Nanomolar inhibitors of the transcription factor STAT5b with high selectivity over STAT5a

Article abstract of DOI:10.1002/anie.201410672

Src homology 2 (SH2) domains play a central role in signal transduction. Although many SH2 domains have been validated as drug targets, their structural similarity makes development of specific inhibitors difficult. The cancer-relevant transcription facto

Full text of DOI:10.1002/anie.201410672

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Chemie
DOI: 10.1002/anie.201410672
Protein–Protein Interactions
Nanomolar Inhibitors of the Transcription Factor STAT5b with High
Selectivity over STAT5a**
Nagarajan Elumalai, Angela Berg, Kalaiselvi Natarajan, Andrej Scharow, and Thorsten Berg*
Abstract: Src homology 2 (SH2) domains play a central role in
signal transduction. Although many SH2 domains have been
validated as drug targets, their structural similarity makes
development of specific inhibitors difficult. The cancer-rele-
vant transcription factors STAT5a and STAT5b are particu-
larly challenging small-molecule targets because their SH2
domains are 93% identical on the amino acid level. Here we
present the natural product-inspired development of the low-
nanomolar inhibitor Stafib-1, as the first small molecule which
inhibits the STAT5b SH2 domain (Ki = 44 nm) with more than
50-fold selectivity over STAT5a. The binding site of the core
moiety of Stafib-1 was validated by functional analysis of point
mutants. A prodrug of Stafib-1 was shown to inhibit STAT5b
with high selectivity over STAT5a in tumor cells. Stafib-1
provides the first demonstration that naturally occurring SH2
domains with more than 90% sequence identity can be
selectively targeted with small organic molecules.
a novel therapeutic approach to combat Bcr-Abl positive
leukemias.[7] Small-molecule inhibitors targeting the SH2
domain of STAT5b with specificity over the STAT5a SH2
domain would be useful for investigating the differential roles
of STAT5a and STAT5b under diverse conditions, and could
serve as lead compounds for drug development.
While a small number of STAT5 SH2 domain inhibitors
have been reported,[8] none of these studies has disclosed
selectivity for one STAT5 protein over the other. We recently
reported Fosfosal as a STAT5b SH2 domain inhibitor (Ki =
17.4 mm)[9] (Figure 1a). Structure–activity relationships of
S
rc homology 2 (SH2) domains are highly homologous
regions, approximately 100 amino acids long, which are found
in a significant subset of signaling proteins. SH2 domains play
a fundamental role in intracellular signaling via recognition of
specific phosphotyrosine-containing peptide motifs.[1] The
conserved nature and similar binding preferences of these
domains pose an enormous challenge for specific inhibitor
development.[2] A particularly challenging example is given
by the SH2 domains of the transcription factors STAT5a and
STAT5b,[3] which are essential for STAT5a/b signaling. These
are 93% identical on the amino acid level (see Figure S1 in
the Supporting Information), and bind to the same peptide
motifs.[4] Consequently, STAT5a and STAT5b are often jointly
referred to as “STAT5”, despite tissue-specific expression
patterns and a number of non-redundant biological func-
tions.[3,5] Antisense oligonucleotides directed against STAT5b,
but not STAT5a, inhibited tumor growth in mice.[6] Most
recently, selective inhibition of Stat5b has been proposed as
Figure 1. Catechol bisphosphate (1) is a selective inhibitor of the
STAT5b SH2 domain. a) Structures of Fosfosal[9] and 1. b) Activities of
1 against the SH2 domains of STAT family members and the tyrosine
kinase Lck analyzed in binding assays based on fluorescence polar-
ization. c) Binding of 1 to the STAT5b SH2 domain as predicted by
docking with AutoDock Vina.
Fosfosal revealed that deleting the carboxylic acid retained
partial activity, but deleting the phosphate led to a complete
loss of activity against STAT5b. In an attempt to increase
activity, we substituted the carboxylic acid group of Fosfosal
with a second phosphate. This structural change is contrary to
standard medicinal chemistry, which aims to replace phos-
phate groups with isosteric groups of lesser charge. However,
the resulting compound, catechol bisphosphate (1), inhibited
STAT5b with an inhibitory constant Ki of only 0.9 Æ 0.1 mm
(Figures 1a,b, Table 1, and see Tables S1 and S2 in the
Supporting Information). Surprisingly, analysis of 1 against
STAT5a in a newly developed binding assay (see Figure S2
and Supporting Methods in the Supporting Information)
revealed 35-fold lower activity against the SH2 domain of
STAT5a (Ki = 34 Æ 3 mm). The SH2 domains of other STAT
family members and the tyrosine kinase Lck[10] were only
[*] M. Sc. N. Elumalai, M. Sc. A. Berg, M. Sc. K. Natarajan,
M. Sc. A. Scharow, Prof. Dr. T. Berg
Institute of Organic Chemistry, University of Leipzig
Johannisallee 29, 04103 Leipzig (Germany)
E-mail: tberg@uni-leipzig.de
Homepage: http://www.uni-leipzig.de/~tberg
[**] This work was supported by the Deutsche Forschungsgemeinschaft
(BE4572/1-1). We extend our thanks to Martin Grꢀber, Stefanie
Jehle, and Stephanie Hꢀfner for experimental support and fruitful
discussions. We extend our thanks to Lothar Hennighausen
(National Institutes of Health, Bethesda, USA) for initial discus-
sions on STATs.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201410672.
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Table 1: Structures and activities of inhibitors against STAT5b and
Table 1: (Continued)
STAT5a.
Compounds
Ki [mm]
Ki [mm]
Selec-
(STAT5b) (STAT5a) tivity
factor
Compounds
Ki [mm]
Ki [mm]
Selec-
(STAT5b) (STAT5a) tivity
factor
0.28Æ
9.3Æ
0.93Æ
12
33
1
34Æ3
24Æ3
n.a.
37
0.02
0.3
0.07
2
3
27Æ4
0.9
n.a.
0.044Æ 2.42Æ
13
14
55
0.001
0.05
n.a.
n.a.
n.a.
n.a.
0.69Æ
19.2Æ
4
5
6
28
0.04
2.7
15
50Æ3
40Æ2
0.80
0.75
0.82Æ
2.5Æ
3
0.05
0.2
16 QDTpYLVLDKWL
0.54Æ
0.41Æ
0.08
0.09
The selectivity factor was calculated as Ki (STAT5a)/Ki (STAT5b).
Conversion of IC50 values into Ki values was carried out as described.[15]
n.a.=not applicable.
0.73Æ
21Æ5
30
0.07
inhibited at higher concentrations. Positioning the two
phosphate groups of 1 in the meta position, as represented
by resorcinol bisphosphate (2), resulted in a strong decrease
of activity against STAT5b (Ki = 27 Æ 4 mm, Table 1), with
concomitant loss of selectivity. Replacement of the two
phosphate groups by methylene diphosphonates, as in the
compound 3, resulted in virtually complete loss of activity
against STAT5b (Table 1 and Table S1).
We carried out molecular docking studies using Auto-
Dock Vina[11] and the previously described homology model
of the STAT5b SH2 domain[9] based on the crystal structure of
STAT5a.[12] The top three docking results placed catechol
bisphosphate into the phosphotyrosine binding pocket of the
STAT5b SH2 domain, consistent with electrostatic interac-
tions between STAT5b Arg618 and both phosphate groups
(Figure 1c). The arginine residue in position 618 of STAT5b is
highly conserved in all SH2 domains and forms essential
electrostatic interactions with the phosphotyrosine moiety of
its natural interaction partners.[13] Alignment of the STAT5b
homology model[9] with the X-ray structure of STAT5a[12]
suggested that small differences in the primary structure of
STAT5a and STAT5b might cause a partial change in
secondary structure in the vicinity of the binding pocket for
1 (see Figure S3 in the Supporting Information). Detailed
comparative structural analysis by crystallographic or NMR-
based methods would be required to identify the subtle
structural differences contributing to specificity.
0.45Æ
16.0Æ
7
8
9
35
17
52
0.04
0.8
0.46Æ
7.8Æ
0.08
1.0
0.21Æ
11Æ2
0.04
10
11
n.a.
n.a.
n.a.
67
0.24Æ
16.1Æ
0.01
1.1
In attempts to further increase the submicromolar affinity
of 1 for STAT5b, we sought inspiration from natural products
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with an explicit or masked 1,2-dihydroxyphenyl motif such as
dopamine, hydrocaffeic acid, piperine, and l-DOPA (see
Scheme S1 in the Supporting Information). Since our syn-
thetic methodology for bisphosphate generation involved
hydrogenolysis of dibenzylphosphate esters,[14] any double
bonds in the natural products are saturated in our derivatives.
The N-acylated dopamine derivative 4 (Ki = 0.69 Æ 0.04 mm),
the hydrocaffeic acid derivative 5 (Ki = 0.82 Æ 0.05 mm), and
the tetrahydropiperine derivative 6 (Ki = 0.73 Æ 0.07 mm) dis-
played similar or marginally higher activities than 1 (Table 1
and Table S1). However, the Fmoc-protected dopamine
bisphosphate 7 (Ki = 0.45 Æ 0.04 mm) and Fmoc-protected l-
Dopa methyl ester bisphosphate 8 (Ki = 0.46 Æ 0.08 mm) are
approximately twice as active as 1. Docking studies suggested
p-stacking interactions with Trp641 as a potential cause, and
suggested that shifting the amide bond of 7 closer to the
catechol moiety might facilitate hydrogen bonds between the
inhibitor and the protein backbone at amino acid positions
642 and 644 (see Figure S4 in the Supporting Information).
Indeed, 9 (Ki = 0.21 Æ 0.04 mm) is twofold more active than 7.
All catechol bisphosphate derivatives, except 5, display strong
selectivity for STAT5b over STAT5a (Table 1 and Table S1).
Before further compound optimization, we verified the
binding site of 1 (Figure 1c). The BODIPY-FL-labeled
derivative 10 (see Scheme S2 in the Supporting Information)
was designed as a tracer molecule for direct binding assays
based on fluorescence polarization, and was found to bind to
wild-type STAT5b with high affinity (Kd = 0.86 Æ 0.08 mm;
Figure 2). Consistent with the binding mode proposed by
docking (Figure 1c), binding of 10 to the STAT5b point
mutant Arg618Ala was markedly reduced as compared to
Stat5b wild-type. Binding to STAT5b Arg618Lys, with
a positive charge one carbon–carbon bond closer to the
protein backbone than in wild-type STAT5b, was partially
reduced. Binding to STAT5b Trp641Ala was significantly
reduced, suggesting that Trp641 is important for binding of
the catechol bisphosphate core. The weak affinity of wild-type
STAT5a for 10 is in line with the results of the competition-
based fluorescence polarization assay (Figure 1b and
Table 1). These results indicate that the catechol bisphosphate
binds to the phosphotyrosine binding pocket of the STAT5b
SH2 domain, confirming its selectivity for STAT5b over
STAT5a.
Figure 2. Validation of the binding mode and specificity of catechol
bisphosphate derivatives. a) Structure of the BODIPY-FL-labeled cat-
echol bisphosphate derivative 10 and principle of the assay. b) 10 nm
of 10 were incubated with the indicated wild-type and mutant STAT5
proteins at the indicated protein concentrations. Binding was detected
by an increase in fluorescence polarization. Kd values for the STAT5b
mutants and STAT5a could not be determined, since their extrapolated
values exceed the highest protein concentration tested (2560 nm).
phosphate groups as in 14 led to a complete loss of activity.
Monophosphorylated 15, which is based on 12, was also
significantly less active, and lost specificity for STAT5b. These
data, as well as data from additional control compounds,
demonstrate the fundamental importance of the bisphoshory-
lated core structure for selective STAT5b inhibition (see
Table S4 in the Supporting Information).
The high activity and selectivity of 13 prompted us to carry
out a comparison with the natural ligand QDTpYLVLDKWL
(16), derived from the EPO receptor.[16] The fluorescence
polarization assays we used for STAT5a and STAT5b are both
based on STAT5a/b binding to the core sequence of this
peptide motif, pYLVLDKWL.[17] Both STAT5a (Kd = 133 Æ
26 nm) and STAT5b (Kd = 103 Æ 13 nm) bind to 5-carboxyl-
fluorescein-pYLVLDKWL with similar affinities. The inhib-
itory constants of the peptide QDTpYLVLDKWL (16)
against the STAT5 proteins were also similar (Ki (STAT5a):
0.41 Æ 0.09 mm, Ki (STAT5b) = 0.54 Æ 0.08 mm), demonstrating
that the natural peptide sequence does not differentiate
between the two STAT5 proteins (Figure 3e). In contrast, 13
discriminates between STAT5a and STAT5b, binding to
STAT5b with higher affinity and ligand efficiency (LE) than
the native EPO-receptor-derived peptide [LE (13) = 0.18; LE
(16) = 0.07].
Replacement of the Fmoc group of 9 with a naphthyl
group resulted in the compounds 11 (Ki = 0.24 Æ 0.01 mm) and
12 (Ki = 0.28 Æ 0.02 mm; Table 1). In order to further improve
the activity of the compounds, we envisioned targeting the
hydrophobic pocket created by Phe633 and Tyr665 by an
aromatic group. We designed compound 13 by extending the
core of 12 via an N-phenyl carboxamide group (Figure 3a,b)
for synthesis in an eight-step procedure (Figure 3c). Com-
pound 12 was chosen over 11 as a template, since it displays
more favorable geometry for extending to the adjacent
hydrophobic pocket. Compound 13 displayed fourfold
higher activity against STAT5b (Ki = 0.044 Æ 0.001 mm) than
12 (Figure 3d, Table 1), and displays 55-fold selectivity for
STAT5b over STAT5a (Ki (STAT5a) = 2.42 Æ 0.05 mm; see
Table S3 in the Supporting Information), and even higher
selectivity against other SH2 domains. Removal of both
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phosphorylation is hampered by the
lack of antibodies able to selec-
tively recognize tyrosine-phos-
phorylated STAT5b but not tyro-
sine-phosphorylated STAT5a (or
vice versa). All current commercial
antibodies recognizing phosphory-
lated STAT5b (pTyr699) and/or
STAT5a (pTyr694) have been
raised against a phosphotyrosine-
containing peptide which is identi-
cal in both proteins. In order to
analyze the relative activity of small
molecules against the individual
STAT5 proteins, we transfected
K562 cells with expression vectors
for the fusion proteins STAT5a-
GFP or STAT5b-GFP. Like endo-
genous STAT5a/b, the fusion pro-
teins are phosphorylated at the
conserved tyrosine residue. How-
ever, the presence of the GFP tag
increases the molecular weight,
allowing the fusion proteins to be
distinguished from endogenous
Stat5 on a Western blot.
To test its activity in cell-based
assays, 13 was converted into the
corresponding cell-permeable piva-
loyloxymethyl (POM) ester 17,
which is designed to liberate 13
inside the cell after cleavage by
intracellular
esterases
(Fig-
ure 4b).[19] To rule out the possibil-
ity that inhibitory effects of 17 on
STAT5b phosphorylation might be
caused by the release of pivalic acid
or formaldehyde generated during
the cleavage, the pivaloyloxyme-
thylester 18, based on the inactive
bisphosphonate 3, was prepared as
a control compound. Neither 17 nor
18 displayed activity against
STAT5b in vitro (see Table S1).
Treatment of Stat5b-GFP-trans-
fected K562 cells with 17 showed
Figure 3. Design, synthesis, and biochemical activity of compound 13. a) Docking of 12 into the
STAT5b SH2 domain with AutoDock Vina. b) Binding mode of 13 as suggested by docking.
c) Synthesis of 13. d) Activities of 13 against the SH2 domains of STAT family members and the
tyrosine kinase Lck. e) Comparison of the activities of 13 and the natural peptide ligand
QDTpYLVLDKWL (16) against STAT5a and STAT5b.
a
dose-dependent decrease of
Tyr699 phosphorylation of
STAT5b-GFP (Figure 4c,d). Time-
course experiments indicated
A key event in STAT signaling is STAT binding to
activated cell surface receptors and/or non-receptor tyrosine
kinases (Figure 4a).[18] Binding is required for STAT phos-
phorylation at the conserved tyrosine residue C-terminal of
their SH2 domain (STAT5a: Tyr694; STAT5b: Tyr699). The
efficiency of STAT SH2 domain inhibitors can thus be
investigated by analyzing the phosphorylation state of STAT
proteins, using phospho-specific antibodies for the conserved
tyrosine. However, analysis of endogenous Stat5a and Stat5b
a steady increase in inhibition during the first hour of
exposure to 17, consistent with cleavage of the POM groups
to liberate the active agent, and inhibition was still observed
after 8 hours (see Figure S5 in the Supporting Information).
The negative control compound 18 did not inhibit phosphor-
ylation. Treatment of Stat5a-GFP-transfected K562 cells with
17 did not affect phosphorylation of Tyr694 of STAT5a-GFP
(Figure 4e,f).
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Figure 4. Selective inhibition of STAT5b tyrosine phosphorylation by 17, the pivaloyloxymethyl ester of 13. a) STAT signaling is induced by activated
cell surface receptors and non-receptor tyrosine kinases. A small-molecule inhibitor of a STAT SH2 domain (the red triangle) inhibits STAT
signaling by inhibiting STAT phosphorylation at the conserved tyrosine residue. The graphic is modified from the literature.[20] b) Structure of 17
and the negative control compound 18. c) Inhibition of STAT5b tyrosine phosphorylation by 17 in K562 cells transfected with STAT5b-GFP. A
separate gel was run to confirm even transfection via the GFP tag. d) Quantification of the pSTAT5b-GFP bands, normalized against total STAT5b-
GFP. Error bars represent the standard deviations from two independent experiments. e) Tyrosine phosphorylation of STAT5a in K562 cells
transfected with STAT5a-GFP is not inhibited by 17. A separate gel was run to confirm even transfection via the GFP tag. f) Quantification of the
pSTAT5a-GFP bands, normalized against total STAT5a-GFP. Error bars represent the standard deviations from two independent experiments.
g) Effect of 17 and 18 on tyrosine phosphorylation of endogenous STAT5. h) Quantification of the endogenous pSTAT5 bands, normalized against
total endogenous STAT5. Error bars represent the standard deviations from four independent experiments.
These data demonstrate that the high specificity of 13
observed in vitro is also maintained under cellular conditions.
Since endogenous STAT5 consists of both STAT5a and
STAT5b, the effect of 17 on endogenous STAT5 (Figure 4g,h)
is lower than the effect on STAT5b-GFP (Figure 4c,d). Based
on the relative degrees of inhibition of endogenous STAT5
and STAT5b-GFP, we conclude that the majority of phos-
phorylated endogenous STAT5 in K562 cells is STAT5b.
Studies quantifying the relative amounts of STAT5a and
STAT5b and their phosphorylation levels in K562 cells have
not been published.
In summary, we have developed 13 as the first small
molecule that can differentiate between the two highly
homologous STAT5 proteins. 13, dubbed Stafib-1 (for “Stat
five b inhibitor-1”), displays low-nanomolar activity against
STAT5b (Ki = 44 Æ 1 nm), with more than 50-fold selectivity
over STAT5a. This is the first report describing strongly
divergent affinities of the SH2 domains of STAT5a/b for
a chemical entity, both in vitro and in cultured human cells.
The binding site of the core of the inhibitors, catechol
bisphosphate (1), on STAT5b was characterized by point
mutants, which represents the first point mutant validation of
a small-molecule inhibitor of a STAT SH2 domain. The
pivaloyloxymethylester of Stafib-1, dubbed Pomstafib-1 (17),
inhibited tyrosine phosphorylation of a STAT5b-GFP fusion
protein with high selectivity over the corresponding STAT5a-
GFP fusion protein in human leukemia cells. 17 will be
a valuable tool for dissecting the functions of STAT5b and
STAT5a in mammalian cells. To the best of our knowledge,
our work represents the first demonstration that small
molecules can differentiate between naturally occurring
SH2 domains which have more than 90% amino acid
sequence identity. We believe that our data will significantly
influence the scientific communityꢀs perception of the poten-
tial of small molecules as potent and selective inhibitors of
highly similar protein–protein interaction domains.
Received: November 2, 2014
Revised: December 21, 2014
Published online: && &&, &&&&
Keywords: inhibitors · natural products · protein–
.
protein interactions · STAT5 · transcription factors
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Protein–Protein Interactions
N. Elumalai, A. Berg, K. Natarajan,
A. Scharow, T. Berg*
&&&& — &&&&
Nanomolar Inhibitors of the
Transcription Factor STAT5b with High
Selectivity over STAT5a
Can you tell them apart? Neither small
molecules nor peptides have previously
been reported to be able to distinguish
between the highly homologous tran-
scription factors STAT5a and STAT5b.
Described herein is the natural product-
inspired development of Stafib-1, as the
first small molecule that can differentiate
between STAT5a and STAT5b in vitro and
in human tumor cells.
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