Novel multiplexed assay for identifying SH2 domain antagonists of STAT family proteins.

Publisher: Public Library of Science Country of Publication: United States NLM ID: 101285081 Publication Model: eCollection Cited Medium: Internet ISSN: 1932-6203 (Electronic) Linking ISSN: 19326203 NLM ISO Abbreviation: PLoS One Subsets: MEDLINE

Original Publication: San Francisco, CA : Public Library of Science

src Homology Domains*; Biological Assay/*methods ; STAT3 Transcription Factor/*antagonists & inhibitors ; STAT3 Transcription Factor/*chemistry ; STAT5 Transcription Factor/*antagonists & inhibitors ; STAT5 Transcription Factor/*chemistry; Amino Acid Sequence ; Cell Nucleus/metabolism ; Digoxigenin/metabolism ; HeLa Cells ; Humans ; Molecular Sequence Data ; Peptides/chemistry ; Peptides/metabolism ; Protein Binding ; Protein Transport ; Recombinant Proteins/metabolism ; STAT3 Transcription Factor/metabolism ; STAT5 Transcription Factor/metabolism ; Structure-Activity Relationship

Some of the signal transducer and activator of transcription (STAT) family members are constitutively activated in a wide variety of human tumors. The activity of STAT depends on their Src homology 2 (SH2) domain-mediated binding to sequences containing phosphorylated tyrosine. Thus, antagonizing this binding is a feasible approach to inhibiting STAT activation. We have developed a novel multiplexed assay for STAT3- and STAT5b-SH2 binding, based on amplified luminescent proximity homogeneous assay (Alpha) technology. AlphaLISA and AlphaScreen beads were combined in a single-well assay, which allowed the binding of STAT3- and STAT5b-SH2 to phosphotyrosine peptides to be simultaneously monitored. Biotin-labeled recombinant human STAT proteins were obtained as N- and C-terminal deletion mutants. The spacer length of the DIG-labeled peptide, the reaction time, and the concentration of sodium chloride were optimized to establish a HTS system with Z' values of greater than 0.6 for both STAT3- and STAT5b-SH2 binding. We performed a HTS campaign for chemical libraries using this multiplexed assay and identified hit compounds. A 2-chloro-1,4-naphthalenedione derivative, Compound 1, preferentially inhibited STAT3-SH2 binding in vitro, and the nuclear translocation of STAT3 in HeLa cells. Initial structure activity relationship (SAR) studies using the multiplexed assay showed the 3-substituent effect on both the activity and selectivity of STAT3 and STAT5b inhibition. Therefore, this multiplexed assay is useful for not only searching for potential lead compounds but also obtaining SAR data for developing new STAT3/STAT5b inhibitors.

Cell Growth Differ. 2001 Jan;12(1):1-7. (PMID: 11205741)
J Biomol Screen. 2007 Jun;12(4):560-7. (PMID: 17478484)
Clin Cancer Res. 2013 Apr 15;19(8):1933-40. (PMID: 23406773)
J Biomol Screen. 2003 Jun;8(3):239-46. (PMID: 12857377)
Methods Mol Biol. 2012;795:191-202. (PMID: 21960224)
Assay Drug Dev Technol. 2010 Jun;8(3):367-79. (PMID: 20230302)
Biochemistry. 2010 Apr 20;49(15):3213-5. (PMID: 20232875)
Methods Mol Biol. 2012;827:253-70. (PMID: 22144280)
Assay Drug Dev Technol. 2007 Aug;5(4):501-13. (PMID: 17767418)
J Mammary Gland Biol Neoplasia. 2006 Jan;11(1):75-87. (PMID: 16947086)
ChemMedChem. 2011 Aug 1;6(8):1459-70. (PMID: 21618433)
Anal Biochem. 2004 Jul 1;330(1):114-8. (PMID: 15183768)
Cancer Res. 2006 Oct 1;66(19):9714-21. (PMID: 17018630)
Chembiochem. 2008 Mar 25;9(5):723-7. (PMID: 18247434)
J Invest Dermatol. 2011 Jan;131(1):108-17. (PMID: 20811392)
J Biomol Screen. 1999;4(2):67-73. (PMID: 10838414)
Chem Biol. 2003 Dec;10(12):1245-53. (PMID: 14700632)
J Biol Chem. 2001 Nov 30;276(48):45443-55. (PMID: 11579100)
Anal Biochem. 2008 Apr 15;375(2):249-54. (PMID: 18258175)
Proc Natl Acad Sci U S A. 2007 May 1;104(18):7391-6. (PMID: 17463090)
Nat Rev Cancer. 2007 Sep;7(9):673-83. (PMID: 17721432)
Cancer Res. 2003 Oct 15;63(20):6763-71. (PMID: 14583472)
Curr Drug Targets Immune Endocr Metabol Disord. 2005 Dec;5(4):449-63. (PMID: 16375697)
ACS Chem Biol. 2007 Dec 21;2(12):787-98. (PMID: 18154266)
Nat Rev Mol Cell Biol. 2002 Sep;3(9):651-62. (PMID: 12209125)
Biochem Biophys Res Commun. 2010 Mar 5;393(2):253-8. (PMID: 20117083)
Cancer Res. 2003 Dec 15;63(24):8757-62. (PMID: 14695191)
Annu Rev Biophys Biomol Struct. 1997;26:259-88. (PMID: 9241420)
Clin Cancer Res. 2002 Apr;8(4):945-54. (PMID: 11948098)
Proc Natl Acad Sci U S A. 2012 Sep 4;109(36):14592-7. (PMID: 22912405)
Biochem Biophys Res Commun. 2009 Mar 13;380(3):627-31. (PMID: 19285012)
Chem Biol. 2006 Nov;13(11):1235-42. (PMID: 17114005)
Oncogene. 2002 Apr 25;21(18):2846-53. (PMID: 11973644)
Bioorg Med Chem Lett. 2003 Feb 24;13(4):633-6. (PMID: 12639546)
FEBS Lett. 1996 Sep 30;394(2):221-6. (PMID: 8843168)
Mol Cell Biol. 1996 Apr;16(4):1622-31. (PMID: 8657137)
PLoS One. 2013;8(1):e54565. (PMID: 23382914)
Curr Cancer Drug Targets. 2006 Mar;6(2):107-21. (PMID: 16529541)
Nat Clin Pract Oncol. 2005 Jun;2(6):315-24. (PMID: 16264989)
Bioorg Med Chem Lett. 2002 Apr 22;12(8):1219-23. (PMID: 11934592)

0 (Peptides)
0 (Recombinant Proteins)
0 (STAT3 Transcription Factor)
0 (STAT3 protein, human)
0 (STAT5 Transcription Factor)
0 (STAT5B protein, human)
NQ1SX9LNAU (Digoxigenin)

Date Created: 20130827 Date Completed: 20140408 Latest Revision: 20211021

20250114

PMC3745430

10.1371/journal.pone.0071646

23977103