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Inhibitors of Stat5 protein signalling

Abbarna A. Cumaraswamy,a Aleksandra Todic,a Diana Resetca,a Mark D. Mindenb and Patrick T. Gunning*a

Received 7th July 2011, Accepted 25th August 2011

DOI: 10.1039/c1md00175b

Evidence shows that signal transducer and activator of transcription 5 (Stat5) protein, a member of the

STAT family of signalling proteins, plays a pivotal role in the progression of many human cancers

including acute myeloid leukemias and prostate cancer. This mini-review outlines progress made

towards identifying agents capable of silencing aberrant Stat5 signalling.

Introduction

Signal transducer and activator of transcription 5 (Stat5) protein,

a member of the STAT family of signalling proteins, plays

a crucial transcriptional role in numerous human cancers and has

become a target for therapeutic intervention. Due to growth

factor receptor mutations, amplified expression or misdirected

kinase activity in the cytoplasm, constitutively activated Stat5 is

present in a diverse number of human cancers, including those of

the breast, prostate, liver, skin, blood, head and neck.1 The

STAT family of proteins is comprised of seven members

including Stat1, Stat2, Stat3, Stat4, Stat5A and 5B, as well as

Stat6. These proteins perform dual roles as both cytosolic sig-

nalling proteins and as nuclear transcription factors that mediate

the expression of specific sets of genes. In particular, expression

of genes involved in proliferation (Bcl-xl, c-Myc, pim-1),

apoptosis (JAB), cell differentiation (p21),2 and inflammation is

mediated. In cancer cells, STAT proteins are routinely hyper-

activated through either somatic mutation or dysregulated

expression of upstream signalling pathways. As a result, several

members of the STAT family, including Stat5, can act as onco-

genes by facilitating the aberrant expression of key proteins

associated with the cancer phenotype. This article will review the

progress made towards the development of Stat5-targeting

molecular therapeutics.

Stat5 proteins are activated/phosphorylated by kinases asso-

ciated with transmembrane receptors, including IL-2, GM-CSF,

erythropoietin, IL-5, IL-7, thrombopoietin, prolactin, and

growth hormone receptors.1 Extracellular ligand binding to these

target receptors induces receptor dimerization and intracellular

activation of receptor-associated kinases, such as JAK. JAK-

mediated phosphorylation of key tyrosine (Y) residues on the

cytoplasmic receptor domain creates docking sites for non-

phosphorylated Stat5 proteins. Stat5 binds to the receptor

aDepartment of Chemistry, University of Toronto, 3359 Mississauga RoadNorth, Mississauga, ON, L5L 1C6, Canada. E-mail: patrick.gunning@utoronto.ca; Tel: +905-828-5354bPrincess Margaret Hospital, Ontario Cancer Institute, 610 UniversityAvenue, Toronto, ON, M5G 2M9, Canada. E-mail: mark.minden@uhn.on.ca; Fax: +416-946-6546; Tel: +416.946.4501

22 | Med. Chem. Commun., 2012, 3, 22–27

through its phosphotyrosine binding, Src Homology 2 (SH2)

domain. Once recruited, Stat5 is phosphorylated by receptor

associated JAK kinases, such as Jak2, which phosphorylate Stat5

proteins at specific Y residues in the C-terminus (Y694 for Stat5a

and Y699 for Stat5b). Phosphorylated Stat5 protein dissociates

from the receptor and forms either homo- or hetero- Stat5–StatX

dimers, through reciprocal SH2 domain-pY interactions. Acti-

vated Stat5 translocates to the nucleus and binds to Stat5

response elements and induces target gene transcription.

In normal cells, Stat5 proteins are transiently activated by

extracellular cytokine or ligand-receptor engagement and rapidly

deactivated by a number of intracellular mechanisms, including

cytosolic and nuclear phosphatases. For example, deactivation

of Stat5 can occur directly via phosphatases such as protein

inhibitors of aberrant STAT proteins (PIAS) or protein tyrosine

phosphatases such as SHP-2, or indirectly by down-regulation of

cytokine signalling with SOCS1, SOCS3 and CIS proteins.1,3

However, in cancer cells Stat5 is constitutively phosphorylated,

leading to elevated expression levels of its target genes, such as

the anti-apoptotic proteins Bcl-xl, Bcl-2, Myc and MCL. Aber-

rant Stat5 activity has been associated with hyperactivated

upstream cytosolic tyrosine kinases including TEL-Jak2 and Bcr-

Abl, mutated FMS-like tyrosine kinase 3 (FLT3), as well as

overactive receptor tyrosine kinases, such as SRC and EGFR.4

Medicinal chemists have attempted to silence aberrant Stat5

activity through a number of direct and indirect molecular

approaches. These therapeutic efforts have included the targeting

of upstream effector proteins or the direct targeting of Stat5

protein. This review will explore the progress made towards

developing effective inhibitors of Stat5 function in human

cancers.

Indirect inhibitors of Stat5 signalling

Stat5’s crucial role in cellular signalling and gene transcription is

mediated by an intricate cascade of protein–protein interactions.

Consequently, there are several junctures at which inhibitors may

disrupt the Stat5 signalling pathway. Notably, most inhibitors of

Stat5 function have not directly targeted Stat5 protein

complexation events, but rather focussed on inhibiting upstream

This journal is ª The Royal Society of Chemistry 2012

Fig. 1 Jak2/Stat5 signalling pathway.

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tyrosine kinases including Bcr/Abl, FLT3, and Jak. In addition

to indirect kinase inhibitors, Stat5 function has been inhibited by

suppression of proteases responsible for protein terminal

truncation.

Suppressing Stat5 function through Bcr/Abl inhibitors

Bcr/Abl is a novel oncogenic protein that arises in cells as a result

of a chromosomal translocation event. This chimeric protein

drives constitutive Abl kinase signalling within the cytoplasm of

the cell. Aberrant Bcr/Abl signalling leads to constitutive Stat5

activation and subsequent dysregulation of downstream targets.5

Bcr/Abl is the primary genetic abnormality leading to Chronic

Myelogenous Leukemia (CML).6 The breakthrough small

molecule inhibitor of Bcr/Abl, imatinib or Gleevec (1, Fig. 2),

was first approved by the FDA in 2001 as a highly selective

inhibitor that provided rapid reduction in the CML clone,

recovery of normal hematopoiesis, and improved survival.

Consequently the drug quickly became the primary agent for

treatment of CML. Imatinib is a highly specific inhibitor of the

Abl tyrosine kinase (Abl, IC50 ¼ 25 nM; cf. c-Src, IC5 > 100

mM),7 and potently inhibits downstream phosphorylation of

Stat5.8 However, acquired imatinib resistance through develop-

ment of novel Bcr/Abl mutations has highlighted the need for

next-generation inhibitors with activity against imatinib resistant

cancers. A promising candidate, PD180970 (2, Fig. 2), contain-

ing a pyridopyrimidinone core is a highly potent and selective

Abl kinase inhibitor that induces apoptosis in imatinib-resistant

leukemia cells. PD180970 has been shown to potently inhibit

Stat5–Stat5 DNA-binding activity with an IC50 of 5 nM.9

Suppressing Stat5 function through FLT3 inhibitors

In addition to Bcr/Abl targeting, Stat5 signalling has been

inhibited through inhibition of the FLT3 receptor tyrosine

Fig. 2 Chemical structures of Bcr/Abl inhibitors.

This journal is ª The Royal Society of Chemistry 2012

kinase. FLT3 is highly expressed in lymphoid and myeloid

progenitor cells.10 Approximately one third of all AML patients

harbour constitutive FLT3 activity due to the development of

internal tandem duplication (ITD) mutations. This dysregulation

of FLT3 leads to aberrant activation of numerous signalling

cascades, including Jak2/Stat5, PI3K/PTEN/Akt/mTOR, and

RAS/Raf/MEK/ERK resulting in the up-regulation of Stat5

transcriptional targets such as Bcl-xl, c-Myc, pim-1, JAB and

p-21.11 Thus, constitutively activated FLT3 increases cancer cell

survival and apoptotic resistance.12 Gain-of-function point

mutations, which are associated with hyperactivation of

Stat5 and increased growth of lethal myeloproliferative disease

cell clones, have been identified in different FLT3 receptor

domains.13 To deal with this, a number of small-molecule FLT3

inhibitors have been developed and advanced to clinical trials

as potential treatments for AML patients. For example, the

indolocarbazole alkaloid, lestaurtinib (3, Fig. 3), also known to

be a potent Jak2 inhibitor, is an orally bioavailable inhibitor

of FLT3 phosphorylation (IC50 � 1.5–3.0 nM). As a result,

lestaurtinib has been shown to inhibit FLT3 proliferation in

MV4-11 leukemia cells and in turn inhibit Stat5 phosphorylation

(IC50 ¼ 2.5–5 nM).14

Multi-kinase inhibitor, sorafenib (4, Fig. 3), has also been

shown to indirectly inhibit Stat5 function. This biaryl urea-based

compound inhibited both FLT3-ITD and wild-type FLT3

phosphorylation in MV4-11 and EOL-1 leukemia cell lines and,

in turn, potently inhibited cell proliferation with IC50 values of

0.87 nM and 2.3 nM, respectively.15 Moreover, sorafenib

produced complete remissions in AML patients, alone or in

combination with other drugs, and is currently being evaluated

for safety and efficacy.16 In addition to sorafenib, multi-kinase

inhibitor, KW-2449 (5, Fig. 3), suppressed FLT3 phosphoryla-

tion and subsequent Stat5 phosphorylation inMolM-14 cell lines

(IC50 values of 13.1 nM and 14.8 nM, respectively).

AC220 (6, Fig. 3), a second-generation FLT3 inhibitor, has

been shown to more potently and selectively inhibit cell proli-

feration in MV4-11 cells (IC50 ¼ 0.56 nM) than first-generation

FLT3 inhibitors, such as 3 and 4.17 In addition, ponatinib (7,

Fig. 3), a potent Bcr-Abl inhibitor from ARIAD Pharmaceuti-

cals (IC50 ¼ 0.5–36 nM), has demonstrated comparable inhibi-

tion against FLT3 in MV4-11 cells (IC50 ¼ 0.3–17 nM) and

subsequent inhibition of Stat5 phosphorylation.18

Despite the identification of chemical compounds with excel-

lent FLT3 inhibitory potency in vitro, clinical trials of these

Fig. 3 Chemical structures of FLT3 Inhibitors.

Med. Chem. Commun., 2012, 3, 22–27 | 23

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inhibitors demonstrated only moderate inhibition and therefore

have not yet resulted in a commercially available FLT3 inhibitor

drug. Three possible explanations have been proposed. Firstly, it

has been shown that FLT3 mutations are not fundamentally

important to the development of AML. Thus, inhibition of

FLT3-ITD mutations are simply selecting for AML subclones

that are less dependent on complex signalling via the bone

marrow stroma.17 Secondly, Sato et al. have recently suggested

that the significant up-regulation of the FLT3 ligand, observed in

patients previously treated with chemotherapy, could drastically

impair the efficacy of FLT3 inhibitors in vivo.19 Finally, limita-

tions in vivomay be due to poor pharmacokinetic profiles of these

drugs. Despite major successes in effectively inhibiting FLT3, the

rapid evolution of resistance to this class of drugs in AML

patients remains an ongoing concern.

Suppressing Stat5 function through Jak2 inhibitors

In addition to FLT3 and Bcr/Abl, Jak2 kinase activates Stat5

protein and has been shown to be the most frequently activated

oncogenic protein in myeloproliferative neoplasms.20 Somatic

mutations in the JAK2 allele can render Jak2 constitutively active

and contribute to a number of myeloproliferative disorders.

Critically, up-regulation of Jak2 results in constitutively active

Stat5 signalling, leading to increased Stat5 transcriptional

activity and over-expression of different pro-survival factors.

A commonly observed somatic mutation in the JAK2 loci in

cancer cells results from a G to T transversion at position 1849,

substituting Phe617 with a Val (Jak2-V617F).21 Jak2 has a multi-

domain structure containing two Jak homology domains (JH),

JH1 and JH2. The C-terminal JH1 domain is a highly conserved

kinase domain and is responsible for Jak2 kinase activity. JH2 is

a pseudokinase domain lacking ATP binding activity and is

responsible for inhibition of the JH1 domain. Gnanasambandan

et al. hypothesised that the V617F mutation resulted in a more

energetically favourable structural conformation, mediated by

a p–p stacking interaction between Phe617 and Phe595. Thus,

constitutive Jak2-V617F activity has been attributed to a stabi-

lized aberrant structure, which reduces the auto-inhibitory

function of the JH2 domain on the JH1 kinase domain.

A number of small-molecule inhibitors have been identified

that selectively modulate the activity of receptor-associated

kinases, like Jak2, and thus inhibit Stat5 protein function

Fig. 4 Chemical structures of Jak2 inhibitors.

24 | Med. Chem. Commun., 2012, 3, 22–27

indirectly. Compounds identified to date have exhibited

encouraging oral bioavailability and have progressed to various

advanced stages of preclinical and clinical development. Quintas-

Cardama and coworkers identified compound 8 (Fig. 4) as

a potent and orally bioavailable Jak1/2 inhibitor with increased

apoptosis in patients with the Jak2-V617F mutation. Preclinical

evaluation in Jak2-V615F+ Ba/F3 cells showed inhibition of Jak1

and Jak2 phosphorylation (IC50 ¼ 126 nM).22 This effective

pan-Jak kinase activity resulted in the inhibition of Stat5 phos-

phorylation (IC50 > 100 nM) and, subsequently, Stat5’s down-

stream transcriptional activity.

CYT387 (9, Fig. 4), an ATP mimetic, has exhibited promising

activity in preclinical studies, inhibiting Jak2 activity in

Jak2V615F+ Ba/F3 cells (IC50 ¼ 1.5 mM).23,24 In addition to

CYT387, TG101348 (10, Fig. 4) has been shown to be a selective

Jak2 inhibitor (IC50 ¼ 3 nM) with a 334 fold selectivity over

Jak3.25 As mentioned previously, compound 3, an ATP mimetic,

and inhibitor of FLT3, potently inhibits both Jak2 and Jak3 in

AML cells thereby silencing Stat5 activity.14

However, while potent small molecule inhibitors of upstream

activators of Stat5 have been forthcoming, such inhibitors have

a number of disadvantages as indirect inhibitors of Stat5 activity.

First, kinase inhibitors often lack target specificity. For example,

compounds targeting Jak2 often exhibit pan-JAK activity. Jak2

inhibitors 8 and 9, both potently inhibit Jak1 kinase activity with

IC50 values of 3.3 nM and 11 nM, respectively. In addition to

pan-JAK kinase activity, JAK inhibitors have exhibited signifi-

cant off-target effects against other kinase families. As previously

mentioned, compound 3 also acts upon FLT3 (IC50 ¼ 2–3 nM).

Off-target reactivity and kinase promiscuity remains a significant

limitation, often resulting in variable clinical outcomes. More-

over, the majority of small-molecule Jak2 inhibitors are not

selective for Jak2 mutants (Jak2-V617F). However, evidence

suggests that malignant cells have increased sensitivity to Jak2

inhibitors, thus deriving a therapeutic window to target cancer

cells selectively. Nevertheless, the lack of selectivity makes these

molecules potent immune-modulators that are generally both

pro-inflammatory and immunosuppressive.26 While Stat5 and

other STAT family proteins are principle targets of Jak2, acti-

vated Jak2 phosphorylates other non-STAT targets feeding into

additional pathways.27 These arguments suggest that achieving

selectivity for Stat5 over other targets is difficult when using

small molecule-based inhibitors to indirectly inhibit Stat5

downstream signalling. Furthermore, Stat5 mutations have been

identified in leukemia Balb/c mouse models that render Stat5

constitutively active, independent of the upstream Jak2 signal-

ling pathway.28 Indirect inhibition of Stat5 by targeting Jak2

accelerates the evolution of mutations and drug resistance,

maintaining the activity of other Jak2-regulated signalling

pathways.

Suppressing Stat5 function through truncation inhibitors

In both healthy and cancerous cells, there naturally exists

a proportion of terminally truncated Stat5 proteins. Although

the concentration of these variants is not sufficient in healthy

cells to exhibit a dominant negative effect over wildtype Stat5,

dominant negative effects have been observed in various cancers,

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including prostate cancer cells (CWR22Rv1, DU145, PC-3,

LNCaP)29 and AML patient samples.30

One of the most commonly over-expressed Stat5 variants,

particularly in prostate cancers, is the N-terminally truncated

Stat5 protein.29 In healthy cells, PIAS3 protein serves to down-

regulate wildtype Stat5 by interacting with the N-terminal

domain within the nucleus and thereby inhibiting DNA binding

(Fig. 1). N-terminal truncation therefore provides a mechanism

by which Stat5 evades PIAS3 interaction and continues to bind

DNA, leading to metastasis, tumour growth, and an increased

histological grade of the cancer. Inhibiting the generation of

these variants could therefore restore PIAS3 inhibition and

provide a potential therapy for these dominant negative effects.

Presently, the proteases responsible for N-terminal truncation

have not been effectively identified and purified in order for the

development of small-molecule inhibitors. However, these

proteases responsible for truncation could provide a novel and

selective therapeutic target for Stat5.

In addition to N-terminal truncation, the C-terminal truncated

Stat5 protein is a commonly occurring variant of Stat5. These 77

or 80 kDa variants typically lack their transactivation domain

(TAD, aa750–772), which is necessary for interaction with co-

activators and gene transcription. The heterodimer formed

between these variants and wildtype Stat5 binds DNA with more

stability than a wildtype homodimer and thereby suppresses the

expression of genes normally regulated by Stat5.31 In healthy

mammary cells, this truncation has been shown to reduce cell

proliferation rates, increase apoptosis by three-fold, and decrease

b-lactoglobulin/luciferase activity.32 Presently, cathepsin G has

been identified as being responsible for this truncation in myeloid

cells (32D, FDC-P1).33 As with the N-terminally truncated Stat5

variants, targeting this protease may provide a mechanism for

inhibiting variant activity in cancerous cells as well as reducing

dominant negative effects in healthy cells. However, recent

evidence suggests that these C-terminally truncated proteins are

in fact artificially generated during sample preparation and do

not occur naturally in vivo.34 The physiological significance of C-

terminal truncation is therefore brought into question. Genera-

tion of N-terminally truncated Stat5, on the other hand, was

shown to occur prior to sample preparation and therefore these

variants do occur in nature.29 Thus, Stat5 truncation at the N-

terminus seems to be a more promising therapeutic target than

C-terminal truncated isoforms, and may provide a more selective

indirect inhibitory method than kinase targeting.

Fig. 5 Schematic of oligonucleotide inhibitors of Stat5.

Direct inhibitors of Stat5 signalling

A significant drawback associated with indirect STAT inhibition

strategies has been the large potential for off-target effects and

subsequent increases in toxicity. This lack of selectivity is often

responsible for poor clinical development of indirect inhibitors,

such as kinase inhibitors. Therefore, it is therapeutically

appealing to target Stat5 protein directly using small-molecule

inhibitors. Presently, two different strategies have been employed

to inhibit Stat5 directly: (A) direct inhibition of Stat5 binding to

DNA and (B) inhibiting Stat5–Stat5 protein–protein interactions

via disruption of the reciprocal phosphotyrosine-SH2 domain

interaction.

This journal is ª The Royal Society of Chemistry 2012

(A) Direct inhibition of Stat5 binding to DNA. Oligonucleo-

tide-based strategies have been investigated for the inhibition of

Stat5–Stat5:DNA binding activity. These can be classified into

two categories: firstly, approaches that target Stat5 protein

expression and, secondly, those that interfere with binding to

nuclear target sequences. Conventional RNAi and antisense

oligodeoxynucleotide (ODN)-based approaches have shown

effective down-regulation of Stat5 protein expression in primary

and tumour cell lines, both in vitro and in vivo.35 Because of the

96% sequence similarity between Stat5a and Stat5b isoforms,36

a single RNAi or ODN-based strategy can be implemented to

inhibit both proteins, but may consequently result in decreased

selectivity or efficacy. For instance, transfection with a short

interfering RNA (siRNA) directed against both Stat5 isoforms

has been shown to effectively knock-down Stat5 protein

expression.37 However, off-target effects have also been shown.38

To improve selective targeting of Stat5, short hairpin RNA

(shRNA) (Fig. 5), which is more stable than siRNA, can be

delivered using a transfected plasmid or viral gene delivery

vehicle before ultimately being processed into siRNA.39 On the

other hand, approaches based on the delivery of antisense ODNs

(Fig. 5) directed against Stat5 isoforms have been shown to be

more sequence specific,40 but require a two-fold larger molar

excess than the corresponding siRNA to achieve effective knock-

down.37

An alternative strategy for inhibiting Stat5 activity, which has

been successfully implemented with many other transcription

factors,40,41 is based on delivering decoy ODNs that mimic

Stat5’s cis-elements on DNA. Decoy ODNs occupy the DNA

binding site and sequester activated Stat5 proteins in the cyto-

plasm, thereby reducing their ability to bind to nuclear tran-

scriptional targets and ultimately inhibiting transcription of

target genes. This approach holds promise for faster kinetics of

Stat5 inhibition than strategies targeting Stat5 isoforms, which

rely on the cellular turn-over rates of the Stat5a and Stat5b

proteins. According to Behbod et al., these turnover rates could

be as little as 6 h, for Stat5a, or as much as 48 h, for Stat5b. While

decoy ODNs provide an effective approach for the inhibition of

transcriptional activity, the delivery of these ODNs by trans-

fection or other methods limits their sub-cellular compartmen-

talization, potentially restricting the inhibition of activated Stat5

in the cytoplasm.42

(B) Disrupting dimerization. The formation of transcrip-

tionally active Stat5 dimers via reciprocal SH2-pTyr interactions

is an important step in the Stat5 signalling pathway. Therefore,

the development of small molecule compounds that bind to the

Med. Chem. Commun., 2012, 3, 22–27 | 25

Fig. 6 Direct inhibitors of Stat5 dimerization.

Fig. 7 Chemical structure of pimozide.

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SH2 domain and inhibit phosphorylation and/or prevent/disrupt

Stat5–Stat5 protein complexation events represent an appealing

molecularly targeted approach. For Stat3, rational drug design

approaches have been aided by the Stat3–Stat3:DNA crystal

structure determination. However, the activated Stat5 crystal

structure has not yet been solved. Nonetheless, progress towards

effective targeting of Stat5’s SH2 domain using small-molecule

inhibitors has been made. Using a high-throughput fluorescence

polarization assay developed by Berg and coworkers, the same

investigators have discovered a series of chromone-derived acyl

hydrazone inhibitors of which N0-((4-Oxo-4H-chromen-3-yl)

methylene) nicotinohydrazide (11, Fig. 6) demonstrated the most

potent and selective inhibition of Stat5 (Stat5, IC50 ¼ 47 mM cf.

Stat3, IC50 ¼ 180 mM and Stat1, IC50 ¼ 130 mM). Treatment of

lymphoma cells (Daudi) with 11 (100–200 mM) inhibited Stat5–

Stat5 dimerization and suppressed Stat5 DNA binding activity.43

Similarly, Gunning and co-workers conducted an in vitro

screen of a focussed library of SH2 domain binding salicylic acid-

containing inhibitors against Stat5, Stat3 and Stat1 for SH2

domain selectivity. Several potent leads were identified with Ki <

10 mM.Most notably, 12 (Fig. 6), 13 (Fig. 6) and 14 (Fig. 6) (Ki¼7.9 mM, Ki ¼ 2.8 mM and Ki ¼ 8.3mM, respectively) exhibited a

3-fold selectivity for Stat5 over Stat3 and Stat1. Of these Stat5

inhibitors, 12 exhibited the highest Stat5 selectivity (Stat5 Ki ¼7.9 mM cf. Stat1, >25 mM and Stat3 >25 mM). When evaluated in

K562 and MV4-11 leukemia cells, these lead agents demon-

strated potent and selective suppression of Stat5 phosphoryla-

tion, inhibition of Stat5’s target genes and induction of apoptosis

(IC50 � 20mM). Compound 12, in particular, showed negligible

cytotoxic effects against healthy bone marrow cells, which do not

harbour constitutively activated Stat5. The compounds identified

in this study represent the most potent, non-phosphorylated

direct small molecule inhibitors of Stat5 to date.44

Finally, a function-based screening approach was used to

discover the drug pimozide (15, Fig. 7), which was found to

significantly inhibit Stat5 gene expression. Further studies in

KU812 and K562 CML cells revealed that both cell lines

exhibited dose-dependent decreases in Stat5 tyrosine phosphory-

lation at 3–10mM doses of 15. Moreover, treatment of CML cells

26 | Med. Chem. Commun., 2012, 3, 22–27

with 15 (10 mM) resulted in decreased viability and induction of

apoptosis. Encouragingly, 15 showed no toxicity against healthy

cells at similar concentrations. Notably, Nelson and co-workers

demonstrated that 15 did not directly inhibit kinases such as Bcr-

Abl or its downstream pathway (MAPK) or Jak2 kinase. Rather,

15 is proposed to interact with Stat5 protein directly. Nelson and

co-workers showed that 15, in combination with imatinib (1), is

effective against Bcr-Abl mutant cancer cells.49

Conclusions

Significant effort has been exerted for the targeting of upstream

effectors of Stat5 function, including, Bcr-Abl, Jak2 and FLT-3.

Most notably, imatinib, the potent Bcr/Abl targeting drug, has

achieved considerable success in the clinic. In general, however,

kinase inhibitors upstream of Stat5 have suffered from the

development of resistance, toxicity as a result of poor kinase

selectivity, as well as cardiovascular toxicity. In an effort to

overcome these issues, downstream targeting of Stat5 protein

and Stat5-DNA binding has been attempted through application

of oligonucleotide-based therapeutics, various truncated Stat5

proteins and small molecule inhibitors. However, to date, the

number of direct inhibitors of Stat5 protein has been limited to

only a handful of examples. Given the recent successes of small

molecule therapeutic approaches against Stat3 complexation

events,45–48 there is much scope for developing improved agents

against Stat5 directly. The prospect for a Stat5-selective thera-

peutic for treatment of leukemias and prostate cancer is highly

appealing.

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