Signal Transducer and Activator of Transcription 6 (STAT6) is a pivotal transcription factor within the JAK-STAT signaling pathway, activated downstream of cytokines IL-4 and IL-13. Upon phosphorylation at Tyr641, STAT6 undergoes reciprocal SH2 domain–mediated dimerization, translocates to the nucleus, and initiates transcription of genes central to Th2-type immune responses. Aberrant STAT6 signaling is implicated in asthma, atopic dermatitis, and other allergic diseases, making it a highly attractive target for therapeutic modulation.

While upstream blockade (e.g., via IL-4Rα antagonists) has shown clinical benefit, direct targeting of STAT6 is emerging as a promising alternative. Recent efforts have focused on a range of STAT6-binding molecules, including small-molecule inhibitors, PROTAC degraders, and peptide mimetics, many of which aim to disrupt the SH2 domain interactions critical for dimerization and function.

STAT6 consists of 847 amino acids and features a modular domain architecture:

1. N-terminal domain (ND, residues 1–130): Supports cooperative DNA binding and tetramerization

2. Coiled-coil domain (CCD): Mediates protein-protein interactions and nuclear import

3. DNA-binding domain (DBD): Recognizes GAS elements via a helix-turn-helix motif

4. Linker domain: Allows conformational flexibility

5. SH2 domain: Binds phosphorylated Tyr641 and mediates dimerization

6. Transactivation domain (TAD): Recruits coactivators (e.g., CBP/p300), subject to post-translational modifications

STAT6-IN-3: A small-molecule inhibitor blocking STAT6 phosphorylation or DNA binding for Th2-driven diseases.

AK-1690: A highly selective STAT6 PROTAC degrader with a DC50 of 1 nM, developed by the University of Michigan/Shaomeng Wang group.

KT-621: An oral STAT6 PROTAC degrader with picomolar potency against IL-4/IL-13 signaling, developed by Kymera Therapeutics for atopic dermatitis and asthma.

I-2: A reference compound used as a control in SPR assays.

To compare the binding site preferences of different STAT6-binding compounds, a SPR-based competitive binding assay was employed. The assay workflow is designed to distinguish whether two molecules bind to the same or distinct sites on STAT6:

Injection 1: Saturate the immobilized STAT6 protein with Compound A; monitor response unit (RU) until plateau

Injection 2: Inject a mixture of Compound A + Compound B

Note on Assay Design: In the second injection of the SPR competitive binding assay, a mixture of Compound A and Compound B is used—rather than Compound B alone—to ensure that the binding site occupied by Compound A remains saturated throughout the test. This setup allows accurate assessment of whether Compound B can bind to an independent site on the target protein. If Compound B binds a different site, the RU signal increases. If the two compounds compete for the same site, no additional binding is detected. This approach ensures reliable differentiation of overlapping vs. non-overlapping binding sites.

AK1690 & STAT6-IN-3: No RU increase → AK1690 and STAT6-IN-3 share the same binding site.

STAT6-IN-3 & IL-4Rα Peptide: RU increases → STAT6-IN-3 and IL-4Rα bind to distinct sites.

Significant RU increase → I-2 binds to a different site than both STAT6-IN-3 and AK1690.

This study demonstrates how SPR-based competition assays can accurately map binding site relationships among STAT6 inhibitors, even across different modalities (small molecules, PROTACs, peptides).