Luciferase- and HaloTag-based reporter assays to measure small-molecule-induced degradation pathway in living cells

Stefan Knapp, Martin P. Schwalm, Krishna Saxena, Susanne Müller

Published: 2024-04-18 DOI: 10.1038/s41596-024-00979-z

NanoLuciferase

HaloTag

protein degradation

live-cell assay

small-molecule degraders

AI 解读

Extraction of nuclei from archived postmortem tissues for single-nucleus sequencing applications

High-quality and robust protein quantification in large clinical/pharmaceutical cohorts with IonStar proteomics investigation

Longitudinal interrogation of sympathetic neural circuits and hemodynamics in preclinical models

Experimental modulation of physiological force application on leg joint neurons in intactDrosophila melanogaster

Combining surface-accessible Ag and Au colloidal nanomaterials with SERS for in situ analysis of molecule–metal interactions in complex solution environments

Generation of mouse–human chimeric embryos

Labeling and tracking of immune cells in ex vivo human skin

Large-scale preparation of fluorescence multiplex host cell reactivation (FM-HCR) reporters

Mutate-and-chemical-shift-fingerprint (MCSF) to characterize excited states in RNA using NMR spectroscopy

Comprehensive multi-attribute method workflow for biotherapeutic characterization and current good manufacturing practices testing

查看全部

Sections

Figures

References

Introduction
Materials
Procedure
Troubleshooting
Timing
Anticipated results
Acknowledgements

Tolvanen, T. A. Current advances in CETSA. Front. Mol. Biosci. 9, 866764 (2022).
Schwalm, M. P. & Knapp, S. BET bromodomain inhibitors. Curr. Opin. Chem. Biol. 68, 102148 (2022).
Hu, Z. & Crews, C. M. Recent developments in PROTAC-mediated protein degradation: From bench to clinic. Chembiochem 23, e202100270 (2022).
Petrylak, D. P. et al. First-in-human phase I study of ARV-110, an androgen receptor (AR) PROTAC degrader in patients (pts) with metastatic castrate-resistant prostate cancer (mCRPC) following enzalutamide (ENZ) and/or abiraterone (ABI). J. Clin. Oncol. 38, 3500–3500 (2020).
Paiva, S. L. & Crews, C. M. Targeted protein degradation: elements of PROTAC design. Curr. Opin. Chem. Biol. 50, 111–119 (2019).
Nemec, V., Schwalm, M. P., Muller, S. & Knapp, S. PROTAC degraders as chemical probes for studying target biology and target validation. Chem. Soc. Rev. 51, 7971–7993 (2022).
Roy, M. J. et al. SPR-measured dissociation kinetics of PROTAC ternary complexes influence target degradation rate. ACS Chem. Biol. 14, 361–368 (2019).
Casement, R., Bond, A., Craigon, C. & Ciulli, A. Mechanistic and structural features of PROTAC ternary complexes. Methods Mol. Biol. 2365, 79–113 (2021).
Li, K. & Crews, C. M. PROTACs: past, present and future. Chem. Soc. Rev. 51, 5214–5236 (2022).
Gadd, M. S. et al. Structural basis of PROTAC cooperative recognition for selective protein degradation. Nat. Chem. Biol. 13, 514–521 (2017).
Yamamoto, J., Ito, T., Yamaguchi, Y. & Handa, H. Discovery of CRBN as a target of thalidomide: a breakthrough for progress in the development of protein degraders. Chem. Soc. Rev. 51, 6234–6250 (2022).
Moehler, T. M., Hillengass, J., Glasmacher, A. & Goldschmidt, H. Thalidomide in multiple myeloma. Curr. Pharm. Biotechnol. 7, 431–440 (2006).
Teo, S. et al. Thalidomide in the treatment of leprosy. Microbes Infect. 4, 1193–1202 (2002).
Li, D. et al. Discovery of a dual WDR5 and Ikaros PROTAC degrader as an anti-cancer therapeutic. Oncogene 41, 3328–3340 (2022).
Domostegui, A., Nieto-Barrado, L., Perez-Lopez, C. & Mayor-Ruiz, C. Chasing molecular glue degraders: screening approaches. Chem. Soc. Rev. 51, 5498–5517 (2022).
Che, Y., Gilbert, A. M., Shanmugasundaram, V. & Noe, M. C. Inducing protein–protein interactions with molecular glues. Bioorg. Med. Chem. Lett. 28, 2585–2592 (2018).
Schwalm, M. P. et al. Tracking the PROTAC degradation pathway in living cells highlights the importance of ternary complex measurement for PROTAC optimization. Cell Chem. Biol. 30, 753–765.e8 (2023).
Mo, X. L. & Fu, H. BRET: NanoLuc-based bioluminescence resonance energy transfer platform to monitor protein–protein interactions in live cells. Methods Mol. Biol. 1439, 263–271 (2016).
Dixon, A. S. et al. NanoLuc complementation reporter optimized for accurate measurement of protein interactions in cells. ACS Chem. Biol. 11, 400–408 (2016).
Schwalm, M. P. et al. A toolbox for the generation of chemical probes for Baculovirus IAP repeat containing proteins. Front Cell. Dev. Biol 10, 886537 (2022).
Vasta, J. D. et al. Quantitative, wide-spectrum kinase profiling in live cells for assessing the effect of cellular ATP on target engagement. Cell Chem. Biol. 25, 206–214 e211 (2018).
Machleidt, T. et al. NanoBRET—a novel BRET platform for the analysis of protein–protein interactions. ACS Chem. Biol. 10, 1797–1804 (2015).
Riching, K. M. et al. Quantitative live-cell kinetic degradation and mechanistic profiling of PROTAC mode of action. ACS Chem. Biol. 13, 2758–2770 (2018).
Owens, D. D. G. et al. A chemical probe to modulate human GID4 Pro/N-degron interactions. Preprint at bioRxiv https://doi.org/10.1101/2023.01.17.524225 (2023).
Mortison, J. D. et al. Rapid evaluation of small molecule cellular target engagement with a luminescent thermal shift assay. ACS Med. Chem. Lett. 12, 1288–1294 (2021).
RA, M. S. et al. Development of the first covalent monopolar spindle kinase 1 (MPS1/TTK) inhibitor. J. Med. Chem. 65, 3173–3192 (2022).
Moon, S. B., Kim, D. Y., Ko, J. H. & Kim, Y. S. Recent advances in the CRISPR genome editing tool set. Exp. Mol. Med. 51, 1–11 (2019).
Degorce, F. et al. HTRF: a technology tailored for drug discovery - a review of theoretical aspects and recent applications. Curr. Chem. Genomics 3, 22–32 (2009).
Shyu, Y. J., Suarez, C. D. & Hu, C. D. Visualization of ternary complexes in living cells by using a BiFC-based FRET assay. Nat. Protoc. 3, 1693–1702 (2008).
Bielefeld-Sevigny, M. AlphaLISA immunoassay platform—the ‘no-wash’ high-throughput alternative to ELISA. Assay Drug Dev. Technol. 7, 90–92 (2009).
Kowarz, E., Loscher, D. & Marschalek, R. Optimized sleeping beauty transposons rapidly generate stable transgenic cell lines. Biotechnol J 10, 647–653 (2015).
Spitzer, J., Landthaler, M. & Tuschl, T. Rapid creation of stable mammalian cell lines for regulated expression of proteins using the Gateway(R) recombination cloning technology and Flp-In T-REx(R) lines. Methods Enzymol. 529, 99–124 (2013).
Chen, Y. H. et al. Rapid lentiviral vector producer cell line generation using a single DNA construct. Mol. Ther. Methods Clin. Dev. 19, 47–57 (2020).
Khan, S. H. Genome-editing technologies: concept, pros, and cons of various genome-editing techniques and bioethical concerns for clinical application. Mol. Ther. Nucleic Acids 16, 326–334 (2019).
Uhlen, M. et al. Proteomics. Tissue-based map of the human proteome. Science 347, 1260419 (2015).
Smith, J. D. et al. Quantitative CRISPR interference screens in yeast identify chemical–genetic interactions and new rules for guide RNA design. Genome Biol. 17, 45 (2016).
Doudna, J. A. & Charpentier, E. Genome editing. The new frontier of genome engineering with CRISPR–Cas9. Science 346, 1258096 (2014).
Desjardins, P. & Conklin, D. NanoDrop microvolume quantitation of nucleic acids. J. Vis. Exp. https://doi.org/10.3791/2565 (2010).
Cheng, Y. & Prusoff, W. H. Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 22, 3099–3108 (1973).

Luciferase- and HaloTag-based reporter assays to measure small-molecule-induced degradation pathway in living cells

推荐阅读