Alexander E Yarawsky, Judith A Ronau, Tiffany A Thibaudeau, Aaron C Ehlinger, Gekleng Chhor, Suki M Hyman, Michelle A Estrada, Vladimir Stojkovic, Michael T DeLion, Anil Vasudevan, Justin M Reitsma, Scott E Warder, Lake N Paul
{"title":"A paradigm shift: analytical ultracentrifugation as a multi-attribute platform method in targeted protein degradation.","authors":"Alexander E Yarawsky, Judith A Ronau, Tiffany A Thibaudeau, Aaron C Ehlinger, Gekleng Chhor, Suki M Hyman, Michelle A Estrada, Vladimir Stojkovic, Michael T DeLion, Anil Vasudevan, Justin M Reitsma, Scott E Warder, Lake N Paul","doi":"10.1007/s00249-025-01735-1","DOIUrl":null,"url":null,"abstract":"<p><p>Targeted protein degradation (TPD) has garnered appreciable interest in drug discovery due to its unique mechanism of action - degradation of a target in an event-driven manner, instead of traditional occupancy-driven inhibitor-based therapies. This is achieved by employing mono- or hetero-bifunctional small molecules known as degraders to induce the proximity of two proteins: a target protein and an E3 ubiquitin ligase, ultimately resulting in clearance of the target protein by the cell's inherent degradation machinery. A critical step in this pathway is ternary complex formation (TCF) between the ligase, degrader molecule, and the target protein. Although a bevy of biochemical, biophysical, cellular and structural approaches have been used to characterize degrader-induced ternary complexes, several knowledge gaps remain, such as stoichiometry and how much ternary complex is formed in solution. Analytical ultracentrifugation (AUC) is a biophysical method that is uniquely suited to address these questions, yet to this point has been surprisingly overlooked as an ideal method to characterize degrader candidates. In this study, we leveraged sedimentation velocity AUC (SV-AUC) to profile the degrader-induced ternary complex formation between Bruton's tyrosine kinase (BTK) and Cereblon (CRBN), allowing for evaluation of multiple attributes including sample purity, percent ternary complex, binding and kinetic rate constants, and hydrodynamics. We show that sedimentation equilibrium AUC (SE-AUC) can further complement the SV-AUC data with accurate molecular weight estimates of the ternary complex to confirm stoichiometry. This work demonstrates that AUC can be used both as a highly informative platform method for rapid characterization of candidate degrader compounds and as a rigorous method for elucidating additional details of the system.</p>","PeriodicalId":548,"journal":{"name":"European Biophysics Journal","volume":" ","pages":""},"PeriodicalIF":2.2000,"publicationDate":"2025-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"European Biophysics Journal","FirstCategoryId":"2","ListUrlMain":"https://doi.org/10.1007/s00249-025-01735-1","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOPHYSICS","Score":null,"Total":0}
A paradigm shift: analytical ultracentrifugation as a multi-attribute platform method in targeted protein degradation.
Targeted protein degradation (TPD) has garnered appreciable interest in drug discovery due to its unique mechanism of action - degradation of a target in an event-driven manner, instead of traditional occupancy-driven inhibitor-based therapies. This is achieved by employing mono- or hetero-bifunctional small molecules known as degraders to induce the proximity of two proteins: a target protein and an E3 ubiquitin ligase, ultimately resulting in clearance of the target protein by the cell's inherent degradation machinery. A critical step in this pathway is ternary complex formation (TCF) between the ligase, degrader molecule, and the target protein. Although a bevy of biochemical, biophysical, cellular and structural approaches have been used to characterize degrader-induced ternary complexes, several knowledge gaps remain, such as stoichiometry and how much ternary complex is formed in solution. Analytical ultracentrifugation (AUC) is a biophysical method that is uniquely suited to address these questions, yet to this point has been surprisingly overlooked as an ideal method to characterize degrader candidates. In this study, we leveraged sedimentation velocity AUC (SV-AUC) to profile the degrader-induced ternary complex formation between Bruton's tyrosine kinase (BTK) and Cereblon (CRBN), allowing for evaluation of multiple attributes including sample purity, percent ternary complex, binding and kinetic rate constants, and hydrodynamics. We show that sedimentation equilibrium AUC (SE-AUC) can further complement the SV-AUC data with accurate molecular weight estimates of the ternary complex to confirm stoichiometry. This work demonstrates that AUC can be used both as a highly informative platform method for rapid characterization of candidate degrader compounds and as a rigorous method for elucidating additional details of the system.
期刊介绍:
The journal publishes papers in the field of biophysics, which is defined as the study of biological phenomena by using physical methods and concepts. Original papers, reviews and Biophysics letters are published. The primary goal of this journal is to advance the understanding of biological structure and function by application of the principles of physical science, and by presenting the work in a biophysical context.
Papers employing a distinctively biophysical approach at all levels of biological organisation will be considered, as will both experimental and theoretical studies. The criteria for acceptance are scientific content, originality and relevance to biological systems of current interest and importance.
Principal areas of interest include:
- Structure and dynamics of biological macromolecules
- Membrane biophysics and ion channels
- Cell biophysics and organisation
- Macromolecular assemblies
- Biophysical methods and instrumentation
- Advanced microscopics
- System dynamics.