Biological Insights: Leveraging CETSA® to Understand Degrader Biology at Molecular and Proteomic Levels

The complexities of degrader discovery demand methods that go beyond traditional binding assays. CETSA® offers a label-free, physiologically relevant approach to uncovering degrader activity and mechanism, addressing critical challenges in drug discovery with precision and depth.

The introduction of targeted protein degraders has reshaped therapeutic strategies by offering a mechanism to selectively remove proteins involved in disease progression. These molecules circumvent traditional inhibition paradigms, introducing an additional layer of complexity into drug discovery workflows. Understanding whether a degrader engages its targets effectively, triggers degradation as intended, and is selective requires robust, high-resolution methods.

The Cellular Thermal Shift Assay (CETSA) has emerged as an indispensable tool in degrader research, providing direct, physiologically relevant data without requiring modifications to the degrader, its targets, or the assay matrix. By measuring thermal stability changes in endogenous proteins, CETSA enables researchers to link target engagement, degradation, and cellular response with unparalleled clarity.

Target Engagement in Live Cellular Systems

Degrader efficacy is contingent on its ability to engage both its protein of interest (POI) and an E3 ligase to form a functional ternary complex. CETSA assesses these interactions directly in live cells. Target engagement is quantified as a thermal stabilization or destabilization, with CETSA offering high-throughput scalability for rapid compound ranking based on binding affinity.

For example, in studies with cereblon (CRBN)-based PROTACs such as BSJ-03-204, CETSA confirmed selective engagement with both CRBN and the POI CDK4. The results demonstrated the target binding and revealed insights into optimal concentrations for effective ternary complex formation for degradation.

Correlating Degradation Efficiency with Engagement

CETSA does more than confirm target binding; it links this interaction in cells to functional degradation. By integrating assays for both target engagement and degradation within the same experimental framework, researchers can correlate binding potency with degradation kinetics.

For instance, in a time-course experiment, CETSA revealed that BSJ-03-204-mediated degradation of CDK4 in live cells peaked at specific concentrations, aligning with the stabilization of the ternary complex. Co-treatment with a proteasome inhibitor demonstrated that observed degradation was indeed proteasome-dependent, providing a definitive mechanistic link between target engagement and protein turnover.

Proteome-Wide Insights: Mechanism of Action and Off-Target Profiling

While targeted assays are critical for confirm target enagement, degraders can have effects that extend beyond their intended targets. Proteome-wide CETSA Explore provides a comprehensive view of these interactions. By quantifying the thermal stability of over 5,000 proteins, it captures both on- and off-target binding events and downstream effects triggered by protein degradation.

This approach was pivotal in assessing the broader impact of BSJ-03-204 in K562 cells, where CETSA identified significant changes in the thermal stability of transcriptional regulators and other proteins downstream of CDK4 degradation. These findings confirmed the degrader’s primary mechanism and highlighted secondary pathways that could be leveraged, or mitigated, in subsequent optimization steps.

Streamlining Degrader Optimization with High-Throughput Screening

The development of degraders is an iterative process that requires rapid feedback on compound modifications. CETSA offers a high-throughput solution, allowing multiple degrader variants to be screened simultaneously for binding and degradation profiles.

For example, a set of PROTACs targeting CDK4 was screened to compare their selectivity and efficacy. By simultaneously measuring thermal stabilization of CRBN and CDK4, and degradation of CDK4 in the same assay, CETSA revealed critical structure-activity relationships (SAR). This data informed the prioritization of compounds with optimal balance between target engagement and degradation efficiency.

Challenges in Targeting the “Undruggable”

CETSA excels where traditional methods falter, such as with targets lacking known binders or functional recombinant protein. For the notoriously challenging target p53, CETSA facilitated assay development directly in a cancer cell line, enabling the identification of both stabilizers and destabilizers. Subsequent high-throughput screening and hit validation yielded novel compounds with functional activity in just 12 weeks. This rapid turnaround exemplifies CETSA’s utility in expediting early discovery for complex targets.

A Platform for Precision Biology

The flexibility of CETSA extends its applicability across degrader discovery pipelines, from hit identification to preclinical validation. By providing quantitative, high-resolution data at every stage, CETSA allows researchers to:

• Validate target engagement and degradation under physiological conditions.
• Profile on- and off-target effects with proteome-wide coverage.
• Iteratively optimize degrader scaffolds with actionable SAR insights.

Degraders represent a leap forward in drug design, but their complexity demands equally advanced tools. CETSA meets this demand with a rigorous, science-driven approach, enabling researchers to untangle the intricacies of degrader biology and confidently make informed decisions.

Ready to deepen your understanding of degrader biology? Let’s explore how CETSA can provide actionable insights for your projects. You are warmly welcome to reach out to us. We are happy to discuss your research challenges and how we can tailor our workflows to your needs.

For additional information, please contact our representative in Japan, Stefan Sandström.