Signal Transducer and Activator of Transcription 6 (STAT6) is a pivotal transcription factor activated downstream of the interleukin-4 (IL-4) and interleukin-13 (IL-13) signaling pathways. It governs the expression of key genes involved in type 2 immune responses, including Th2 cell differentiation, IgE class switching, and M2 macrophage polarization. Dysregulated STAT6 activity has been implicated in a range of diseases, from asthma and atopic dermatitis to fibrosis and immune-evasive tumors.

With the clinical advancement of STAT6-targeting drugs like Kymera’s KT-621 and Recludix’s REX-8756, the demand for precise STAT6 binding profiling is rising—supporting target validation, mechanism studies, and therapeutic development.

We developed TR-FRET assays to evaluate compound binding to STAT6 and its disruption of STAT6–DNA interactions. To assess selectivity, we profiled compounds across the broader STAT family, including STAT1, STAT3, STAT4, and STAT5A/B. For degrader molecules, we also performed binary and ternary complex formation assays to quantify target–ligase engagement and cooperativity.

Figure 1.TR-FRET-based biochemical assays for STAT family profiling.

Figure 2. TR-FRET-based binary and ternary complex formation for STAT6 degraders.

To accurately characterize binding kinetics and selectivity of STAT6-targeting compounds, we employ both Surface Plasmon Resonance (SPR) and spectral shift–based assays. This dual-platform strategy allows us to balance sensitivity, throughput, and molecular mechanism insights, especially when dealing with weak-to-moderate SH2-domain interactions and structurally diverse chemotypes such as degraders or allosteric inhibitors.

To accurately characterize binding kinetics and selectivity of STAT6-targeting compounds, we employ both Surface Plasmon Resonance (SPR) and spectral shift–based assays. This dual-platform strategy allows us to balance sensitivity, throughput, and molecular mechanism insights, especially when dealing with weak-to-moderate SH2-domain interactions and structurally diverse chemotypes such as degraders or allosteric inhibitors.

For STAT6, SPR provides real-time, label-free kinetic profiling and is particularly well-suited for measuring binary binding affinity (KD) and association/dissociation rates (ka/kd). To support selectivity profiling, we conducted a STAT family SPR panel using compound SI-109, comparing its binding across STAT1–STAT6.

Figure 3. SPR binding analysis of SI-109 across STAT family members.

SpS (spectral shift)-based binding assays quantify interaction between CRBN, PROTACs (AK-1690, I-2), and STAT6. Ternary complex formation significantly enhances binding affinity compared to binary CRBN–PROTAC binding, with a 99-fold cooperativity for AK-1690 and 9.2-fold for I-2. Assay performed in 384-well format.

Figure 4. Sps-based binary and ternary complex formation assays for STAT6 degraders.

To confirm functional degradation of STAT6 inside cells, we employed a multi-assay validation strategy combining HiBiT-tag quantification, flow cytometry, and Western blotting. This integrated approach ensures high sensitivity, reproducibility, and orthogonal validation of degradation activity.

This is particularly critical for STAT6, where transcriptional feedback, protein turnover rate, and cell-type specificity can influence readouts. Multi-assay confirmation ensures observed degradation is real, robust, and mechanistically consistent.

Dose-dependent degradation of HiBiT-tagged STAT6 was observed in engineered HEK293 cells (left) and endogenous STAT6 degradation in Jurkat T cells (right) following compound treatment.

Figure 5. STAT6 degradation measured by HiBiT assay.

STAT6 protein levels were detected by flow cytometry following treatment with degrader I-2 at increasing concentrations.

Figure 6. Flow cytometry analysis of STAT6 levels in CD3⁺ T cells.

To independently validate STAT6 degradation and assess endogenous protein levels across cell types, we performed Western blot analysis in both cell lines (Jurkat, A549) and primary cells (PBMCs). As a gold-standard method, Western blot provides direct visualization of target protein abundance, supporting the results from HiBiT and flow cytometry with orthogonal evidence.

Figure 7. Western blot confirmation of STAT6 degradation in multiple cell types.

To fully understand the behavior of STAT6-targeting degraders, we performed two studies: Endogenous STAT6 protein turnover, to establish the baseline stability of the protein; Kinetic degradation profiling, to quantify how quickly and effectively the compound drives STAT6 degradation across doses.

STAT6 stability was measured in Jurkat cells following treatment with CHX (a translation inhibitor). Protein levels were monitored over time, and nonlinear regression analysis estimated a half-life of ~7.1 hours, indicating that STAT6 is a relatively stable transcription factor under basal conditions.

Using a STAT6-HiBiT knock-in Jurkat cell line, we quantified degradation over 72 hours following treatment with increasing concentrations of compound I-2. These data confirm that the compound accelerates STAT6 loss well beyond its natural turnover rate.

Figure 8. STAT6 endogenous half-life and degradation kinetics analysis.

Demonstrating target degradation is only part of the story—verifying downstream functional impact is critical to validating STAT6 as a therapeutic target. As a key transcription factor in the IL-4/IL-13 signaling axis, STAT6 drives expression of multiple disease-relevant genes in immune cells, including CCL17 (TARC) and CD23, and regulates its activity through phosphorylation-dependent nuclear translocation.

To evaluate whether compound-induced STAT6 degradation leads to functional suppression, we employed reporter gene assays, cytokine release assays by ELISA, CD23 expression and pSTAT6 detection by flow cytometry.

Figure 9. Reporter assay confirmation of STAT6 functional inhibition.

Figure 10. Functional inhibition of STAT6 signaling in primary immune cells.

Through a comprehensive, multi-platform approach, we demonstrated our ability to systematically evaluate STAT6-targeting compounds across biochemical, biophysical, cellular, and functional dimensions. As STAT6 continues to gain momentum as a therapeutic target in inflammation and oncology, we are ready to support your program at every stage—from target validation and SAR refinement, to mechanism-of-action studies and preclinical candidate selection.