Small-molecule hydrophobic tagging–induced degradation of halotag fusion proteins

ABSTRACT The ability to regulate any protein of interest in living systems with small molecules remains a challenge. We hypothesized that appending a hydrophobic moiety to the surface of a

protein would mimic the partially denatured state of the protein, thus engaging the cellular quality control machinery to induce its proteasomal degradation. We designed and synthesized

bifunctional small molecules to bind a bacterial dehalogenase (the HaloTag protein) and present a hydrophobic group on its surface. Hydrophobic tagging of the HaloTag protein with an

adamantyl moiety induced the degradation of cytosolic, isoprenylated and transmembrane HaloTag fusion proteins in cell culture. We demonstrated the _in vivo_ utility of hydrophobic tagging

by degrading proteins expressed in zebrafish embryos and by inhibiting _Hras1_G12V-driven tumor progression in mice. Therefore, hydrophobic tagging of HaloTag fusion proteins affords

small-molecule control over any protein of interest, making it an ideal system for validating potential drug targets in disease models. Access through your institution Buy or subscribe This

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AND HALOTAG-BASED REPORTER ASSAYS TO MEASURE SMALL-MOLECULE-INDUCED DEGRADATION PATHWAY IN LIVING CELLS Article 18 April 2024 MECHANISM-BASED TRAPS ENABLE PROTEASE AND HYDROLASE SUBSTRATE

DISCOVERY Article Open access 16 February 2022 RAPID AND DIRECT CONTROL OF TARGET PROTEIN LEVELS WITH VHL-RECRUITING DTAG MOLECULES Article Open access 18 September 2020 REFERENCES *

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We wish to acknowledge financial support from the US National Institutes of Health (R01AI084140) and to thank the members of the Crews lab for critical reading of the manuscript. S. Stricker

at the Max Planck Institute for Molecular Genetics kindly provided Mouse _Ror2_, and R. Weinberg at the Massachusetts Institute of Technology (MIT) contributed Addgene plasmid 9051 from

which _Hras1_G12V was obtained. G. Daley at MIT kindly provided the retroviral pEYK3.1 vector. T.W.C. was the Canadian Institutes of Health Research Jean-François St-Denis Fellow in Cancer

Research and a Bisby Fellow. T.B.S. is a recipient of an American Cancer Society fellowship. AUTHOR INFORMATION Author notes * Timothy W Corson Present address: Current address: Department

of Ophthalmology, Indiana University School of Medicine, Indianapolis, Indiana, USA., * Taavi K Neklesa and Hyun Seop Tae: These authors contributed equally to this work. AUTHORS AND

AFFILIATIONS * Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA Taavi K Neklesa, Hyun Seop Tae, Michael J Stulberg, Timothy W Corson,

 Thomas B Sundberg, Scott A Holley & Craig M Crews * Department of Chemistry, Yale University, New Haven, Connecticut, USA Ashley R Schneekloth, Kanak Raina & Craig M Crews *

Department of Pharmacology, Yale University, New Haven, Connecticut, USA Craig M Crews Authors * Taavi K Neklesa View author publications You can also search for this author inPubMed Google

Scholar * Hyun Seop Tae View author publications You can also search for this author inPubMed Google Scholar * Ashley R Schneekloth View author publications You can also search for this

author inPubMed Google Scholar * Michael J Stulberg View author publications You can also search for this author inPubMed Google Scholar * Timothy W Corson View author publications You can

also search for this author inPubMed Google Scholar * Thomas B Sundberg View author publications You can also search for this author inPubMed Google Scholar * Kanak Raina View author

publications You can also search for this author inPubMed Google Scholar * Scott A Holley View author publications You can also search for this author inPubMed Google Scholar * Craig M Crews

View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS T.K.N., H.S.T., A.R.S. and C.M.C. designed the research. T.K.N., H.S.T., A.R.S., M.J.S.,

T.W.C., T.B.S. and K.R. performed the experiments. T.K.N., H.S.T., S.A.H. and C.M.C. analyzed the data. T.K.N., H.S.T. and C.M.C. wrote and edited the manuscript. CORRESPONDING AUTHOR

Correspondence to Craig M Crews. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. SUPPLEMENTARY INFORMATION SUPPLEMENTARY TEXT AND FIGURES

Supplementary Methods and Supplementary Results (PDF 4459 kb) RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Neklesa, T., Tae, H., Schneekloth, A. _et

al._ Small-molecule hydrophobic tagging–induced degradation of HaloTag fusion proteins. _Nat Chem Biol_ 7, 538–543 (2011). https://doi.org/10.1038/nchembio.597 Download citation * Received:

22 October 2010 * Accepted: 13 April 2011 * Published: 03 July 2011 * Issue Date: August 2011 * DOI: https://doi.org/10.1038/nchembio.597 SHARE THIS ARTICLE Anyone you share the following

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