Abstract
Assessment of small molecules that promote selective protein degradation (degraders) requires detailed characterization and measurement of protein levels in cells. Here we describe ratio-metric methods based on a dual fluorescent GFP/mCherry reporter system to quantify cellular protein levels. We further develop a kinetic framework for the analysis of such data. We describe two methods of generating the stable GFP-protein of interest (POI)/mCherry reporter cell lines, alternative readout methods by FACS and Laser Scanning Cytometry as well as the corresponding tools used for processing and analysis of such data. Finally, we show that the commonly used half-maximal degradation constant (DC50) or maximum degradation efficacy (Dmax) metrics are time-dependent and propose a time-invariant Michaelis-Menten-like analysis of degradation kinetics with analogous key parameters Km app and Vmax app.
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Acknowledgments
The authors gratefully acknowledge the generous financial support of the following sources: NIH grant NCI R01CA214608 (grant to E. S. F). We would like to thank members of Fischer and Ebert Lab at Dana-Farber Cancer Institute for useful discussions on development and optimization of these protocols.
Conflict of Interest: E.S.F. is a founder, scientific advisory board (SAB), and equity holder in Civetta Therapeutics, Jengu Therapeutics (board member), and Neomorph, Inc. E.S.F. is an equity holder in C4 Therapeutics and a consultant to Novartis, Sanofi, EcoR1 capital, Deerfield and Astellas. The Fischer lab receives or has received research funding from Novartis, Ajax and Astellas.
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Nowak, R.P., Yue, H., Park, E.Y., Fischer, E.S. (2021). Methods for Quantitative Assessment of Protein Degradation. 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_13
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DOI: https://doi.org/10.1007/978-1-0716-1665-9_13
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