Src homology 2 (SH2) domains play a central role in signal transduction. Although many SH2 domains have been validated as drug targets, their structural similarity makes development of specific inhibitors difficult. The cancer‐relevant transcription factors STAT5a and STAT5b are particularly challenging small‐molecule targets because their SH2 domains are 93 % identical on the amino acid level. Here we present the natural product‐inspired development of the low‐nanomolar inhibitor Stafib‐1, as the first small molecule which inhibits the STAT5b SH2 domain (K_i=44 nM) with more than 50‐fold selectivity over STAT5a. The binding site of the core moiety of Stafib‐1 was validated by functional analysis of point mutants. A prodrug of Stafib‐1 was shown to inhibit STAT5b with high selectivity over STAT5a in tumor cells. Stafib‐1 provides the first demonstration that naturally occurring SH2 domains with more than 90 % sequence identity can be selectively targeted with small organic molecules.

We present the concept, synthesis, and kinetic characterization of PYRROC as the first functionalized cycloalkyne which cannot form isomers in the reaction with azides. In aqueous buffer, PYRROC displays unprecedented rate accelerations in SPAAC of three to four orders of magnitude, leading to rate constants exceeding 400 M⁻¹ s⁻¹.

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 (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.