Synthesis and in vitro evaluation of a peptidomimetic inhibitor targeting the Src Homology 2 (SH2) domain of STAT6

Article abstract of DOI:10.1021/ml4003919

An improved synthesis of a phosphopeptidomimetic prodrug targeting the Src Homology 2 (SH2) domain of signal transducer and activator of transcription 6 (STAT6) is reported. In our convergent methodology, we employed a phosphotyrosine surrogate active ester harboring pivaloyloxymethyl groups, which efficiently coupled to tert-butylglycinyl proline diarylamide. Biological evaluation of 1 has not been reported. We show that it inhibits STAT6 phosphorylation in intact human bronchial epithelial cells, suggesting potential application in the treatment of asthma.

Full text of DOI:10.1021/ml4003919

Letter
pubs.acs.org/acsmedchemlett
Synthesis and in Vitro Evaluation of a Peptidomimetic Inhibitor
Targeting the Src Homology 2 (SH2) Domain of STAT6
Pietro Morlacchi, Pijus K. Mandal, and John S. McMurray*
Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, 1901 East Road, Houston, Texas
77054, United States
S
* Supporting Information
ABSTRACT: An improved synthesis of a phosphopeptidomi-
metic prodrug targeting the Src Homology 2 (SH2) domain of
signal transducer and activator of transcription 6 (STAT6) is
reported. In our convergent methodology, we employed a
phosphotyrosine surrogate active ester harboring pivaloylox-
ymethyl groups, which efficiently coupled to tert-butylglycinyl
proline diarylamide. Biological evaluation of 1 has not been
reported. We show that it inhibits STAT6 phosphorylation in
intact human bronchial epithelial cells, suggesting potential
application in the treatment of asthma.
KEYWORDS: Peptidomimetic inhibitor, SH2, STAT6, prodrug
sthma is a complex inflammatory disease of the lungs
characterized by mucus production, airway hyperrespon-
production, eosinophil migration, and AHR. Furthermore,
intranasal administration of STAT-6-IP inhibited many features
of allergic airway disease symptoms in a mouse asthma model
induced by ragweed pollen.8 These reports provide proof of
concept that steric block of the SH2 domain of STAT6
prevents recruitment to IL-4Rα, phosphorylation of Tyr641,
and subsequent transcriptional activity leading to asthma
symptoms.
In the early 2000s Tularik, Inc. (now part of Amgen, Inc.)
published identical US and World patents on small molecule
phosphopeptide mimetics targeting the SH2 domain of
STAT6.9,10 Although extensive structure−affinity relationship
studies were reported, the inventors described the synthesis of
only one compound with the potential to inhibit STAT6
phosphorylation in intact cells (1, Figure 1). In this compound,
phosphotyrosine was replaced with the conformationally
constrained 4-phosphoryloxycinnamic acid unit, and the
phosphate was replaced with the noncleavable phosphonodi-
fluoromethyl group.11
A prodrug strategy was employed in which the negative
charges of the phosphonate were capped with carboxyesterase-
labile pivaloyloxymethyl (POM)12 groups to allow passage into
cells. The theory was that, on entering cells, esterases will cleave
the POM groups, thereby freeing the phosphonate to bind to
the phosphotyrosine binding pocket on the SH2 domain of
STAT6. In the patents the synthesis of 1 was inefficient, and
the last step did not have yield data nor was the final compound
characterized by NMR, although the molecular weight was
A
siveness (AHR), eosinophil recruitment, and T-Helper cell 2
(Th2) activation which results in immunoglobulin class
switching to IgE (reviewed in refs 1−3). Activated Th2 cells
release cytokines, especially inerleukin-4 (IL-4) and interleukin-
13 (IL-13), which then bind to receptors at the cell surface and
recruit Janus kinases 1 and 3 (JAK1 and JAK3), and the
tyrosine kinase 2 (Tyk2), leading to phosphorylation of IL-
4Rα, the subunit common to both cytokine receptors.
Cytoplasmic signal transducer and activator of transcription 6
(STAT6) is recruited to the phosphorylated receptor via its Src
homology 2 (SH2 domain) and is phosphorylated on Tyr641
by the associated JAK kinases. Phosphorylated STAT6
(pSTAT6) dimerizes via reciprocal SH2 domain interactions,
translocates to the nucleus, binds to specific DNA promoter
sequences, and participates in the expression of genes leading to
asthma and AHR. Elevated pSTAT6 levels have been found in
the bronchial epithelium of asthma patients,4 and STAT6
knockout mice do not develop AHR or lung pathology
associated with asthma.5 Therefore, inhibiting the activity of
STAT6 is a potential modality for asthma treatment.
Of the steps in the IL4/13-JAK-STAT6 signaling cascade,
blocking association of STAT6 with IL-4Rα by targeting the
SH2 domain is an attractive strategy. A cell-penetrating
phosphopeptide derived from Tyr631 of IL-4Rα, a docking
site for STAT6, inhibited STAT6 phosphorylation stimulated
by IL-4 in Ramos cells.6 Another cell-penetrating phosphopep-
tide, STAT-6-IP, derived from the phosphorylation site of
STAT6, Tyr641, inhibited in vitro IL-4 and IL-13 expression
from splenocytes from mice challenged with ovalbumin
(OVA).7 Importantly, in vivo intranasal administration
inhibited OVA-induced lung inflammation and mucus
Received: September 30, 2013
Accepted: December 4, 2013
Published: December 4, 2013
© 2013 American Chemical Society
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Letter
a
Scheme 2. Improved Synthesis of STAT6 Inhibitor, 1
Figure 1. Structure of the Tularik inhibitor, 1.
confirmed by mass spectrometry. More importantly, no data on
the biological activity of this compound was reported in patent
or peer-reviewed literature. Given the potential impact of
inhibiting STAT6 activity by targeting the SH2 domain, our
laboratory developed an improved synthesis of 1 and found it
to be a relatively potent inhibitor of STAT6 phosphorylation in
IL-4 and IL-13 stimulated immortalized bronchial epithelial
cells, Beas-2B. Our improved synthesis scheme and biological
evaluation of 1 are reported here (see also Supporting
Information).
The Tularik researchers used a convergent scheme in which
the phosphotyrosine surrogate, 4′-phosphonodifluoromethyl-
cinnamate (2), was coupled to a modified dipeptide 3 followed
by installation of the POM groups (Scheme 1). Coupling of 2
a
Reagents and conditions: (a) 4-iodoaniline, EDC, DCM, rt, 4 h, 88%;
(b) Ph3 Bi, Cu(OAc)2, TEA, DCM, overnight, rt, 48%; (c) (i) TFA/
DCM (1:1), 0.5 h, rt; (ii) Fmoc-Tle-OH, EDC, TEA, DCM, 6 h, 78%;
(d) 20% piperidine/DMF, 47%; (e) 3, NMM, DMAP (cat), DMF, rt 2
h, 66%.
a
Scheme 1. Published Synthesis of 1
the free phosphonate group in 2. Further, POM groups are
typically installed on the silver salts of phosphonates in toluene,
which is a poor solvent for peptides and peptidic compounds.
In addition to POM protection, the carboxyl group of 9 is
derivatized as the pentachlorophenyl (Pcp) ester, a known
activator of carboxyl groups. The Pcp ester possesses sufficient
stability to serve as a protecting group for the POM installation
as we described.13,14 Our overall yields for the synthesis of the
protected phosphotyrosine surrogate 13 were 30−40%13,14 in
contrast to 13% for the overall synthesis of 2.9,10 This strategy
resulted in marked improvements in the synthesis of 1.
For the synthesis of 3, we followed almost the same
procedure as that in the patent with some modifications. Boc-
Pro-OH (5) was coupled with 4-iodo-aniline in the presence of
EDC to get 6. The yield of the triphenylbismuth-mediated
replacement of the amide proton13 of 6 to give 7 was 48%.
Yields of coupling of sterically hindered Fmoc-tert-buytlglycine
to the deprotected proline amide were higher with HBTU than
with EDC. The Fmoc-protected dipeptide was obtained in 78%
yield after flash chromatography. The Fmoc group was
removed with piperidine, and 3 was obtained in 47% yield
after reverse-phase HPLC purification. One of the key aspects
of our method is high yield coupling of the bis-POM protected
pTyr surrogate to the dipeptide, depicted in Scheme 2. The
building block 9 was coupled to the dipeptide 3 in the presence
of N-methylmorpholine (NMM) and catalytic DMAP to give 1
in 66% yield after purification by reverse-phase HPLC.
a
Reagents and conditions: (a) HBTU, HOBt, DIPEA, DMF,
overnight, rt, 11%; (b) (i) NaOH, (ii) AgNO3; (iii) iodomethyl
pivalate, toluene.
and 3 was carried out in the presence of HBTU, HOBt, and
DIEA to give intermediate 4 in 11% yield. The POM groups
were installed by converting the phosphonate oxygens to silver
salts and treating with iodomethyl pivalate to get the desired
STAT6 inhibitor 1. As mentioned, no yield was given for this
step.
Our laboratory reported a convergent method that was used
to synthesize phosphatase-stable, cell-permeable peptidomi-
metic prodrugs targeted to the SH2 domain of STAT3.14 The
key to our synthesis is the use of the fully protected
phosphonodifluoromethylcinnamate, 9 (Scheme 2B). Blocking
the phosphonate oxygens with POM groups prior to coupling
to 3 prevents unproductive depletion of coupling agent due to
Because the biological activity has not been reported,
prodrug 1 was evaluated for its ability to inhibit STAT6
Tyr641 phosphorylation in intact Beas-2B cells. Compound 1
was added to cells from a DMSO stock solution. Cells were
preincubated with 1 for 2 h and were then stimulated with IL-4
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ACS Medicinal Chemistry Letters
Letter
or IL-13 for 1 h. Levels of phospho-STAT6(Y641) and total
STAT6 proteins were estimated by Western blotting of the cell
lysates (Figure 2A). Prodrug 1 inhibited pSTAT6 phosphor-
phosphorylation of STAT6 in human bronchial epithelial cells
stimulated with either IL-4 or IL-13. Key to the synthesis is
having the phophonate oxygens protected prior to coupling to
the amine. Furthermore, preactivation of the cinnamate
building block as pentachlorophenyl ester improved coupling
yields compared to the published method. As mentioned, this
class of inhibitor has not been evaluated and shown to inhibit
STAT6 to date. Given the importance of this transcription
factor in asthma and allergic lung disease, our promising results
suggest that targeting the SH2 domain with phosphatase-stable,
cell-permeable phosphopeptide mimetic prodrugs is a potential
treatment modality for this disease.
ASSOCIATED CONTENT
* Supporting Information
■
S
Procedures for the synthesis, NMR, and biological character-
ization of 1. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: jmcmurra@mdanderson.org; phone: 713-745-3763.
■
Present Address
P.M.: Department of Genomic Medicine, The University of
Texas M. D. Anderson Cancer Center, 1901 East Road,
Houston, Texas 77054.
Author Contributions
P.M. and P.K.M. contributed equally. The manuscript was
written through contributions of all authors. Dr. Pietro
Morlacchi performed biological evaluation of compound 1
and contributed to the preparation of the manuscript. Dr. Pijus
Mandal synthesized compound 1 and contributed to
preparation of the manuscript. Dr. John S. McMurray
supervised the work and contributed to preparation of the
manuscript.
Figure 2. Biological evaluation of 1 in Beas-2B cells. (A) Western
blotting showing the inhibitory effect of 1 on STAT6(Y641)
phosphorylation in Beas-2B cells stimulated with IL-4 or IL-13 and
relative quantification of protein-band intensites by densitometric
analysis. (B) Time course of pSTAT6(Y641) inhibition of 1 in IL-4
stimulated Beas-2B cells. (C). Effect of 1 on Beas-2B cell proliferation
after 72 h as determined by MTS assay.
Funding
We are grateful to the American Asthma Foundation for
financial support of this work (Grant # 11-0360). We also
acknowledge the Cancer Center Support Grant (P30
CA16672-38) for support of the NMR laboratory and the
Translational Chemistry Core Facility who provided mass
spectrometry.
ylation at 1−10 μM in both IL-4 and IL-13 stimulated Beas-2B
cells. This result suggests that 1 enters cells, the POM groups
are cleaved, and the resulting phosphonate binds to the SH2
domain of STAT6 thereby preventing recruitment to the
cytokine receptors and subsequent phosphorylation by JAKs.
The free phosphate version of 1 was reported to inhibit the
binding of a fluorescently tagged phosphopeptide at <1 μM
using a solid phase competition assay.9 Prodrug 1 at
concentrations up to 10 μM was found not to be toxic to
Beas-2B cells in a 72 h MTS assay (Figure 2C). The duration of
pSTAT6 inhibition was assayed by treating Beas-2B cells with a
single dose of 1 (10 μM). Cells were incubated for predefined
time intervals and were stimulated with IL-4 for 1 h prior to cell
lysis. Western blot analysis showed that nearly complete
inhibition of phospho-STAT6(Y641) occurred at 2 h and was
maintained up to 48 h (Figure 2B). The reduction in pSTAT6
was not due to toxicity as the MTS assay showed not effect on
proliferation.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
Beas-2B cells were a kind gift from Walter Hittelman.
■
ABBREVIATIONS
■
DMAP, 4-dimethylaminopyridine; EDC, N-ethyl-N′-(3-
dimethylaminopropyl)carbodiimide hydrochloride; HBTU, O-
(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluoro-
phosphate; NMM, N-methylmorpholine; NMP, N-methylpyr-
rolidone; POM, pivaloyloxymethyl
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