title: Novel multiplexed assay for identifying SH2 domain antagonists of STAT family proteins.
abstract: 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.
authors: Center for Drug Discovery, Graduate School of Pharmaceutical Sciences, University of Shizuoka, Suruga-ku, Shizuoka, Japan.
authors: Kazuyuki
authors: K
authors: Takakuma
authors: Naohisa
authors: N
authors: Ogo
authors: Yutaka
authors: Y
authors: Uehara
authors: Susumu
authors: S
authors: Takahashi
authors: Nao
authors: N
authors: Miyoshi
authors: Akira
authors: A
authors: Asai
mesh_terms: D000595:Amino Acid Sequence; D001681:Biological Assay / Q000379:methods*; D002467:Cell Nucleus / Q000378:metabolism; D004076:Digoxigenin / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D011994:Recombinant Proteins / Q000378:metabolism; D050796:STAT3 Transcription Factor / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D050799:STAT5 Transcription Factor / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D018909:src Homology Domains
chemical_list: D010455:Peptides; D011994:Recombinant Proteins; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D050799:STAT5 Transcription Factor; C494095:STAT5B protein, human; D004076:Digoxigenin
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