Identification of Purine-Scaffold Small-Molecule Inhibitors of Stat3

pubs.acs.org/acsmedchemlett

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, 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 (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 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. KEYWORDS Stat3, small-molecule inhibitors, molecular modeling, 3D-QSAR pharmacophore model, purine scaffold, cell viability, antitumor cell effects

T

he signal transducer and activator of transcription (STAT) family of proteins are cytoplasmic transcription factors with important roles in mediating cellular responses, including cell growth and differentiation, and inflammation 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 therefore 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 homology (SH) 2 domain to the cognate receptor phosphotyrosine (pTyr) peptide motifs. The binding to membrane-bound receptors represents an initial key step for STAT phosphorylation 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 phosphorylated STAT monomers engage in dimerization through

r 2010 American Chemical Society

a reciprocal pTyr-SH2 domain interaction, creating transcriptionally active STAT:STAT dimers that in the nucleus regulate gene expression by binding to specific DNA-response elements in the promoters of target genes.1 Given its importance in STAT activation 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 dimerization-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-molecule inhibitors and genetic modulators4,19 provide the proof of concept for the potential therapeutic application of Stat3inhibitory drugs, no small-molecule Stat3-inhibitory agent has thus far advanced to the clinic. To expedite the identification of clinically useful Stat3 inhibitors, the leading Stat3 dimerization-disrupting small-molecules 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

79

DOI: 10.1021/ml100224d |ACS Med. Chem. Lett. 2011, 2, 79–84

pubs.acs.org/acsmedchemlett

cify the relative positions of the functional groups in space. Our pharmacophore model describes an essentially planar, hetero-trisubstituted scaffold decorated with optimal recognition 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 (KD500

147.8 ( 5.1

74.5 ( 8.4

65.1 ( 8.3

62.5 ( 3.4

S3I-V3-31

>500

>500

145.2 ( 4.4

175.7 ( 5.3

68.6 ( 1.8

100.2 ( 2.2

S3I-V3-32 S3I-V3-34

>500 >500

>500 >500

100.4 ( 3.2 66.1 ( 2.1

77.7 ( 2.7 77.2 ( 1.9

80.2 ( 5.1 65.3 ( 2.3

45.6 ( 3.1 41.1 ( 4.6

S3I-V4-01

>500

>500

82.4 ( 1.4

197.6 ( 2.1

75.4 ( 3.4

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 immunoblotting, 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, 905569-5425; e-mail, [email protected]. J.T.: tel, 407-2667031; fax ,407-384-2062; e-mail, [email protected].

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.).

REFERENCES (1) (2) Figure 3. Purine scaffolds selectively suppress viability of malignant cells that harbor persistently active Stat3. Human breast (MDA-MB-231), pancreatic (Panc-1), and prostate (DU145) cancer cells, the v-Src transformed (NIH3T3/v-Src) fibroblasts or their normal counterparts (NIH3T3), and the murine thymic stromal epithelial cells (TE-71) were treated or not treated with 30-500 μM of the indicated agents for 48 h and assessed for viability using CyQuant cell proliferation kit. Values, mean ( SD of 3 independent experiments each in triplicate.

(3) (4) (5)

modeling of purine scaffolds as a novel class of Stat3 inhibitors, and which are the first of their kind. Novel purine-scaffold small-molecules bind to Stat3, presumably the SH2 domain, and selectively inhibit Stat3 activation in vitro, thereby suppressing the viability of tumor cells that are dependent on oncogenic Stat3 signaling. Although the present purinescaffold inhibitors add to the list of Stat3 dimerization

r 2010 American Chemical Society

(6)

(7)

83

Darnell, J. E., Jr. Transcription factors as targets for cancer therapy. Nat. Rev. Cancer 2002, 2, 740–749. Turkson, J.; Jove, R. STAT proteins: novel molecular targets for cancer drug discovery. Oncogene 2000, 19, 6613–6626. Turkson, J. STAT proteins as novel targets for cancer drug discovery. Expert Opin. Ther. Targets 2004, 8, 409–422. Yue, P.; Turkson, J. Targeting STAT3 in cancer: how successful are we? Expert Opin. Invest. Drugs 2009, 18, 45–56. Turkson, J.; Kim, J. S.; Zhang, S.; Yuan, J.; Huang, M.; Glenn, M.; Haura, E.; Sebti, S.; Hamilton, A. D.; Jove, R. Novel peptidomimetic inhibitors of signal transducer and activator of transcription 3 dimerization and biological activity. Mol. Cancer Ther. 2004, 3, 261–269. Turkson, J.; Ryan, D.; Kim, J. S.; Zhang, Y.; Chen, Z.; Haura, E.; Laudano, A.; Sebti, S.; Hamilton, A. D.; Jove, R. Phosphotyrosyl peptides block Stat3-mediated DNA-binding activity, gene regulation and cell transformation. J. Biol. Chem. 2001, 276, 45443–45455. Siddiquee, K.; Zhang, S.; Guida, W. C.; Blaskovich, M. A.; Greedy, B.; Lawrence, H. R.; Yip, M. L.; Jove, R.; McLaughlin,

DOI: 10.1021/ml100224d |ACS Med. Chem. Lett. 2011, 2, 79–84

pubs.acs.org/acsmedchemlett

(8)

(9)

(10)

(11)

(12)

(13)

(14)

(15)

(16)

(17)

(18)

(19)

(20)

(21)

(22)

M. M.; Lawrence, N. J.; Sebti, S. M.; Turkson, J. Selective chemical probe inhibitor of Stat3, identified through structure-based virtual screening, induces antitumor activity. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 7391–7396. Siddiquee, K. A. Z.; Gunning, P. T.; Glenn, M.; Katt, W. P.; Zhang, S.; Schroeck, C.; Sebti, S. M.; Jove, R.; Hamilton, A. D.; Turkson, J. An Oxazole-Based Small-Molecule Stat3 Inhibitor Modulates Stat3 Stability and Processing and Induces Antitumor Cell Effects. ACS Chem. Biol. 2007, 2, 787–798. Chen, J.; Bai, L.; Bernard, D.; Nikolovska-Coleska, Z.; Gomez, C.; Zhang, J.; Yi, H.; Wang, S. Structure-Based Design of Conformationally Constrained, Cell-Permeable STAT3 Inhibitors. ACS Med. Chem. Lett. 2010, 1, 85–89. Lin, L.; Hutzen, B.; Zuo, M.; Ball, S.; Deangelis, S.; Foust, E.; Pandit, B.; Ihnat, M. A.; Shenoy, S. S.; Kulp, S.; Li, P. K.; Li, C.; Fuchs, J.; Lin, J. Novel STAT3 phosphorylation inhibitors exhibit potent growth-suppressive activity in pancreatic and breast cancer cells. Cancer Res. 2010, 70, 2445–2454. Bhasin, D.; Cisek, K.; Pandharkar, T.; Regan, N.; Li, C.; Pandit, B.; Lin, J.; Li, P. K. Design, synthesis, and studies of small molecule STAT3 inhibitors. Bioorg. Med. Chem. Lett. 2008, 18, 391–395. Song, H.; Wang, R.; Wang, S.; Lin, J. A low-molecular-weight compound discovered through virtual database screening inhibits Stat3 function in breast cancer cells. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 4700–4705. Schust, J.; Sperl, B.; Hollis, A.; Mayer, T. U.; Berg, T. Stattic: a small-molecule inhibitor of STAT3 activation and dimerization. Chem. Biol. 2006, 13, 1235–1242. Ren, Z.; Cabell, L. A.; Schaefer, T. S.; McMurray, J. S. Identification of a high-affinity phosphopeptide inhibitor of stat3. Bioorg. Med. Chem. Lett. 2003, 13, 633–636. McMurray, J. S.; Mandal, P. K.; Liao, W. S.; Ren, Z.; Chen, X. Inhibition of Stat3 by cell-permeable peptidomimetic prodrugs targeted to its SH2 domain. Adv. Exp. Med. Biol. 2009, 611, 545–546. Zhang, X.; Yue, P.; Fletcher, S.; Zhao, W.; Gunning, P. T.; Turkson, J. A novel small-molecule disrupts Stat3 SH2 domain-phosphotyrosine interactions and Stat3-dependent tumor processes. Biochem. Pharmacol. 2010, 79, 1398–1409. Gunning, P. T.; Glenn, M. P.; Siddiquee, K. A.; Katt, W. P.; Masson, E.; Sebti, S. M.; Turkson, J.; Hamilton, A. D. Targeting protein-protein interactions: suppression of Stat3 dimerization with rationally designed small-molecule, nonpeptidic SH2 domain binders. ChemBioChem 2008, 9, 2800–2803. Gunning, P. T.; Katt, W. P.; Glenn, M.; Siddiquee, K.; Kim, J. S.; Jove, R.; Sebti, S. M.; Turkson, J.; Hamilton, A. D. Isoform selective inhibition of STAT1 or STAT3 homo-dimerization via peptidomimetic probes: structural recognition of STAT SH2 domains. Bioorg. Med. Chem. Lett. 2007, 17, 1875–1878. Turkson, J.; Zhang, S.; Palmer, J.; Kay, H.; Stanko, J.; Mora, L. B.; Sebti, S.; Yu, H.; Jove, R. Inhibition of constitutive signal transducer and activator of transcription 3 activation by novel platinum complexes with potent anti-tumor activity. Mol. Cancer Ther. 2004, 3, 1533–1542. Becker, S.; Groner, B.; Muller, C. W. Three-dimensional structure of the Stat3beta homodimer bound to DNA. Nature 1998, 394, 145–151. Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor, R. Development and validation of a genetic algorithm for flexible docking. J. Mol. Biol. 1997, 267, 727–748. Fletcher, S.; Turkson, J.; Gunning, P. T. Molecular approaches towards the inhibition of the signal transducer and activator

r 2010 American Chemical Society

(23)

(24) (25)

(26)

(27)

(28)

(29)

84

of transcription 3 (Stat3) protein. ChemMedChem 2008, 3, 1159–1168. Fletcher, S.; Shahani, V. M.; Gunning, P. T. Facile and efficient access to 2,6,9-tri-substituted purines through sequential N9, N2 Mitsunobu reactions. Tetrahedron Lett. 2009, 50 (29), 4258–4261. Fletcher, S.; Shahani, V. M.; Lough, A. J.; Gunning, P. T. Tetrahedron 2010, DOI: 10.1016/j.tet.2010.03.118. Turkson, J.; Zhang, S.; Mora, L. B.; Burns, A.; Sebti, S.; Jove, R. A novel platinum compound inhibits constitutive Stat3 signaling and induces cell cycle arrest and apoptosis of malignant cells. J. Biol. Chem. 2005, 280, 32979–32988. Coleman, D. R.; Ren, Z.; Mandal, P. K.; Cameron, A. G.; Dyer, G. A.; Muranjan, S.; Campbell, M.; Chen, X.; McMurray, J. S. Investigation of the binding determinants of phosphopeptides targeted to the SRC homology 2 domain of the signal transducer and activator of transcription 3. Development of a high-affinity peptide inhibitor. J. Med. Chem. 2005, 48, 6661– 6670. Mandal, P. K.; Limbrick, D.; Coleman, D. R.; Dyer, G. A.; Ren, Z.; Birtwistle, J. S.; Xiong, C.; Chen, X.; Briggs, J. M.; McMurray, J. S. Conformationally constrained peptidomimetic inhibitors of signal transducer and activator of transcription 3: evaluation and molecular modeling. J. Med. Chem. 2009, 52, 2429–2442. Galmarini, C. M.; Mackey, J. R.; Dumontet, C. Nucleoside analogues and nucleobases in cancer treatment. Lancet Oncol. 2002, 3, 415–424. Parker, W. B.; Secrist, J. A., 3rd; Waud, W. R. Purine nucleoside antimetabolites in development for the treatment of cancer. Curr. Opin. Invest. Drugs 2004, 5, 592–596.

DOI: 10.1021/ml100224d |ACS Med. Chem. Lett. 2011, 2, 79–84