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

Journal name:
Nature Chemical Biology
Volume:
7,
Pages:
538–543
Year published:
DOI:
doi:10.1038/nchembio.597
Received
Accepted
Published online

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 Hras1G12V-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.

At a glance

Figures

  1. Hydrophobic tagging strategy using the HaloTag fusion protein system.
    Figure 1: Hydrophobic tagging strategy using the HaloTag fusion protein system.

    (a) Chemical structures of the representative HaloTag ligands: HyT5 (1), HyT12 (2), HyT13 (3), HyT16 (4), HyT21 (5) and HyT22 (6). (b) HEK 293T cells expressing HA–HaloTag–luciferase were treated with the indicated compounds at 1 μM for 24 h, at which point luciferase assays were performed. Error bars, s.e.m.

  2. HyT13 leads to degradation of HaloTag fusion proteins.
    Figure 2: HyT13 leads to degradation of HaloTag fusion proteins.

    (a) Flp-In 293 cells expressing HA–EGFP–HaloTag were treated with the indicated concentrations of HyT13 for 24 h. The lysates were probed with HA-specific and β-actin–specific antibodies. (b) The same cell line as in a was treated for the indicated times with 1 μM HyT13. The rightmost sample was treated with HyT13 for 24 h, after which HyT13-free medium was provided for 24 h. (c) The same cell line as in a was pretreated with proteasome inhibitors MG132 (10 μM) and YU101 (10 μM) for 1 h before addition of 1 μM HyT13. The lysates were prepared from cells 6 h after HyT13 addition. (d) HeLa cells stably expressing EGFP–HaloTag were treated with vehicle or 1 μM HyT13 for 24 h, whereupon the intracellular GFP fluorescence was quantified by flow cytometry. MFI, mean fluorescence intensity. (e) HEK 293T cells stably expressing the indicated transmembrane HA–HaloTag fusion proteins were treated with 1 μM HyT13 for 24 h. Shown are representative images from at least three experiments; bands were quantified, and the mean degradation ± s.e.m. is shown. (f) Zebrafish embryos were injected with HA–HaloTag–Smad5 cRNA and treated with 10 μM HyT13 for 24 h. Shown are representative images from at least three experiments; bands were quantified and mean degradation ± s.e.m. is shown. Full gels are available in the Supplementary Results.

  3. Functional validation of HaloTag degradation by HyT13.
    Figure 3: Functional validation of HaloTag degradation by HyT13.

    (a) NIH-3T3 cells expressing either HA–HaloTag–Hras1G12V or HA–HaloTagD106A–Hras1G12V were treated with vehicle or 1 μM HyT13 for 24 h. The lysates were prepared for immunoblotting, and the blots were probed with HA-specific and β-actin–specific antibodies. Full gels are available in the Supplementary Results. (b) Focus formation assay was performed with NIH-3T3 cells infected with HA–HaloTag–Hras1G12V or HA–HaloTagD106A–Hras1G12V. The cells were treated with vehicle or 1 μM HyT13. Scale bars, 5 mm. (c) Quantification of foci as described in b. The number of foci per cm2 was counted from three separate plates. Error bars, s.e.m. (d) HA–HaloTag–Hras1G12V-expressing NIH-3T3 cells were injected into the flank of nude mice on day 0. The mice were administered i.p. injections of vehicle or HyT13 daily from day 0. Error bars, s.e.m. (n = 7 for each treatment group).

  4. Schematic of HyT13 mediated degradation of HaloTag fusion proteins.
    Figure 4: Schematic of HyT13 mediated degradation of HaloTag fusion proteins.

    A fusion protein composed of a protein of interest and the HaloTag protein is degraded upon HyT13 treatment by the proteasome.

Compounds

53 compounds View all compounds
  1. 3-(1-(2-(2-(2-(2-Aminoethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)-N-(24-chloro-11-oxo-3,6,9,15,18-pentaoxa-12-azatetracosyl)propanamide
    Compound 1 3-(1-(2-(2-(2-(2-Aminoethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)-N-(24-chloro-11-oxo-3,6,9,15,18-pentaoxa-12-azatetracosyl)propanamide
  2. N-(2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)-2,2-diphenylacetamide
    Compound 2 N-(2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)-2,2-diphenylacetamide
  3. 2-((3r,5r,7r)-Adamantan-1-yl)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)acetamide
    Compound 3 2-((3r,5r,7r)-Adamantan-1-yl)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)acetamide
  4. N-(2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)-2-(9H-fluoren-9-yl)acetamide
    Compound 4 N-(2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)-2-(9H-fluoren-9-yl)acetamide
  5. N-(2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)-2,2-dicyclohexylacetamide
    Compound 5 N-(2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)-2,2-dicyclohexylacetamide
  6. (S)-N-(2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)-2-(4-isobutylphenyl)propanamide
    Compound 6 (S)-N-(2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)-2-(4-isobutylphenyl)propanamide
  7. 2-(2-((6-Chlorohexyl)oxy)ethoxy)ethanamine
    Compound 8 2-(2-((6-Chlorohexyl)oxy)ethoxy)ethanamine
  8. tert-Butyl (2-(2-hydroxyethoxy)ethyl)carbamate
    Compound 9 tert-Butyl (2-(2-hydroxyethoxy)ethyl)carbamate
  9. 1-Chloro-6-iodohexane
    Compound 10 1-Chloro-6-iodohexane
  10. tert-Butyl (2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamate
    Compound 11 tert-Butyl (2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamate
  11. 1-Adamantaneacetic acid
    Compound 12 1-Adamantaneacetic acid
  12. 2,2-Dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecan-16-oic acid
    Compound 13 2,2-Dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecan-16-oic acid
  13. tert-Butyl (24-chloro-11-oxo-3,6,9,15,18-pentaoxa-12-azatetracosyl)carbamate
    Compound 14 tert-Butyl (24-chloro-11-oxo-3,6,9,15,18-pentaoxa-12-azatetracosyl)carbamate
  14. N-(24-Chloro-11-oxo-3,6,9,15,18-pentaoxa-12-azatetracosyl)pent-4-ynamide
    Compound 15 N-(24-Chloro-11-oxo-3,6,9,15,18-pentaoxa-12-azatetracosyl)pent-4-ynamide
  15. tert-Butyl (2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamate
    Compound 16 tert-Butyl (2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamate
  16. tert-Butyl (2-(2-(2-(2-(4-(28-chloro-3,15-dioxo-7,10,13,19,22-pentaoxa-4,16-diazaoctacosyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)carbamate
    Compound 17 tert-Butyl (2-(2-(2-(2-(4-(28-chloro-3,15-dioxo-7,10,13,19,22-pentaoxa-4,16-diazaoctacosyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)carbamate
  17. Ethyl 2-(4-((3r,5r,7r)-adamantan-1-yl)phenoxy)acetate
    Compound 18 Ethyl 2-(4-((3r,5r,7r)-adamantan-1-yl)phenoxy)acetate
  18. 2-(4-((3r,5r,7r)-Adamantan-1-yl)phenoxy)acetic acid
    Compound 19 2-(4-((3r,5r,7r)-Adamantan-1-yl)phenoxy)acetic acid
  19. 3-(1-(1-(4-((3r,5r,7r)-Adamantan-1-yl)phenoxy)-2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl)-1H-1,2,3-triazol-4-yl)-N-(24-chloro-11-oxo-3,6,9,15,18-pentaoxa-12-azatetracosyl)propanamide
    Compound 20 3-(1-(1-(4-((3r,5r,7r)-Adamantan-1-yl)phenoxy)-2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl)-1H-1,2,3-triazol-4-yl)-N-(24-chloro-11-oxo-3,6,9,15,18-pentaoxa-12-azatetracosyl)propanamide
  20. N-(2-(2-(2-(2-(4-(28-Chloro-3,15-dioxo-7,10,13,19,22-pentaoxa-4,16-diazaoctacosyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)-3,3,3-triphenylpropanamide
    Compound 21 N-(2-(2-(2-(2-(4-(28-Chloro-3,15-dioxo-7,10,13,19,22-pentaoxa-4,16-diazaoctacosyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)-3,3,3-triphenylpropanamide
  21. N-(2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)-3,3,3-triphenylpropanamide
    Compound 22 N-(2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)-3,3,3-triphenylpropanamide
  22. tert-Butyl (6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)amino)-6-oxohexyl)carbamate
    Compound 23 tert-Butyl (6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)amino)-6-oxohexyl)carbamate
  23. N-(2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)-6-(3,3,3-triphenylpropanamido)hexanamide
    Compound 24 N-(2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)-6-(3,3,3-triphenylpropanamido)hexanamide
  24. N-(24-Chloro-11-oxo-3,6,9,15,18-pentaoxa-12-azatetracosyl)-3,3,3-triphenylpropanamide
    Compound 25 N-(24-Chloro-11-oxo-3,6,9,15,18-pentaoxa-12-azatetracosyl)-3,3,3-triphenylpropanamide
  25. (S)-N-(2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)-2-(3,4,5-trimethoxyphenyl)butanamide
    Compound 26 (S)-N-(2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)-2-(3,4,5-trimethoxyphenyl)butanamide
  26. 2-(4-((3r,5r,7r)-Adamantan-1-yl)phenoxy)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)acetamide
    Compound 27 2-(4-((3r,5r,7r)-Adamantan-1-yl)phenoxy)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)acetamide
  27. (1S,4R)-N-(2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptane-1-carboxamide
    Compound 28 (1S,4R)-N-(2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptane-1-carboxamide
  28. N-(2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)-6-fluoro-2-naphthamide
    Compound 29 N-(2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)-6-fluoro-2-naphthamide
  29. 2-(4-((3r,5r,7r)-Adamantan-1-yl)phenoxy)-N-(24-chloro-11-oxo-3,6,9,15,18-pentaoxa-12-azatetracosyl)acetamide
    Compound 30 2-(4-((3r,5r,7r)-Adamantan-1-yl)phenoxy)-N-(24-chloro-11-oxo-3,6,9,15,18-pentaoxa-12-azatetracosyl)acetamide
  30. N-(2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)-2-(((2S,5R)-2-isopropyl-5-methylcyclohexyl)oxy)acetamide
    Compound 31 N-(2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)-2-(((2S,5R)-2-isopropyl-5-methylcyclohexyl)oxy)acetamide
  31. (R)-N-(2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)-2-(2-fluoro-[1,1'-biphenyl]-4-yl)propanamide
    Compound 32 (R)-N-(2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)-2-(2-fluoro-[1,1'-biphenyl]-4-yl)propanamide
  32. 2-(2,2,4,7-Tetramethyl-3,4-dihydroquinolin-1(2H)-yl)ethyl (2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamate
    Compound 33 2-(2,2,4,7-Tetramethyl-3,4-dihydroquinolin-1(2H)-yl)ethyl (2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamate
  33. 2-(10H-Phenoxazin-10-yl)ethyl (2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamate
    Compound 34 2-(10H-Phenoxazin-10-yl)ethyl (2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamate
  34. (R)-N-(2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)-3,3,3-trifluoro-2-methoxy-2-phenylpropanamide
    Compound 35 (R)-N-(2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)-3,3,3-trifluoro-2-methoxy-2-phenylpropanamide
  35. (R)-1-(2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)-3-(3-methyl-1,1-diphenylbutan-2-yl)urea
    Compound 36 (R)-1-(2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)-3-(3-methyl-1,1-diphenylbutan-2-yl)urea
  36. 2-(Bis((R)-1-phenylethyl)amino)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)acetamide
    Compound 37 2-(Bis((R)-1-phenylethyl)amino)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)acetamide
  37. tert-Butyl 2-(3-(3-(3,4-dimethoxyphenyl)propanoyl)phenoxy)acetate
    Compound 38 tert-Butyl 2-(3-(3-(3,4-dimethoxyphenyl)propanoyl)phenoxy)acetate
  38. (R)-tert-Butyl 2-(3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)acetate
    Compound 39 (R)-tert-Butyl 2-(3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)acetate
  39. tert-Butyl 2-(3-((S)-1-(4-((3S,5S,7S)-adamantan-1-yl)phenoxy)-3-(3,4-dimethoxyphenyl)propyl)phenoxy)acetate
    Compound 40 tert-Butyl 2-(3-((S)-1-(4-((3S,5S,7S)-adamantan-1-yl)phenoxy)-3-(3,4-dimethoxyphenyl)propyl)phenoxy)acetate
  40. 2-(3-((S)-1-(4-((3S,5S,7S)-Adamantan-1-yl)phenoxy)-3-(3,4-dimethoxyphenyl)propyl)phenoxy)acetic acid
    Compound 41 2-(3-((S)-1-(4-((3S,5S,7S)-Adamantan-1-yl)phenoxy)-3-(3,4-dimethoxyphenyl)propyl)phenoxy)acetic acid
  41. 2-(3-((S)-1-(4-((3S,5S,7S)-Adamantan-1-yl)phenoxy)-3-(3,4-dimethoxyphenyl)propyl)phenoxy)-N-(24-chloro-11-oxo-3,6,9,15,18-pentaoxa-12-azatetracosyl)acetamide
    Compound 42 2-(3-((S)-1-(4-((3S,5S,7S)-Adamantan-1-yl)phenoxy)-3-(3,4-dimethoxyphenyl)propyl)phenoxy)-N-(24-chloro-11-oxo-3,6,9,15,18-pentaoxa-12-azatetracosyl)acetamide
  42. N-(2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)-4-pentylbicyclo[2.2.2]octane-1-carboxamide
    Compound 43 N-(2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)-4-pentylbicyclo[2.2.2]octane-1-carboxamide
  43. 1-((3s,5s,7s)-Adamantan-1-yl)-3-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)urea
    Compound 44 1-((3s,5s,7s)-Adamantan-1-yl)-3-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)urea
  44. 1-((3s,5s,7s)-Adamantan-1-yl)-3-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)thiourea
    Compound 45 1-((3s,5s,7s)-Adamantan-1-yl)-3-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)thiourea
  45. (3r,5r,7r)-1-(2-Iodoethyl)adamantane
    Compound 46 (3r,5r,7r)-1-(2-Iodoethyl)adamantane
  46. (R)-4-((3R,5R,7R)-Adamantan-1-yl)-N-((1S,2S)-1-hydroxy-1-phenylpropan-2-yl)-N,2-dimethylbutanamide
    Compound 47 (R)-4-((3R,5R,7R)-Adamantan-1-yl)-N-((1S,2S)-1-hydroxy-1-phenylpropan-2-yl)-N,2-dimethylbutanamide
  47. (R)-4-((3R,5R,7R)-Adamantan-1-yl)-2-methylbutanoic acid
    Compound 48 (R)-4-((3R,5R,7R)-Adamantan-1-yl)-2-methylbutanoic acid
  48. (R)-4-((3R,5R,7R)-Adamantan-1-yl)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-methylbutanamide
    Compound 49 (R)-4-((3R,5R,7R)-Adamantan-1-yl)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-methylbutanamide
  49. N-((1S,2S)-1-Hydroxy-1-phenylpropan-2-yl)-N-methyl-3-phenylpropanamide
    Compound 50 N-((1S,2S)-1-Hydroxy-1-phenylpropan-2-yl)-N-methyl-3-phenylpropanamide
  50. (S)-4-((3S,5S,7S)-Adamantan-1-yl)-2-benzyl-N-((1S,2S)-1-hydroxy-1-phenylpropan-2-yl)-N-methylbutanamide
    Compound 51 (S)-4-((3S,5S,7S)-Adamantan-1-yl)-2-benzyl-N-((1S,2S)-1-hydroxy-1-phenylpropan-2-yl)-N-methylbutanamide
  51. (S)-4-((3S,5S,7S)-Adamantan-1-yl)-2-benzylbutanoic acid
    Compound 52 (S)-4-((3S,5S,7S)-Adamantan-1-yl)-2-benzylbutanoic acid
  52. (S)-4-((3S,5S,7S)-Adamantan-1-yl)-2-benzyl-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)butanamide
    Compound 53 (S)-4-((3S,5S,7S)-Adamantan-1-yl)-2-benzyl-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)butanamide
  53. 2-((3r,5r,7r)-Adamantan-1-yl)-N-(24-chloro-11-oxo-3,6,9,15,18-pentaoxa-12-azatetracosyl)acetamide
    Compound 54 2-((3r,5r,7r)-Adamantan-1-yl)-N-(24-chloro-11-oxo-3,6,9,15,18-pentaoxa-12-azatetracosyl)acetamide

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Author information

  1. These authors contributed equally to this work.

    • Taavi K Neklesa &
    • Hyun Seop Tae

Affiliations

  1. 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
  2. Department of Chemistry, Yale University, New Haven, Connecticut, USA.

    • Ashley R Schneekloth,
    • Kanak Raina &
    • Craig M Crews
  3. Department of Pharmacology, Yale University, New Haven, Connecticut, USA.

    • Craig M Crews
  4. Current address: Department of Ophthalmology, Indiana University School of Medicine, Indianapolis, Indiana, USA.

    • Timothy W Corson

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.

Competing financial interests

The authors declare no competing financial interests.

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    Supplementary Methods and Supplementary Results

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