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...
moreThe 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 (K i = 44 nM) to Stafib-2 (K i = 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. Transcription factors orchestrate cellular signalling by regulating transcription of their target genes, thus allowing precise regulation of cellular phenotype 1. They do not possess enzymatic activities, making their functional manipulation with cell-permeable small molecules more challenging. The transcription factors STAT5a and STAT5b in particular are highly homologous 2 and are frequently referred to jointly as " STAT5 " , implying that they carry out identical functions. However, while some protein functions are indeed redundant, others are not. For example, although both STAT5a and STAT5b are constitutively activated in numerous human cancers, including human leukaemias harbouring the Philadelphia chromosome 3 which leads to expression of the Bcr-Abl fusion protein, the inhibition of STAT5b was shown to reduce tumour cell proliferation more than the inhibition of STAT5a did 4, 5. Small-molecule inhibitors which differentiate between the two STAT5 proteins would be highly beneficial for clarifying their individual roles. The most effective and selective approach by which to inhibit STAT proteins involves functional inhibition of the protein-protein interaction domain, the Src homology 2 (SH2) domain 6, 7. However, for most STAT5 inhibitors developed to date, including chromone-based compounds 8, 9 , fos-fosal 10 , salicylic acid-based STAT5 inhibitors 11, 12 , an adenosine-5′-monophosphate derivative 13 , and an osmium complex 14 , selectivity for one STAT5 protein over the other was either minimal or not reported. We recently presented catechol bisphosphate (1, Fig. 1a) and its derivatives Stafib-1 (2, Table 1) 15 and Capstafin 16 as selective inhibitors of the STAT5b SH2 domain. 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. The isolated STAT SH2 domains are mostly insoluble; virtually all research studies to date have used STAT constructs containing multiple STAT domains 17, 18. The molecular weight of the proteins expressed by these constructs is too high for deducing detailed structural information of the STAT-ligand complex by solution-state NMR methods. We have employed solid-state NMR spectroscopy to verify and characterize the binding of catechol bisphos-phate to STAT5b, an interaction which provides the foundation of our docking-based model for the binding of Stafib-1 to STAT5b 15. Following structure-guided optimization of Stafib-1, we performed a comparative analysis
