Identification of purine-scaffold small-molecule inhibitors of stat3 activation by QSAR studies

Article abstract of DOI:10.1021/ml100224d

To facilitate the discovery of clinically useful Stat3 inhibitors, computational analysis of the binding to Stat3 of the existing Stat3 dimerization disruptors and quantitative structure-activity relationships (QSAR) were pursued, by which a pharmacophore model was derived for predicting optimized Stat3 dimerization inhibitors. The 2,6,9-trisubstituted-purine scaffold was functionalized in order to access the three subpockets of the Stat3 SH2 domain surface and to derive potent Stat3-binding inhibitors. Select purine scaffolds showed good affinities (K_D, 0.8-12 μM) for purified, nonphosphorylated Stat3 and inhibited Stat3 DNA-binding activity in vitro and intracellular phosphorylation at 20-60 μM. Furthermore, agents selectively suppressed viability of human prostate, breast and pancreatic cancer cells, and v-Src-transformed mouse fibroblasts that harbor aberrant Stat3 activity. Studies herein identified novel small-molecule trisubstituted purines as effective inhibitors of constitutively active Stat3 and of the viability of Stat3-dependent tumor cells, and are the first to validate the use of purine bases as templates for building novel Stat3 inhibitors.

Full text of DOI:10.1021/ml100224d

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Identification of Purine-Scaffold Small-Molecule
Inhibitors of Stat3 Activation by QSAR Studies
Vijay M. Shahani,†,‡,§ Peibin Yue,§, Sina Haftchenary,†,‡ Wei Zhao, Julie L. Lukkarila,†,‡
Xiaolei Zhang, Daniel Ball,‡ Christina Nona,‡ Patrick T. Gunning,*,†,‡ and James Turkson*,
Departments of †Chemistry, and ‡Chemical and Physical Sciences, University of Toronto, 3359 Mississauga Road North,
Mississauga, ON, L5L 1C6, Canada, and Burnett School of Biomedical Sciences, University of Central Florida
College of Medicine, 6900 Lake Nona Boulevard, Orlando, Florida 32827, United States
ABSTRACT To facilitate the discovery of clinically useful Stat3 inhibitors, com-
putational analysis of the binding to Stat3 of the existing Stat3 dimerization
disruptors and quantitative structure-activity relationships (QSAR) were pursued,
by which a pharmacophore model was derived for predicting optimized Stat3
dimerization inhibitors. The 2,6,9-trisubstituted-purine scaffold was functiona-
lized in order to access the three subpockets of the Stat3 SH2 domain surface and
to derive potent Stat3-binding inhibitors. Select purine scaffolds showed good
affinities (KD, 0.8-12 μM) for purified, nonphosphorylated Stat3 and inhibited
Stat3 DNA-binding activity in vitro and intracellular phosphorylation at 20-60 μM.
Furthermore, agents selectively suppressed viability of human prostate, breast and
pancreatic cancer cells, and v-Src-transformed mouse fibroblasts that harbor
aberrant Stat3 activity. Studies herein identified novel small-molecule trisubsti-
tuted purines as effective inhibitors of constitutively active Stat3 and of the viability
of Stat3-dependent tumor cells, and are the first to validate the use of purine bases
as templates for building novel Stat3 inhibitors.
KEYWORDS Stat3, small-molecule inhibitors, molecular modeling, 3D-QSAR
pharmacophore model, purine scaffold, cell viability, antitumor cell effects
he signal transducer and activator of transcription
(STAT) family of proteins are cytoplasmic transcrip-
tion factors with important roles in mediating cellular
a reciprocal pTyr-SH2 domain interaction, creating transcrip-
tionally active STAT:STAT dimers that in the nucleus regulate
gene expression by binding to specific DNA-response ele-
ments in the promoters of target genes.1
T
responses, including cell growth and differentiation, and in-
flammation and immune responses.1 Normally, STATs are
activated upon receptor stimulation by cytokines, growth
factors, and other polypeptide ligands. However, constitutive
activation of the family member Stat3 is highly prevalent in
human tumors.2,3 Compelling evidence demonstrates that
aberrantly active Stat3 induces malignant transformation
and tumorigenesis,3 by serving as a master regulator of
tumor-promoting molecular events in cells. Stat3 has there-
fore become a valid target for anticancer drug design.
Presently, there are several efforts to discover and develop
novel Stat3 inhibitors for therapeutic application and for
probing Stat3-mediated molecular and tumor processes.1-4
Protein:protein interactions are a critical molecular event
in the induction of signal transduction pathways. During
activation, the STAT proteins are recruited via the Src homol-
ogy (SH) 2 domain to the cognate receptor phosphotyrosine
(pTyr) peptide motifs. The binding to membrane-bound
receptors represents an initial key step for STAT phos-
phorylation and activation, bringing the proteins into close
proximity to tyrosine kinases for the phosphorylation of the
critical tyrosine residue (Y705 for Stat3).1,3,4 Tyrosine phos-
phorylated STAT monomers engage in dimerization through
Given its importance in STATactivation and function, the
initial pTyr-SH2 domain interaction is one of the targeting
sites in many drug discovery programs to identify Stat3
inhibitors as novel anticancer agents. Many diverse dimer-
ization-disrupting small-molecule Stat3 inhibitors have been
reported, in some cases with cellular activities and evidence
of in vivo efficacy.5-18 While the studies using small-mole-
cule inhibitors and genetic modulators4,19 provide the proof
of concept for the potential therapeutic application of Stat3-
inhibitory drugs, no small-molecule Stat3-inhibitory agent
has thus far advanced to the clinic.
To expedite the identification of clinically useful Stat3 in-
hibitors, the leading Stat3 dimerization-disrupting small-mole-
cules were subjected to computational (genetically optimized
ligand docking, GOLD) analysis for deriving a 3D quantative
structure activity relationship (QSAR) pharmacophore model
to predict optimized Stat3 inhibitors. The crystal structure of
Received Date: September 24, 2010
Accepted Date: October 19, 2010
Published on Web Date: October 25, 2010
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cify the relative positions of the functional groups in space.
Our pharmacophore model describes an essentially planar,
hetero-trisubstituted scaffold decorated with optimal rec-
ognition elements and confined within the specified
pharmacophore plot (Figure 1C). Computational docking
studies identified 2,6,9-trisubstituted purine scaffolds as a
promising choice of structural skeleton for projecting
functionality into the three vertices of the pharmacophore
plot (Figure 1D and 1E).
Forty-seven pharmacophore-directed 2,6,9-trisubstituted
purine inhibitors were synthesized (Table 1), as previously
reported23,24 and evaluated by surface plasmon resonance
(SPR) analysis16 for their interactions (as analyte) with Stat3
(target). Over one-third showed high affinity binding to Stat3
(KD<10 μM), including S3I-V3-32, S3I-V4-01, S3I-V3-31, S3I-
S2-36, S3I-S2-30, S3I-V2-66 and S3I-S2-38 (Table 1, SPR).
Details of the QSAR study and the discussion of the SAR are
presented in the Supporting Information.
Our study shows that 2,6,9-trisubstituted purine scaffolds
interact with Stat3, presumably at the three subpockets
on the Stat3 SH2 domain surface (Figures 1A and 1D). The
interaction with Stat3 is expected to disrupt Stat3 binding to
its cognate pTyr peptide and hence, Stat3:Stat3 dimeri-
zation.6-8,16 Further evaluation of purine-scaffold molecules
in in vitro Stat3 DNA-binding assay/electrophoretic mobility
shift assay (EMSA) analysis, as previously reported,16 de-
monstrated that the select agents, S3I-V3-29, S3I-V3-30, S3I-
V3-31, S3I-V3-32, S3I-V3-34, S3I-V4-01, and S3I-S2-21, which
showed good binding affinity for Stat3 (Table 1, SPR ), also
strongly inhibited Stat3 activity, with IC50 values of 27-84 μM
(Table 1, EMSA). Data from the EMSA analysis did not always
show a good correlation to the SPR analysis. We note that
although Stat3 dimerization disruptors may exhibit high
potency in inhibiting dimerization, the activities in DNA-
binding assay/EMSA analysis have generally been weaker.16
The reasons for this difference may be the presence of signi-
ficantly large number of protein “targets” in the nuclear extract
preparations utilized in the DNA-binding assay and the poten-
tial that disrupting preformed Stat3:Stat3 dimers, as in the
DNA-binding assay is a more challenging task, as has pre-
viously been suggested.5,6 Immunoblotting analysis showed a
moderate to strong inhibition by S3I-V3-31, S3I-V3-32, S3I-V3-
34, and S3I-V4-01 of constitutive Stat3 phosphorylation in the
v-Src transformed mouse fibroblasts (NIH3T3/v-Src) and
the human breast cancer line, MDA-MB-231 that harbor
aberrant Stat3 activity3,4 following treatments for 12, 24, or
48 h (Figure 2A(i) and Figure 2A(ii), pY705, lanes 2, 3, 5, 6, 8,
9, 11, 12, 15, and 18, compared to lanes 1, 4, 7, 10, 13, and 16,
and data not shown). By contrast, same treatments have no
effect on the constitutive levels of phospho- ErkMAPK (pErk1/2)
or Src (pSrc) (Figure 2A). The inhibition of Stat3 phosphoryla-
tion, however, showed different kinetics among the purine
compounds studied. Thus, additional time-course study was
performed to provide more insight into the effects of the
inhibitors. In NIH3T3/v-Src fibroblasts, treatment with S3I-
V3-31 or S3I-V3-32 consistently inhibited Stat3 activity as
early as 15-60 min (Figure 2A(iii), right two panels). Data
also shows the Stat3 phosphorylation, which was inhibited
early, appeared to be temporarily restored during prolonged
Figure 1. GOLD docking and quantitative structure-activity rela-
tionship studies, and pharmacophore modeling of the binding of
Stat3 to small-molecule dimerization inhibitors. (A) Stat3 SH2
domain binding sites and key amino acid residues, (B) Stat3
inhibitors GOLD-docked and overlaid with the Stat3 SH2 domain,
(C) Pharmacophore plot identifying the optimal distances for the
projection of functionality from the centroid unit, (D) A GOLD
docked purine derivative superimposed over the pharmacophore
plot and previously docked Stat3 inhibitors, and (E) Low energy
GOLD-docked 8aa in the Stat3 SH2 domain (pdb: 1BG1).
the Stat3:Stat3-DNA ternary complex20 revealed the structural
composition and topology of the SH2 domain binding “hot-
spot”, identifying three subpockets on the SH2 domain protein
surface that are solvent-accessible, labeled A, B and C (Figure 1A).
The GOLD analysis21 of all known Stat3 inhibitors within the
Stat3 SH2 domain yielded homologous binding configurations
(Figure 1B), with inhibitors consistently occupying at least
two of the three main subpockets (Figure 1B). Most notably,
all compounds interacted with subpocket A, which hosts the
key pTyr705 group, and is broadly composed of the polar
residues Lys591, Ser611, Ser613 and Arg609,22 and were found
to engage these residues through predominantly hydrogen
bond donor (HBD) or hydrogen bond acceptor (HBA) groups.
In developing our pharmacophore model from known
active compounds, we considered only the functional groups
pertinent to effective interaction with the three main binding
centers. We prepared a simple pharmacophore plot identify-
ing the optimal positioning of functionality crucial for bind-
ing to the Stat3-SH2 domain (Figure 1C). The centroid unit,
shown as a blue sphere, represents the potential posi-
tioning of the scaffold core from which functionality can
be optimally appended to access A, B and C. We then
considered the 3D pharmacophore, where we would spe-
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Table 1. Structures and Activities of Purine Scaffold Moleculesa
a *KD, affinity determined by SPR; #IC50, concentration at which DNA-binding activity is inhibited by 50%.
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Figure 2. Immunoprecipitation and immunoblotting analysis for the effects of agents on intracellular Stat3, ErkMAPK, Src, and Stat1
activation. Immunoblotting analysis of whole-cell lysates of equal total protein prepared from NIH3T3/v-Src and MDA-MB-231 cells (A) or
from EGF-stimulated NIH3T3/hEGFR fibroblasts (B(i)), or of Stat1 immune complexes prepared from EGF-stimulated NIH3T3/hEGFR
fibroblasts (B(ii)) treated or untreated with the indicated agents at 40 or 50 μM for the indicated times and probing for pY705Stat3, Stat3,
pErk1/2, Erk1/2, pSrc, Src, pStat1, Stat1, or pTyr (clone 4G10). Positions of proteins in gel are labeled; control lanes (0), whole-cell lysates
from 0.05% DMSO-treated cells. Data are representative of 3-4 independent determinations.
treatment (Figure 2A(iii), NIH3T3/v-Src). Similarly, in the MDA-
MB-231 line, Stat3 phosphorylation was consistently inhi-
bited by S3I-V3-31 as early as 15-30 min following treatment,
with evidence of restoration during treatment for 6 h
(Figure 2A(iii), left, top two panels), while inhibition of Stat3
activation by S3I-V3-32 was strong at 6 h following treat-
ment (Figure 2A(iii), left, bottom two panels). While data
together suggests different kinetics of inhibition of intracellular
constitutive Stat3 phosphorylation by purine scaffolds, data
shows that, by 48 h following treatment, Stat3 phosphoryla-
tion remains low.
Moreover, studies with S3I-V3-34 and S3I-V4-01 for effects
on ligand-stimulated STAT activation showed a selective
inhibition of the epidermal growth factor (EGF)-induced
Stat3 phosphorylation in mouse fibroblasts overexpressing
the human EGF receptor (NIH3T3/hEGFR), as assessed by
immunoblotting of whole-cell lysates (Figure 2B(i), pY705-
Stat3, lanes 3 and 4, compared to lane 2). The block of
ligand-induced Stat3 phosphorylation suggests that purine
agents could interact with the inactive Stat3 monomers,
presumably the SH2 domain, and thereby occlude Stat3
binding to the EGFR receptor and, hence, block de novo Stat3
phosphorylation, as well as blocking Stat3:Stat3 dimerization,
as has been previously reported for other SH2 domain-
binding Stat3 inhibitors.6-8 By contrast, there was no effect
on the phosphorylation of Erk (pErk1/2) and Src (pSrc) in the
same study (Figure 2B(i)). Importantly, similar treatments
showed no significant effect on EGF-induced phosphorylation
of the STAT family member, Stat1, as assessed by immuno-
blotting analysis (Figure 2B(i), pStat1) and by way of Stat1
immune complex precipitation, with general pTyr immu-
noblotting analysis (Figure 2B(ii)). Thus, although purine
scaffolds have typically been employed to target the ATP
pocket of aberrant kinases, we speculate that the size
of the appendages and the lack of HBA with the agents in
the present study will preclude access to the majority of the
confined active sites, while conferring high selectivity for
the Stat3 SH2 domain. Aberrant Stat3 activity promotes
cancer cell growth and survival, and tumor angiogenesis
and metastasis.3-8,12,25 CyQuant cell proliferation assay
shows the human prostate (DU145), breast (MDA-MB-231),
and pancreatic cancer (Panc-1) lines and the v-Src-trans-
formed mouse fibroblasts (NIH3T3/v-Src) that harbor con-
stitutively active Stat3 are sensitive to select purine-scaffold
small-molecules, including S3I-V3-30, S3I-V3-31, S3I-V3-32,
S3I-V3-34, and S3I-V4-01 which decrease malignant cell viabi-
lity (Figure 3), with IC50 values of 41-80 μM concentrations
(Table 2). The viability of normal mouse fibroblasts (NIH3T3)
and the murine thymus epithelial stromal cells (TE-71)
that do not harbor aberrant Stat3 activity,16 however, are not
affected by agents, with IC50 values greater than 500 μM
(Figure 3 and Table 2). Thus, select purine-scaffold small
molecules selectively inhibit Stat3 activation in malignant cells,
and induce the loss of viability of tumor cells that harbor
persistently active Stat3. Altogether, data presented indicate
that select purine compounds, including S3I-V3-31, S3I-V3-
32, S3I-V3-34 and S3I-V4-01, have an effect on Stat3
activity and induce loss of viability of malignant cells that
harbor constitutively active Stat3. Although Stat3 activity
appears to be temporarily restored following inhibition, the
early initial inhibition of aberrant Stat3 activity is sufficient
to promote the loss of viability of malignant cells harboring
persistently active Stat3.
Herein is presented the application of QSAR to study the
binding of Stat3 dimerization inhibitors to the Stat3 SH2
domain and the identification by QSAR pharmacophore
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Table 2. Cell Viability Assay
IC50a (μM)
Panc-1
NIH3T3
TE-71
MDA-MB-231
DU145
NIH3T3/v-Src
S3I-V3-30
S3I-V3-31
S3I-V3-32
S3I-V3-34
S3I-V4-01
420
>500
>500
>500
>500
>500
>500
>500
>500
>500
147.8 ( 5.1
145.2 ( 4.4
100.4 ( 3.2
66.1 ( 2.1
82.4 ( 1.4
74.5 ( 8.4
175.7 ( 5.3
77.7 ( 2.7
77.2 ( 1.9
197.6 ( 2.1
65.1 ( 8.3
68.6 ( 1.8
80.2 ( 5.1
65.3 ( 2.3
75.4 ( 3.4
62.5 ( 3.4
100.2 ( 2.2
45.6 ( 3.1
41.1 ( 4.6
145.6 ( 6.1
a IC50 values: Drug concentration at which 50% of cell viability is inhibited.
inhibitors,9,11,12,14,17,18,26,27 they are more attractive, given
the existing clinical application of nucleoside analogs.28,29
The present purine scaffold provides a suitable platform to
develop optimized and clinically viable Stat3 inhibitors.
SUPPORTING INFORMATION AVAILABLE Information on
compound synthesis, chemical data, and the molecular modeling
method, detailed results, the experimental procedures for SPR,
DNA-binding activity/EMSA, immunoprecipitation and immuno-
blotting, and Cyquant cell proliferation assays. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author: *P.T.G.: tel, 905-828-5354; fax, 905-
569-5425; e-mail, patrick.gunning@utoronto.ca. J.T.: tel, 407-266-
7031; fax ,407-384-2062; e-mail, jturkson@mail.ucf.edu.
Author Contributions: § These two authors contributed equally
to this work.
ACKNOWLEDGMENT This work was supported by the Na-
tional Cancer Institute Grants CA106439 (J.T.) and CA128865
( J.T.), and by the Leukemia and Lymphoma Society of Canada
(P.T.G.) and the University of Toronto (P.T.G.).
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