Nanomolar-potency small molecule inhibitor of STAT5 protein

Article abstract of DOI:10.1021/ml500165r

We herein report the design and synthesis of the first nanomolar binding inhibitor of STAT5 protein. Lead compound 13a, possessing a phosphotyrosyl-mimicking salicylic acid group, potently and selectively binds to STAT5 over STAT3, inhibits STAT5-SH2 doma

Full text of DOI:10.1021/ml500165r

Letter
pubs.acs.org/acsmedchemlett
Nanomolar-Potency Small Molecule Inhibitor of STAT5 Protein
Abbarna A. Cumaraswamy,†,‡ Andrew M. Lewis,†,‡ Mulu Geletu,†,‡ Aleksandra Todic,† Diego B. Diaz,†
Xin Ran Cheng,‡ Carla E. Brown,† Rob C. Laister,∥ David Muench,§ Kagan Kerman,‡
H. Leighton Grimes,§ Mark D. Minden,∥ and Patrick T. Gunning*,†
†Department of Chemistry, University of Toronto Mississauga, Mississauga, Ontario L5L 1C6, Canada
‡Department of Physical & Environmental Sciences, University of Toronto at Scarborough, Toronto, Ontario M1C 1A4, Canada
§Division of Immunobiology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio 45229, United States
∥Princess Margaret Cancer Centre, Ontario Cancer Institute, 610 University Avenue, Toronto, Ontario M5G 2M9, Canada
S
* Supporting Information
ABSTRACT: We herein report the design and synthesis of
the first nanomolar binding inhibitor of STAT5 protein. Lead
compound 13a, possessing a phosphotyrosyl-mimicking
salicylic acid group, potently and selectively binds to STAT5
over STAT3, inhibits STAT5−SH2 domain complexation
events in vitro, silences activated STAT5 in leukemic cells, as
well as STAT5′s downstream transcriptional targets, including
MYC and MCL1, and, as a result, leads to apoptosis. We
believe 13a represents a useful probe for interrogating STAT5
function in cells as well as being a potential candidate for
advanced preclinical trials.
KEYWORDS: STAT5, small-molecule inhibitor, protein−protein interactions, anticancer drug, leukemia cells
umerous inhibitors have been developed to target the
JAK-STAT signaling pathway, a major driving force in
hetero-8,9 STAT5X-STATX dimers through reciprocal phos-
photyrosine−SH2 interactions. Activated STAT5 dimers trans-
locate to the nucleus, where they bind to STAT5 DNA
response elements inducing transcription of genes involved in
proliferation (Bcl-xl, c-Myc, pim-1), cell differentiation (p21),
cell survival (MCL-1) inflammation (Osm), and apoptosis
(JAB).10 In contrast, mutations within cytosolic kinases (TEL-
JAK2, Bcr-Abl, FLT-3) as well as overactive receptor-associated
tyrosine kinases (SRC, EGFR) induce constitutive phosphor-
ylation of STAT5 proteins, increasing the production of
antiapoptotic genes, which can contribute to driving the cancer
phenotype.11
Approaches aimed at directly targeting STAT5 have been
limited to a chromone-derived acyl hydrazine inhibitor,
identified through a high-throughput fluorescence polarization
(FP) screen. While this agent exhibited promising in vitro
disruption of the STAT5:EPOR phosphopeptide interaction,
higher concentrations were required to inhibit STAT5 in
cells.12 A STAT5 function-based screening approach identified
N
hematopoietic malignancies. While emphasis has focused on
identifying effective upstream kinase inhibitors for suppressing
STAT activity, inhibitors have suffered from poor kinase
selectivity,1 cardiovascular toxicity,2,3 and, in some clinical cases,
acquired resistance.4 Multiple kinase inhibitors have been used
in combination to try to combat resistance.5,6 An alternative
strategy to reduce off-target toxicity is to target proteins
immediately downstream of the kinases, such as the Signal
Transducer and Activator of Transcription (STAT) 5 protein.
STAT5 is thus a compelling molecular target for therapeutic
intervention.
In normal cells, the activation of STAT5 proteins is tightly
regulated by cytokines (IL-2, IL-5, IL-7, GM-CSF, eryth-
ropoietin (EPO), thrombopoietin, and prolactin) and growth
factors.7 Binding of these extracellular ligands to their target
receptors induces the activation of receptor-associated JAK
kinases that phosphorylate key tyrosine residues within the
receptor, providing docking sites for the SRC homology 2
(SH2) domains of the inactive cytoplasmic STAT5 monomers.
STAT5 is then phosphorylated at specific tyrosine residues,
either Y694 (STAT5A) or Y699 (STAT5B) of the C-terminus.
Phosphorylated STAT5 monomers form either homo- or
Received: April 27, 2014
Accepted: September 19, 2014
© XXXX American Chemical Society
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ACS Medicinal Chemistry Letters
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the FDA approved neuroleptic drug, pimozide, as a potent
inhibitor of STAT5 phosphorylation, resulting in the down-
regulation of STAT5 target gene expression in Ba/f3 FLT3
ITD cells. The authors believe pimozide is an effector of
negative regulators that modulate STAT5 phosphorylation.13,14
We have previously prepared a library of phosphopeptide-
mimicking salicylic acid-based compounds to target STAT3′s
SH2 domain. Screening against STAT5 via FP15 identified
compounds 1 and 2 as STAT5 binders with 2−3-fold selectivity
over STAT3 (Figure 1). In whole cells, 1 showed selective
Figure 2. (A) STAT5a’s SH2 domain with three binding pockets:
hydrophilic, red; amphiphilic, green; amphiphilic, blue; (B) in silico
docking of 1 interacting with R618, S622, and N639; 2 interacting
with R618 and S622, as well as a cationic−π interaction of the R1
benzyl with K644.
protein structure, allowing for ligand flexibility using a
Lamarckian Genetic Algorithm (GA) with the global and
adaptive local search parameters through 50 trials of the “long”
GA runs. 1 and 2 occupied two amphiphilic pockets. The first
pocket contained residues N639, L640, and W641 (blue region,
Figure 2A,B), which interact with the sulfonamide mesityl and
toluyl of 1 and 2, respectively. The adjacent pocket containing
W631, L643, and K644 (green) was found to interact with the
R1 substituents of both leads. Interestingly, for 2 (R1 = Ph),
docking poses showed favorable cationic−π interactions with
K644. In contrast, the binding of 1 (R1 = cyclohexylphenyl) was
dominated by van der Waal’s interactions with L643. Since the
K644 residue is unique to STAT5, we elected to prepare
analogs possessing hydrophobic, electron rich, aromatic R1
substituents to derive selectivity. The R3 substituents were
carried forward from previous SAR studies, owing to their
favorable biological profiles.
To survey the R1 binding pocket, we explored 24 substituents
of varying size and chemical diversity, including furan (8a,m),
thiophene (8b,n), imidazole (8c,o), cyclopropyl (8d,p), and
bicyclo[2.2.1]heptane (8e,q) heterocycles, naphthyl, and
phenyl; these were chosen for their small size and rich
electronic character, instead of the cyclohexylphenyl moiety of
1 (Table 1). The R3 position was left either as a mesityl or
pentafluorobenzyl substituent. The tolyl group of 2 was not
retained due to poor solubility.
The library was screened through a previously reported high-
throughput STAT5B FP assay which measures the disruption
of phosphopeptide−STAT5B SH2 domain interactions.15 FP
measurements were taken every 15 min for an hour against the
fluorescein conjugated phosphopeptide−STAT5B complex to
verify that the phosphopeptide probe (5-FAM-GpYLVLDKW)
was not being displaced over time.
Time intervals identified the optimal time point at which the
maximum change in fluorescence polarization was observed. In
general, the smaller heterocyclic R1 substituents had no
observable activity at inhibitor concentrations up to 100 μM.
In contrast, only aromatic derivatives 8j, 8l, 8v, and 8x
demonstrated similar potencies relative to the parent leads, with
Ki values of 6.62, 4.77, 12.36, and 11.51 μM, respectively
(Supporting Information Figure S4 and Table S1). We noted
that the electron rich, hydrophobic di-tert butyl benzyl groups
of 8l and 8x consistently engaged in cation−π interactions with
Figure 1. STAT5 inhibitors BP-1-108 (1) and SF-1-088 (2).
suppression of STAT5 Tyr phosphorylation (IC50 ∼ 20 μM)
and STAT5 target genes, MYC and CCND1, at μM
concentrations (∼40 μM).16 However, progress toward a
potent and selective nanomolar (nM) inhibitor of STAT5 has
been slow. Herein, we describe the identification of the first
nM, STAT5-selective inhibitor using in silico-directed efforts.17
Scaffolds 1 and 2 were selected as a starting point for
structural optimization. Interestingly, both leads possessed
opposing lipophilic trends for the substituents found at the R1
position. Increasing lipophilicity and bulk at R1 seems to favor
selectivity for the STAT3 protein. However, bulky hydrophobic
substituents on the R2 position seem to dictate the largest
selectivity for the STAT5 protein, suggesting structural
variability within the STAT3 and STAT5 SH2 domains.16 To
exploit these observed differences, we probed the SH2 domain
pocket using computational docking simulations.
While the activated STAT5−STAT5 dimer structure has yet
to be solved, the pY binding pocket of the SH2 domain was
identified by comparing and contrasting the structural
architectures and binding sites of 121 SH2 domain-containing
proteins.18 SH2 domains are defined by an antiparallel β-sheet
flanked by two α helices. The pY binds in a pocket located
within the βB, C, and D strands. Specifically, a conserved
R(XXX) residue on the βB strand participates in electrostatic
interactions with pY.19 Notably, STAT3′s structure was
resolved in both the unphosphorylated (PDB: 3CWB) and
phosphorylated (PDB: 1BG1) states.20,21 Superimposition of
structures revealed that STAT3′s SH2 domain structure is not
significantly altered upon phosphopeptide binding.22 Thus, we
reasoned that the unphosphorylated STAT5 structure (PDB:
1Y1U) is suitable for in silico-based drug design. Analogous to
the canonical pY-SH2 domain binding, the STAT5 pY likely
docks proximal to the conserved R618 (βB strand), making H-
bonding/electrostatic interactions with nearby polar residues,
K600 (αA), T628 (βC), and S622 (βB and βC) Figure 2A.
Next, using AutoDock4.2, we performed global searches of
the conformational space along with careful local searches to
derive the best conformational fit within the STAT5 SH2
domain. Docking simulations were carried out with a rigid
B
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ACS Medicinal Chemistry Letters
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Table 1. Focused Library of 24 Derivatives with Small
Heterocycles and Substituted Phenyl Groups at the R1
Position, with Corresponding Ki Values Determined through
FP
Figure 3. (A) 13a docked with STAT5b; (B) FP binding traces for
13a against STAT5b and STAT3 protein; (C) SPR curves for 13a
against STAT5b and STAT3 (5, 1.67, 0.56, 0.19 μM).
K644, more so than the corresponding heterocyclic derivatives.
The naphthyl derivatives, 8j and 8v, favored π−π stacking
interactions with W641 and not with K644 (Supporting
Information Figures S9−12). Introduction of small heterocycles
abolished activity. To investigate whether the N-boc R2
substituent of 2 might contribute to STAT5 selectivity, we
prepared a new series of tetrapodal inhibitors. Di-tert-butyl
benzyl and naphthyl substituents at R1 were retained for SAR
analysis. In silico experiments showed that the boc group in 2
interacted with residues L643, W631, and W641 (Figure 2B,
green region). Since this region contained predominantly
electron-rich aromatic residues (W), we hypothesized that
agents equipped with electron-deficient R2 aromatic groups
such as a p-chlorobenzyl may favor strong π−π stacking
interactions. Thus, a focused library 13a−d was prepared and
evaluated via FP against both STAT5 and STAT3 at 1−60,000
nM (Supporting Information Figure S5). Most encouragingly,
13a was found to potently disrupt phosphopeptide−STAT5B
interactions, Ki = 145 nM. Moreover, 13a was 1000-fold more
selective for STAT5B cf. STAT3, with a STAT3 Ki = 143 μM
(Figure 3B). In silico docking simulations revealed that 13a, via
the p-chlorobenzyl and pentafluorobenzene groups, made
favorable contacts with the two adjacent amphipathic pockets
(Figure 3A, and Figure S13).
120 representative kinases covering the diversity of the kinase
families (DiscoveRx). Ultrasensitive quantitative PCR (qPCR)
was used to measure levels of immobilized kinases after
treatment with 13a at 5 μM. Encouragingly, 13a showed
negligible effects against the bank of kinases and, more notably,
against upstream STAT5 activating kinases, JAK1/2, ABL, and
FLT-3 (Supporting Information Figure S1). These data suggest
that inhibition of pSTAT5 is due to interaction with STAT5′s
SH2 domain and not through inhibition of upstream kinases.
Next, 13a was assessed for whole cell potency against STAT5
transformed CML and AML cell lines, K562 (Bcr-Abl) and
MV-4;11 (FLT3-ITD), respectively. Cell viability was assessed
following treatment at various concentrations of inhibitor
(0.78−50 μM) using a CellTiter-Blue cell viability assay (72 h).
As compared to first generation STAT5 inhibitors (8j, 8l, 8v,
8x), IC50 values for 13a−d were 2−3-fold higher in potency,
with activities ranging from 3 to 20 μM (Supporting
Information Figures S14, S15). Encouragingly, 13a displayed
the most potent activity in FLT3-ITD driven MV4;11 cells,
IC50 = 3.5 μM. We next evaluated 13a-mediated inhibition of
STAT5 phosphorylation levels. K652 leukemia cells were
treated with 13a for 5 h, the cells were harvested, and the levels
of phosphorylated STAT5 (Y694) were determined (Figure
4A). 13a decreased pSTAT5 in a dose dependent manner and
ablated pSTAT5 above 10 μM with no change in the total
STAT5 concentration or cleavable PARP-1. However, K562
cells were found to undergo cell death (cleaved PARP-1,
Caspase-3) at 24 h at 15 μM, indicating that 13a might induce
apoptosis as a result of STAT5 inactivation (Figure 4B). To
investigate selectivity, MDA-MB-231 breast cancer cells, which
harbor high pSTAT3 and negligible pSTAT5 activity
(Supporting Information, Figure S18), were assessed for
differential pSTAT inhibition by 13a (Figure 4C). Encourag-
ingly, pSTAT3 was not inhibited at doses corresponding to
pSTAT5 inhibition within the leukemic cell line and neither
In addition to FP, surface plasmon resonance (SPR) binding
experiments were conducted to measure the kinetic association
and dissociation using a ProteOn XPR36 (Biorad) with full-
length His-tagged, STAT5, and STAT3 (SignalChem)
immobilized on a biosensor chip. Most encouragingly,
inhibitors exhibited potent nM binding affinities for STAT5,
and selectivity for the STAT5 cf. STAT3 (Supporting
Information Figure S2). Compound 13a exhibited the most
potent KD (koff /kon) of 42 4 nM, with 7-fold selectivity for
STAT5 cf. STAT3, KD = 287
29 nM (Figure 3C). As
assessed by FP and SPR, 13a represents the first nM STAT5-
binding, STAT5-selective inhibitor.
To determine the selectivity of 13a for STAT5, we screened
for off-target kinase activity, a possible alternative target for an
effector of STAT5 phosphorylation. 13a was assessed against
C
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while having no effect on pSTAT3. Thus, 13a represents a
useful probe for interrogating the STAT5 function in diseased
cells as well as being a candidate for advanced preclinical trials.
ASSOCIATED CONTENT
■
S
* Supporting Information
Inhibitor synthesis, characterization, and experimental proce-
dures for FP, SPR, cytotoxicity, and Western Blot studies. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: patrick.gunning@utoronto.ca.
Author Contributions
‡A.A.C., A.M.L., and M.G. contributed equally to this work.
Funding
Figure 4. (A) Inhibition of pSTAT5 with 13a (15 μM) in K562 cells;
(B) Initiation of apoptosis after 24 h and knockdown of MCL-1 (15
μM); (C) 13a shows no effect on pSTAT3 in MDA-MB-231 cells up
to 20 μM; (D) Knockdown of downstream target c-Myc after 5 h.
Financial support was provided by a Canada Research Chair
(PTG), an NSERC Discovery Grant (PTG), NIH
R21CA186945 (PTG, HLG), the Princess Margaret Founda-
tion (MDM), an Ontario Graduate Scholarship (AAC), an
NSERC Graduate Scholarship (CEB), and an NSERC USRA
(DBD).
total STAT3 or STAT5 protein levels were affected. It was
shown that 13a was 3-fold less cytotoxic in MDA-MB-231
(IC50 = 10 μM) than in the high pSTAT5 leukemic cell line
(Supporting Information Figure S16). Downstream of STAT5,
we assessed for modulation of the STAT5 transcriptional
targets, MCL-1, CYCLIN D1/D2, and MYC. We reasoned that
13a should decrease gene expression and induce apoptosis by
24 h. K562 cells were dosed with 13a at the same
concentrations observed for selective STAT5 inhibition. At 5
h, we observed dose-dependent decreases in MYC and
complete knockdown of MCL-1 observed at 24 h at 15 μM
(Figure 4D).
Notes
The authors declare no competing financial interest.
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