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Successful development of bifunctional small molecule Degraders (a general term encompassing PROTAC® molecules, Degronimids, SNIPERs and uSMITE® molecules) and molecular glues requires a robust screening cascade. While the same is true for any drug discovery/medicinal chemistry program, the distinct mode of action of Degraders compared to standard small molecule inhibitors, requires a set of assays that address a specific and differentiated set of questions.
Multiple orthogonal assays, exploring target engagement by the Degrader and ternary complex formation, target protein ubiquitination and degradation, as well as the downstream effect of protein knockdown, will maximize the chance of successful Degrader development. An example assay workflow that aims to address these different aspects of Degrader activity is shown below.
Target Engagement
A starting point of bifunctional Degrader design is the identification of a suitable ‘exit vector’ on a ‘warhead’ ligand for your target protein of interest (POI) and E3 ligase ligand. This exit vector is the point of attachment for the linker group and should accommodate linker attachment without significant loss of ligand-protein binding affinity.
Explore target engagement of candidate Degraders with FRET/FP Assays to establish whether your Degrader molecules retain binary binding affinity. Cell-based FRET/FP assays, can also provide information relating to cellular permeability.
Ternary Complex Formation and Ubiquitination Assays
The formation of a ternary complex between the POI, Degrader and E3 ligase is fundamental to the underlying Degrader mechanism of action. Ternary complex formation enables transfer of ubiquitin from the ligase to the POI. Bio-Techne-brand R&D SystemsTM provides a range of products to help you explore ternary complex formation including in vitro ubiquitination assays and assay components, antibodies and Tandem Ubiquitin Binding Entities (TUBEs).
Learn More About Ubiquitination-related Products from R&D Systems
Assays for Degradation
Is the protein of interest degraded in cells following Degrader treatment? The traditional method for answering this question is a western blot assay. This is despite well-known drawbacks associated with traditional western blotting, including poor throughput, reproducibility, and quantification. Instead, Simple Western™ is a high-throughput, fully automated, and fully quantitative capillary electrophoresis approach to western blotting that can be effectively applied to Degrader development workflows.
Products for Exploring Protein Degradation
Simple Western Assays for Targeted Protein Degradation
Simple Western Assays for Targeted Protein Degradation
The activity of small molecule Degraders can be readily profiled using high throughput Simple Western systems. The highly reproducible and quantitative results generated by Simple Western allow for easy determination of DC50 values (the concentration of Degrader that induces 50% degradation of the target protein) within a short time frame. Fully automated Simple Western assays allow separation and analysis of proteins by size (or charge) from 2 kDa to 440 kDa in as little as 3 hours. Western blotting antibodies may be used for detection, as well as any antibody in the large and expanding database of Simple Western validated antibodies.
Western Blot and Simple Western Validated Antibodies
Western Blot and Simple Western Validated Antibodies
Bio-Techne provides a wide range of primary antibodies validated for western blot and Simple Western assays. We also offer enzyme- and fluorochrome-conjugated secondary antibodies as well as antibodies for the most commonly used epitope tags for the detection of tagged proteins by Western blot. We offer a variety of size options, including bulk and sample sizes to ensure that you can find what you need for your research.
Targeted Protein Degradation Assay Controls
Targeted Protein Degradation Assay Controls
Controls should be included to assess whether target degradation and pharmacological effect are ‘on-mechanism’ i.e. mediated by the ubiquitin-proteasome system (UPS). This can be achieved via co-treatment with a proteasome inhibitor, such as Bortezomib (#7282), which should block Degrader-mediated target protein degradation. A useful additional control is co-treatment with a NEDD8-activating enzyme inhibitor, for example MLN 4924 (#6499) to confirm that an active E3 ligase is involved in degradation.
Downstream Pharmacology
Exploring the resulting phenotype following successful Degrader-mediated protein degradation enables an understanding of the biological role of the POI. A relevant question to ask is: “does degradation offer a more desirable or differentiated phenotype compared to inhibition?” Our downstream pharmacology assays provide researchers with methods to explore and understand the biological consequences of targeted protein degradation.
Products for Exploring Downstream Pharmacology
In Situ Hybridization with RNAscopeTM
In Situ Hybridization with RNAscopeTM
RNAscope in situ hybridization technology is a powerful tool that allows you to spatially map and quantify specific target mRNAs in intact cells and tissues. Detect up to 12 RNA targets simultaneously using the RNAscope HiPlex assay or combine the assay with IHC or IF on the same slide to investigate mRNA and protein co-expression. Catalog probes are available for over 23,000 targets in 140+ species and can also be made-to-order. Discover how your Degrader affects downstream gene expression using RNAscope.
Proteome ProfilerTM Antibody Arrays
Proteome ProfilerTM Antibody Arrays
Proteome Profiler Antibody Arrays are a simple, high throughput and cost-effective tool for early-stage analyte profiling. Our antibody arrays have superior specificity, low background noise and no cross-reactivity means that you can count on this assay for clear and consistent data. Use our antibody arrays as part of your PROTAC® assay cascade to explore the downstream consequences of Degrader-induced protein knockdown.
Related Resources for Targeted Protein Degradation Assays
Webinar: Advanced TPD Research with Automated Western Blotting
Webinar: Advanced TPD Research with Automated Western Blotting
Dr Gary Allenby, CSO at Aurelia Bioscience, shares how their research team uses Simple Western systems to rapidly and reproducibly quantify protein knockdown following treatment with Degraders.
Application Note: Automated Profiling of PROTAC®-induced Degradation
Application Note: Automated Profiling of PROTAC®-induced Degradation
Learn from Protein Simple how fully automated Simple Westerns can be used to profile degradation of cereblon neosubstrates using cereblon-recruiting Degraders and IMiDs.
PROTAC® is a registered trademark of Arvinas Operations, Inc., and is used under license.
