Fig. 5: Results of screening the PhP library against the STAT5B N-terminal domain.
From: Mapping protein binding sites by photoreactive fragment pharmacophores
a Structures and predicted binding modes of PhP065 and PhP097 against the dimerization interfaces of the STAT5B N-terminal domain. PhP065 binds close to the cavity formed in the handshake dimer interface by H-bonds with the Q36 and K70 residues. PhP097 binds at the tip of the α6 and α7 helices next to the Ni2+-mediated tetramer interface with an H-bond to the T58 sidechain and further hydrophobic contacts. b In microscale thermophoresis, PhP065 and PhP097 bind directly to STAT5B-NTD, with Kd values of 2.58 and 18.06 µM, respectively (black curves). Furthermore, the fragments inhibit the viability of the established leukemia cell lines MV4-11 (red) and MOLM-13 (blue) with IC50 values of 76.15 and 100.1 µM (PhP065), and 56.62 and 68.22 µM (PhP097), respectively. c In our modified workflow, the iridium-based photocatalyst Ir-G2-PEG3-COOH is first attached to the target protein. After initial non-covalent binding, the crosslinking of the photoaffinity-labeled fragments is enhanced by Dexter-energy transfer from the photocatalyst. d Structure of the photocatalyst Ir-G2-PEG3-COOH41. e Application of the photocatalyst results in higher labeling efficiency and, by transition, a higher number of primary hits (>1% labeling). In the Kd plots, data are presented as mean values ± SD, calculated from two biologically independent samples, each with three repetitions. In the cellular IC50 plots, data are presented as mean values ± SEM, separately for two biologically independent samples, each with three repetitions. Source data are provided in the Source Data file.
