Biaryl Phosphates and Phosphonates as Selective Inhibitors of the Transcription Factor STAT4

Abstract

The transcription factor STAT4 has been implicated in the pathogenesis of autoimmune diseases, including inflammatory bowel disease, multiple sclerosis, rheumatoid arthritis, and diabetes mellitus. Here, we report p-biaryl phosphates and phosphonates as the first small-molecule inhibitors of STAT4. The most potent p-biaryl phosphate inhibited the protein-protein interaction domain of STAT4, the SH2 domain, with submicromolar potency (K_i = 0.35 µM) and 14-fold selectivity over the closely related family member STAT3, which has the same core peptide binding motif as STAT4. Further development resulted in the phosphatase-stable inhibitor Stafori-1, which protected STAT4 but not STAT3, against thermal denaturation in cell lysates. Its cell-permeable prodrug Pomstafori-1 selectively inhibited STAT4 phosphorylation in cultured human cells at low micromolar concentrations. Our data open up the possibility of exploring STAT4 as a target protein for small molecules in the treatment of unmet medical needs.

Keywords: Biological activity; Inhibitors; Protein–protein interactions; SH2 domains; Transcription factors.

Figure 1

a) Structure of para‐biphenyl phosphate (1) and b) its activity profile in FP assays against STAT proteins. Error bars represent standard deviations (n = 3).

Scheme 1

a) Synthesis of biaryl precursors 4c–m. Details of the Suzuki couplings are provided in the Supporting Information. Compounds 4a and 4b were obtained from commercially available 4′‐hydroxybiphenyl‐2‐carboxylic acid via esterification. b) Atherton‐Todd phosphorylation and debenzylation provide biaryl phosphates 6a–m.

Figure 2

a) Structure and activity profile of 6m in FP assays against STAT proteins. Error bars represent standard deviations (n = 3). b) Docking of 6m into the AlphaFold2^{[ 17} , ^{18 ]} model of the STAT4 SH2 domain using AutoDock FR.^{[ 19 ]} The side chains of Lys580, Arg584, Arg598, and Phe622 were defined as flexible. The figure was generated using PyMOL.^{[ 20 ]} c)–e) Representative ITC data for binding between 6m and c) wild‐type STAT4, d) STAT4 Lys580Ala, and e) STAT4 Arg598Ala (n = 4 for each). Error bars in c)–e) represent integration errors assigned by the data analysis software NITPIC for the depicted individual experiments.^{[ 21 ]}

Figure 3

a) Representative ITC data for binding between 8k and wild‐type STAT4 (n = 3). Error bars represent integration errors assigned by the data analysis software NITPIC for the depicted individual experiment.^{[ 21 ]} b) Activity profile of 8k in FP assays against STAT proteins. Error bars represent standard deviations (n = 3).

Figure 4

Thermal stability of STAT4 in lysates from NK‐92 cells in the presence of a) DMSO, b) 8a (100 µM), c) 8k (100 µM), as well as the peptide Ac‐GpYLPQNID at d) 100 µM and e) 500 µM. f) Quantitation of the STAT4 band intensities for repeated experiments as shown in a)–e) (n = 3 in each case). Error bars represent standard deviations. p‐values refer to Student's t‐test, two‐tailed, two‐sample equal variance. Uncropped blots are shown in Figure S7 in the Supporting Information.

Figure 5

a) Induction of STAT4 Tyr693 phosphorylation upon activation of the IL‐12 receptor. The graphic is modified from the literature.^{[ 26 ]} b) Structure of prodrugs 9 and 10. c) Inhibition of IL‐12‐induced STAT4 Tyr693 phosphorylation in NK‐92 cells by 9 but not 10. d) Quantitation of band intensity from repeat experiments (9, n = 4; 10, n = 2). Effect of 9 on e) STAT3 Tyr705 phosphorylation in HCC‐827 cells with f) quantitation of band intensity from repeat experiments (n = 3), and g) STAT5 phosphorylation of Tyr694 (STAT5a)/Tyr699 (STAT5b) in K562 cells with h) quantitation of band intensity from repeat experiments (n = 3). Error bars represent standard deviations. Uncropped blots are shown in Figure S8 in the Supporting Information.

Figure 6

Prodrug 9 inhibits IL‐12/IL‐18‐mediated STAT4 phosphorylation and IFN‐γ induction in NK‐92 cells. Uncropped blots are shown in Figure S9 in the Supporting Information.