Abstract
Assessing the specificity of PROTACs and confirming their proposed mechanism of action are critical for a robust targeted protein degradation program. Owing to their novel mechanism, new assays are needed to meet these goals. We and others have shown that a common explanation of PROTAC efficacy is the ability of the PROTAC to form a ternary complex between the E3 ubiquitin ligase and the target protein. In this chapter, we provide a simple in vitro method to quickly and inexpensively assess this property of PROTAC molecules. We provide detailed instructions for the purification of the specific E3 ubiquitin ligase VHL and then a generic protocol which can be adapted to any E3 ligase and substrate protein combination. This accessible method to study the ternary complex can strengthen any PROTAC-focused medicinal chemistry effort.
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Acknowledgements
We thank Craig M. Crews for mentorship and supervision of this assay design. DPB is supported by the National Cancer Institute fellowship F99/K00 CA212229. BES (T32 GM007753) and ADB (T32 GM008152) are supported by Medical Scientist Training Program grants from the National Institute of General Medical Sciences at their respective institutions.
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Bondeson, D.P., Smith, B.E., Buhimschi, A.D. (2021). An In Vitro Pull-down Assay of the E3 Ligase:PROTAC:Substrate Ternary Complex to Identify Effective PROTACs . In: Cacace, A.M., Hickey, C.M., Békés, M. (eds) Targeted Protein Degradation. Methods in Molecular Biology, vol 2365. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1665-9_7
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DOI: https://doi.org/10.1007/978-1-0716-1665-9_7
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