STAT family proteins are important mediators of cell signaling and represent therapeutic targets for the treatment of human diseases. Most STAT inhibitors target the protein–protein interaction domain, the SH2 domain, but specificity for a single STAT protein is often limited. Recently, we developed catechol bisphosphates as the first inhibitors of STAT5b demonstrated to exhibit a high degree of selectivity over the close homologue STAT5a. Here, we show that the amino acid in position 566 of the linker domain, not the SH2 domain, is the main determinant of specificity. Arg566 in wild-type STAT5b favors tight binding of catechol bisphosphates, while Trp566 in wild-type STAT5a does not. Amino acid 566 also determines the affinity for a tyrosine-phosphorylated peptide derived from the EPO receptor for STAT5a and STAT5b, demonstrating the functional relevance of the STAT5 linker domain for the adjacent SH2 domain. These results provide the first demonstration that a residue in the linker domain can determine the affinity of nonpeptidic small-molecule inhibitors for the SH2 domain of STAT proteins. We propose targeting the interface between the SH2 domain and linker domain as a novel design approach for the development of potent and selective STAT inhibitors. In addition, our data suggest that the linker domain could contribute to the enigmatically divergent biological functions of the two STAT5 proteins.

Fosfosal is the O‐phosphorylated derivative of salicylic acid, with documented clinical use as a prodrug for the treatment of inflammatory diseases. We recently discovered that fosfosal itself inhibits the protein‐protein interaction domain, the SH2 domain, of the tumor‐related transcription factor STAT5b. Here, we demonstrate that fosfosal is selective for STAT5b over its close homologue STAT5a. This selectivity is mediated by the STAT5b residue Arg566, located in the SH2 domain‐adjacent linker domain. Our data provide further evidence for the role of the STAT linker domain in determining the activity of small molecules against the SH2 domain. We present a refined binding model for fosfosal and STAT5b, which can serve as the basis for the development of fosfosal‐based STAT5b inhibitors.

The transcription factors STAT5a and STAT5b are constitutively active in many human tumors. Combined inhibition of both STAT5 proteins is a valuable approach with promising applications in tumor biology. We recently reported resorcinol bisphosphate as a moderately active inhibitor of the proteinprotein interaction domains, the SH2 domains, of both STAT5a and STAT5b. Here, we describe the development of resorcinol bisphosphate to Stafiba, a phosphatase-stable inhibitor of STAT5a and STAT5b with activity in the low micromolar concentration range. Our data provide insights into the structure-activity relationships of resorcinol bisphosphates and the corresponding bisphosphonates for use as inhibitors of both STAT5a and STAT5b.