The inhibitory
effect of compounds was expressed as percent inhibition of ExoS activity over
vehicle control.
Chemical Synthesis of Compound F2. A 5.1 mg sample of F1 was dissolved in
1.0 mL of hydrofluoric acid (48%, Sigma-Aldrich) and incubated in an ice bath
for 2 h. The solution was adjusted to a pH of 5 using a saturated NaOH aqu-
eous solution. The solution was dried under a vacuum using a rotary-evapora-
tor at 30 °C. Precooled (20 °C) ethanol was added to the remaining residue.
The solid residue was filtered and the filtrate was dried to give 3.1 mg of a
brown solid product. Yield: 89%.
ESI-NEG: [M-H] 314.1
1
H-NMR (400 MHz, CD
3
OD): 8.028.24 (d, 4H), 6.30 (m, 1H), 5.355.38
(m, 2H), 2.63 (s, 3H).
Crystallography. The 14-3-3ζ protein (10 mgmL in 20 mM Tris/HCl, pH 8.5,
100 mM NaCl) was crystallized by the hanging drop vapor diffusion method
using conditions similar to those previously described (22) (25% PEG3350,
100 mM Tris-HCl, pH 8.5, 10 mM MgCl
2
, 1 mM NiCl
2
, 1% glycerol at 16 °C).
The 14-3-3ζ crystals were soaked with 5 mM F1 compound for 3 d. The crystals
were then momentarily immersed into well solution containing either 25%
ethylene glycol or glycerol as a cryoprotectorant and frozen by plunging into
liquid nitrogen. The crystals were then stored in a liquid nitrogen dewar until
data collection at the SET-CAT synchrotron beamline at the Advanced Photon
Source of Argonne National Laboratory. Structural determination of the
14-3-3ζF1 complex proceeded by molecular replacement with the program
PHASER (29) using the 14-3-3 coordinates of a 14-3-3/ExoS complex (PDB
2O02) (30). Refinement of the model proceeded with manual manipulation
using the graphic programs COOT (31) and O (32) and computational manip-
ulation with the program CNS (33). On the basis of evident electron density,
we modeled the F1 modified Lys120, whose topology and parameter files for
refinement were obtained from the Dundee PRODRG2 Server (34) (http://
davapc1.bioch.dundee.ac.uk/prodrg /).
Mass Spectrometry. Covalent adduct formation between 14-3-3ζ-K120 and
fragmented F1 was verified by performing MALDI-TOF-M S analysis of V8-pro-
tease (New England BioLab) digested peptide fragments of the F1-protein
crystals with or without X-ray exposure.
Detailed information on mass spectrometry, isothermal titration
calorimetry, mutagenesis analysis is included in the SI Materials and
Methods as well as additio nal information on FOBISIN 104107.
ACKNOWLEDGMENTS. We thank Paul R. Thompson for critical comments. The
Department of Biochemistry at the Emory University School of Medicine sup-
ported the use of the SER-CAT synchrotron beamline at the Advanced Photon
Source of Argonne National Laboratory, local X-ray facility and MALDI-TOF
mass spectrometry and HTS was performed at the Emory Chemical Biology
Discovery Center. This work was supported in part by the National Institutes
Zhao et al. PNAS September 27, 2011 vol. 108 no. 39 16215
BIOCHEMISTRY
Page 4
of Health Grants P01 CA116676 (to F.R.K and H.F.), GM068680 and GM092035
(to X.C.), AI030162 (to J.T.B.), Georgia Cancer Coalition (to F.R.K. and H.F.),
Georgia Research Alliance (to X.C. and H.F.), and Chinese Academy of Sciences
K. C. Wong Awards (to L.Z. and H.F.).
1. Aitken A (2006) 14-3-3 proteins: a historic overview. Semin Cancer Biol 16:162172.
2. Dougherty MK, Morrison DK (2004) Unlocking the code of 14-3-3. J Cell Sci
117:18751884.
3. Fu H, Subramanian RR, Masters SC (2000) 14-3-3 proteins: structure, function, and
regulation. Annu Rev Pharmacol Toxicol 40:617647.
4. Fantl WJ, et al. (1994) Activation of Raf-1 by 14-3-3 proteins. Nature 371:612614.
5. Fu H, et al. (1994) Interaction of the protein kinase Raf-1 with 14-3-3 proteins. Science
266:126129.
6. Irie K, et al. (1994) Stimulatory effects of yeast and mammalian 14-3-3 proteins on the
Raf protein kinase. Science 265:17161719.
7. Michaud NR, Fabian JR, Mathes KD, Morrison DK (1995) 14-3-3 is not essential for
Raf-1 function: identification of Raf-1 proteins that are biologically activated in a
14-3-3- and Ras-independent manner. Mol Cell Biol 15:33903397.
8. Muslin AJ, Tanner JW, Allen PM, Shaw AS (1996) Interaction of 14-3-3 with signaling
proteins is mediated by the recognition of phosphoserine. Cell 84:889897.
9. Yaffe MB, et al. (1997) The structural basis for 14-3-3:phosphopeptide binding
specificity. Cell 91:961971.
10. Ganguly S, et al. (2005) Melatonin synthesis: 14-3-3-dependent activation and inhibi-
tion of arylalkylamine N-acetyltransferase mediated by phosphoserine-205. Proc Natl
Acad Sci USA 102:12221227.
11. Coblitz B, Wu M, Shikano S, Li M (2006) C-terminal binding: an expanded repertoire
and function of 14-3-3 proteins. FEBS Lett 580:15311535.
12. Pozuelo Rubio M, et al. (2004) 14-3-3-affinity purification of over 200 human phospho-
proteins reveals new links to regulation of cellular metabolism, proliferation and
trafficking. Biochem J 379:395408.
13. Meek SE, Lane WS, Piwnica-Worms H (2004) Comprehensive proteomic analysis of
interphase and mitotic 14-3-3-binding proteins. J Biol Chem 279:3204632054.
14. Jin J, et al. (2004) Proteomic, functional, and domain-based analysis of in vivo 14-3-3
binding proteins involved in cytoskeletal regulation and cellular organization. Curr
Biol 14:14361450.
15. Zippo A, et al. (2009) Histone crosstalk between H3S10ph and H4K16ac generates a
histone code that mediates transcription elongation. Cell 138:11221136.
16. Masters SC, Fu H (2001) 14-3-3 proteins mediate an essential anti-apoptotic signal. J
Biol Chem 276:4519345200.
17. Petosa C, et al. (1998) 14-3-3zeta binds a phosphorylated Raf peptide and an
unphosphorylated peptide via its conserved amphipathic groove. J Biol Chem
273:1630516310.
18. Wang B, et al. (1999) Isolation of high-affinity peptide antagonists of 14-3-3 proteins
by phage display. Biochemistry 38:1249912504.
19. Du Y, Masters SC, Khuri FR, Fu H (2006) Monitoring 14-3-3 protein interactions with a
homogeneous fluorescence polarization assay. J Biomol Screen 11:269276.
20. Kim YC, et al. (1998) Synthesis and structure-activity relationships of pyridoxal-6-
arylazo-5 phosphate and phosphonate derivatives as P2 receptor antagonists. Drug
Develop Res 45:5266.
21. Fu H, Coburn J, Collier RJ (1993) The eukaryotic host factor that activates exoenzyme S
of Pseudomonas aeruginosa is a member of the 14-3-3 protein family. Proc Natl Acad
Sci USA 90:23202324.
22. Liu D, et al. (1995) Crystal structure of the zeta isoform of the 14-3-3 protein. Nature
376:191194.
23. Obsil T, Ghirlando R, Klein DC, Ganguly S, Dyda F (2001) Crystal structure of the
14-3-3zeta:serotonin N-acetyltransferase complex. a role for scaffolding in enzyme
regulation. Cell 105:257267.
24. Yang X, et al. (2006) Structural basis for protein-protein interactions in the 14-3-3 pro-
tein family. Proc Natl Acad Sci USA 103:1723717242.
25. Macdonald N, et al. (2005) Molecular basis for the recognition of phosphorylated and
phosphoacetylated histone h3 by 14-3-3. Mol Cell 20:199211.
26. Hoijemberg PA, Karlen SD, Sanrame CN, Aramendia PF, Garcia-Garibay MA (2009)
Photolysis of an asymmetrically substituted diazene in solution and in the crystalline
state. Photochem Photobiol S 8:961969.
27. Subramanian RR, Masters SC, Zhang H, Fu H (2001) Functional conservation of 14-3-3
isoforms in inhibiting bad-induced apoptosis. Exp Cell Res 271:142151.
28. Knight DA, Finck-Barbancon V, Kulich SM, Barbieri JT (1995) Functional domains
of Pseudomonas aeruginosa exoenzyme S. Infect Immun 63:31823186.
29. Storoni LC, McCoy AJ, Read RJ (2004) Likelihood-enhanced fast rotation functions.
Acta Crystallogr D 60:432438.
30. Ottmann C, et al. (2007) Phosphorylation-independent interaction between 14-3-3
and exoenzyme S: from structure to pathogenesis. Embo J 26:902913.
31. Emsley P, Cowtan K (2004) COOT: model-building tools for molecular graphics. Acta
Crystallogr D 60:2126 2132.
32. Jones TA, Zou JY, Cowan SW, Kjeldgaard M (1991) Improved methods for building
protein models in electron density maps and the location of errors in these models.
Acta Crystallogr A 47:110119.
33. Brunger AT, et al. (1998) Crystallography and NMR system: a new software suite for
macromolecular structure determination. Acta Crystallogr D 54:905921.
34. Schuttelkopf AW, van Aalten DM (2004) PRODRG: a tool for high-throughput crystal-
lography of protein-ligand complexes. Acta Crystallogr D 60:1355
1363.
35. Annan RS, Carr SA (1996) Phosphopeptide analysis by matrix-assisted laser desorption
time-of-flight mass spectrometry. Anal Chem 68:34133421.
36. Neubauer G, Mann M (1999) Mapping of phosphorylation sites of gel-isolated proteins
by nanoelectrospray tandem mass spectrometry: potentials and limitations. Anal
Chem 71:235242.
37. Kinumi T, Niwa H, Matsumoto H (2000) Phosphopeptide sequencing by in-source decay
spectrum in delayed extraction matrix-assisted laser desorption ionization time-of-
flight mass spectrometry. Anal Biochem 277:177186.
38. Jagannadham MV, Nagaraj R (2008) Detecting the site of phosphorylation in phospho-
peptides without loss of phosphate group using MALDI TOF mass spectrometry. Ana-
lytical Chemistry Insights 3:2129.
16216 www.pnas.org/cgi/doi/10.1073/pnas.1100012108 Zhao et al.
Page 5
  • Source
    • "The obtained KD values from for new identified isatin inhibitors from SPRi assay and IC50 from ELISA presented in (Table 2). The new identified inhibitors show a comparable kinetics from SPRi and IC50 when compared to those from literatures (Zhao et al., 2011, Yan et al., 2012). This difference between KD and IC50 is may be due to that IC50 was obtained for protein-protein inhibition instead of direct measurement ligands affinity toward the target proteins. "
    [Show abstract] [Hide abstract] ABSTRACT: In order to increase the scope and utility of small molecule microarray (SMMs) we have combined SMMs and SPRi to screen small molecule antagonists against protein targets. Several small molecules, including immunosuppressive drugs (rapamycin and FK506) and reported inhibitors (FOBISIN and Blapsin) of 14-3-3ζ proteins have been used to validate this technology. Furthermore, a small library of isatin derivatives have been synthesized and screened on developed platform against 14-3-3ζ protein. Three molecules, derived from the endogenous intermediate isatin termed, FZIB-35, FZIB-36 and FZIB-38 were identified as novel inhibitors which shows significant interaction with 14-3-3ζ. A mutation in the binding groove of 14-3-3ζ, (K49E), almost abolishes the binding of these compounds to 14-3-3ζ protein. To exclude the probability of false positives, two more purified proteins (PtpA and BirA) were also tested. Furthermore, in order to confirm the binding pocket specificity, competition assay against R18 peptide was also carried out on presented platform. We show that SMMs in combination with SPRi is a powerful method to identify lead compounds in high throughput manner without the need to develop an activity based assay.
    Full-text · Article · Jan 2015 · Arabian Journal of Chemistry
  • Source
    • "Briefly, the molecular dynamics package Xplor-NIH 2.29 [24,25] and docking, minimization, and rigid body/torsion dynamics protocols were performed in a similar manner as described previously [26]. As the starting structure for the docking calculations, the X-ray structure of 14-3-3ζ (PDB code 3RDH) was used [13]. The calculations are based on 119 distance restraints derived from PDB file plus 4 additional distance restraints (Lys9–Glu81, N-terminus–Glu81, Asp21–Lys85, and Glu5–Lys74; so-called " R-6 summed " ) from chemical cross-linking [7]. "
    [Show abstract] [Hide abstract] ABSTRACT: Protein-protein interaction was investigated using a protein nanoprobe capable of photo-initiated cross-linking in combination with high-resolution and tandem mass spectrometry. This emerging experimental approach introduces photo-analogs of amino acids within a protein sequence during its recombinant expression, preserves native protein structure and is suitable for mapping the contact between two proteins. The contact surface regions involved in the well-characterized interaction between two molecules of human 14-3-3ζ regulatory protein were used as a model. The employed photo-initiated cross-linking techniques extend the number of residues shown to be within interaction distance in the contact surface of the 14-3-3ζ dimer (Gln8-Met78). The results of this study are in agreement with our previously published data from molecular dynamic calculations based on high-resolution chemical cross-linking data and Hydrogen/Deuterium exchange mass spectrometry. The observed contact is also in accord with the 14-3-3ζ X-ray crystal structure (PDB 3dhr). The results of the present work are relevant to the structural biology of transient interaction in the 14-3-3ζ protein, and demonstrate the ability of the chosen methodology (the combination of photo-initiated cross-linking protein nanoprobes and mass spectrometry analysis) to map the protein-protein interface or regions with a flexible structure.
    Full-text · Article · Jun 2014 · International Journal of Molecular Sciences
  • Source
    • "For example, 14-3-3 protein epsilon shielded MEKK1 from caspase-3 that could split the kinase by the binding of 14-3-3 protein epsilon to the N-terminus regulated domain of MEKK1, thus inhibiting cell apoptosis. And, the 14-3-3 protein epsilon also participated in protein-protein interactions, for example, it interacted with Bad (a pro-apoptotic member of Bcl-2 proteins family) in a phosphoserine-dependent manner and inhibited the effect on promoting the cell apoptosis [4]. The other protein regulated by 5'-AMP was AI, which was the focal enzyme of the urea cycle hydrolyzing L-arginine to urea and L-ornithine. "
    [Show abstract] [Hide abstract] ABSTRACT: Understanding the protection mechanism of 5'-AMP requires comprehensive knowledge of the proteins expressed during the period that the body is exposed to irradiation. Proteomics provides the tools for such analyses. Here, the experimental ICR mice were divided into three groups (normal group, model group and 5'-AMP + irradiation group). After different treatment, the hepatic total protein of each animal in three groups was separated by two-dimensional gel electrophoresis (2-DE). 2-DE analysis revealed fifty-eight protein spots were differentially expressed in comparison to three groups. From 58 protein spots, we selected nine spots to identify by MALDI-TOF-MS and received credible results. They were determined to be type I arginase, annexin A5, regucalcin, catalase, Tpm3 protein, Pdia4 protein, 14-3-3 protein epsilon, NAD-Malate dehydrogenase and heat shock protein 90. Considering the characteristic of these proteins, we proposed a possible protection pathway.
    Full-text · Article · Dec 2013 · International Journal of Molecular Sciences
Show more