Abstract
The transcription factor STAT5b is a target for tumour therapy. We recently reported catechol bisphosphate and derivatives such as Stafib-1 as the first selective inhibitors of the STAT5b SH2 domain. Here, we demonstrate STAT5b binding of catechol bisphosphate by solid-state nuclear magnetic resonance, and report on rational optimization of Stafib-1 (Ki = 44 nM) to Stafib-2 (Ki = 9 nM). The binding site of Stafib-2 was validated using combined isothermal titration calorimetry (ITC) and protein point mutant analysis, representing the first time that functional comparison of wild-type versus mutant protein by ITC has been used to characterize the binding site of a small-molecule ligand of a STAT protein with amino acid resolution. The prodrug Pomstafib-2 selectively inhibits tyrosine phosphorylation of STAT5b in human leukaemia cells and induces apoptosis in a STAT5-dependent manner. We propose Pomstafib-2, which currently represents the most active, selective inhibitor of STAT5b activation available, as a chemical tool for addressing the fundamental question of which roles the different STAT5 proteins play in various cell processes.
Highlights
Transcription factors orchestrate cellular signalling by regulating transcription of their target genes, allowing precise regulation of cellular phenotype[1]
Since binding of 1 to STAT5b Arg[618] in the Src homology 2 (SH2) domain had been indicated in a fluorescence polarization (FP)-based assay[15, 19], we decided to investigate the mode of STAT5b-binding of 1 using solid-state NMR spectroscopy
The use of solid-state NMR for the characterization of binding between a small molecule and an SH2 domain has not previously been reported. 13C direct polarization (DP) / magic-angle spinning (MAS) spectra of 13C6-1 revealed that the three 13C resonances of 13C6-1 split into sets of two in the presence of STAT5b (Fig. 1b)
Summary
Transcription factors orchestrate cellular signalling by regulating transcription of their target genes, allowing precise regulation of cellular phenotype[1]. They do not possess enzymatic activities, making their functional manipulation with cell-permeable small molecules more challenging. Despite overwhelming evidence for the biological significance of STAT proteins as therapeutic targets, the development of small-molecule STAT inhibitors has been hampered by the absence of structural characterization of complexes between STATs and small molecules by X-ray crystallography or NMR spectroscopy. We have employed solid-state NMR spectroscopy to verify and characterize the binding of catechol bisphosphate to STAT5b, an interaction which provides the foundation of our docking-based model for the binding of Stafib-1 to STAT5b15. We present the design and binding mode validation of the optimized STAT5b inhibitor Stafib-2, and its prodrug Pomstafib-2, for use as a research tool in cell-based assays
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