Targeted Degradation of Proteins by PROTACs

Eun Ryoung Jang1, Wooin Lee1, Kyung Bo Kim1

1 University of Kentucky, Lexington, Kentucky
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch090242
Online Posting Date:  April, 2010
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Abstract

In recent years, small interference RNAs (siRNAs) have greatly enhanced our understanding of protein functions by allowing knockdown of targeted proteins at the mRNA level. Similarly, in an effort to achieve degradation of targeted proteins at the post‐translational level, chimeric small molecules called “PROTACs” (PROteolysis TArgeting Chimeric molecules) have been developed. The PROTAC approach utilizes chimeric small molecules which recruit targeted proteins to the ubiquitin‐proteasome pathway, a major intracellular protein degradation system. Unlike conventional small molecules that bind to protein and inhibit its function, the PROTAC approach induces destruction of target protein via the ubiquitin‐proteasome system. This article presents a typical strategy for PROTAC design and preparation and biological characterization. Curr. Protoc. Chem Biol. 2:71‐87. © 2010 by John Wiley & Sons, Inc.

Keywords: PROTAC; ubiquitin; proteasome; small molecule

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of E3 Ligase Recognition Motif (H2N‐Leu‐Ala‐ProOH‐Tyr‐Ile‐OBzl)
  • Basic Protocol 2: Attachment of an Amine (or Free Acid) Handle to a Small‐Molecule Ligand in Preparation for Synthesis of a PROTAC Molecule
  • Basic Protocol 3: Assembly of PROTACs: Linking DHT‐NH2 to the pVHL Recognition Motif (Pentapeptide)
  • Basic Protocol 4: Assays for PROTAC‐Induced Targeted Protein Degradation: Immunoblot Analyses
  • Alternate Protocol 1: Validation of PROTAC's Mode of Action Using Cell Lines
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Synthesis of E3 Ligase Recognition Motif (H2N‐Leu‐Ala‐ProOH‐Tyr‐Ile‐OBzl)

  Materials
  • NH 2‐Isoleucine‐O‐Bzl (Advanced ChemTech; http://www.advancedchemtech.com/)
  • FmocHN‐Tyr(O‐t‐Bu)‐CO 2H (Advanced ChemTech)
  • HBTU (2‐(1H‐Benzotriazol‐1‐yl)‐1,1,3,3‐tetramethyluronium hexafluorophosphate) (Advanced ChemTech)
  • HOBt (N‐Hydroxybenzotriazole; Advanced ChemTech)
  • Methylene chloride (CH 2Cl 2; Fluka; dried over calcium hydride)
  • DIPEA (N,Ndiisopropylethylamine; Sigma‐Aldrich)
  • Flash silica gel 60 (particle size 0.040‐0.063 mm; Merck)
  • Ethyl acetate (Fluka)
  • n‐Hexane (Fluka)
  • Piperidine (Sigma‐Aldrich)
  • Anhydrous dimethylformamide (DMF; Sigma‐Aldrich)
  • FmocHN‐ OHPro‐CO 2H (Advanced ChemTech)
  • FmocHN‐Leu‐CO 2H (Advanced ChemTech)
  • FmocHN‐Ala‐CO 2H (Advanced ChemTech)
  • TBAF (t‐butyl ammonium fluoride; Sigma‐Aldrich)
  • TFA (trifluoroacetic acid, Sigma‐Aldrich)
  • Anydrous nitrogen or argon gas
  • Round‐bottom flasks
  • High‐vacuum source
  • 60 F254 precoated silica gel TLC plates (Merck)
  • Rotary evaporator (Buchi)
  • Chromatography columns

Basic Protocol 2: Attachment of an Amine (or Free Acid) Handle to a Small‐Molecule Ligand in Preparation for Synthesis of a PROTAC Molecule

  Materials
  • FmocHN‐Gly‐CO 2H (Advanced ChemTech)
  • Methylene chloride (CH 2Cl 2; Fluka; dried over calcium hydride)
  • Anhydrous dimethylformamide (DMF; Sigma‐Aldrich)
  • Oxalyl chloride (ClC(=O)C(=O)Cl; Sigma‐Aldrich)
  • Anhydrous nitrogen
  • Dihydrotestosterone (DHT, Sigma‐Aldrich)
  • Dimethylaminopyridine (DMAP, Aldrich)
  • Flash silica gel 60 (particle size 0.040 to 0.063 mm; Merck)
  • Ethyl acetate (Fluka)
  • n‐Hexane (Fluka)
  • TBAF (t‐butyl ammonium fluoride) (Sigma‐Aldrich)
  • Disuccinimidyl suberate (Pierce)
  • Round‐bottom flasks
  • High‐vacuum source
  • Rotary evaporator (Buchi)
  • Chromatography columns

Basic Protocol 3: Assembly of PROTACs: Linking DHT‐NH2 to the pVHL Recognition Motif (Pentapeptide)

  Materials
  • DHT‐Gly‐monosuccinimidyl suberate ( protocol 2)
  • Anhydrous dimethylformamide (DMF; Sigma‐Aldrich)
  • pVHL‐recognizing pentapeptide ( protocol 1)
  • Ninhydrin
  • Phenol
  • Potassium cyanide
  • Ethanol
  • Pyridine
  • Methylene chloride (CH 2Cl 2; Fluka; dried over calcium hydride)
  • Methanol
  • 60 F254 precoated silica gel TLC plates (Merck)
  • Glass vial
  • High‐vacuum source
  • Additional reagents and equipment for mass spectrometry (Carr and Annan, ) and HPLC (Boysen and Hearn, )

Basic Protocol 4: Assays for PROTAC‐Induced Targeted Protein Degradation: Immunoblot Analyses

  Materials
  • LNCaP cells (ATCC, cat. no. CRL‐1740)
  • Cell culture medium (see recipe)
  • PROTAC ( protocol 3)
  • Negative PROTAC control (OHPro→norLeu; synthesized following protocol 1, except using norleucine instead of hydroxyproline)
  • Vehicle (DMSO)
  • Lysis buffer (see recipe)
  • Protein Assay Dye Reagent Concentrate (BioRad, cat. no. 500‐0006)
  • Laemmli Sample Buffer 2× Concentrate (Sigma‐Aldrich, cat. no. S3401)
  • 10% to 12% SDS‐PAGE gel (Gallagher, )
  • Prestained SDS‐PAGE Standards, Low Range (BioRad, cat. no. 161‐0305)
  • Blocking solution: 5% (w/v) blocking grade nonfat dry milk (BioRad, cat. no. 170‐6404) in PBS (see recipe for PBS)
  • Anti‐AR antibody (Upstate Biotechnology Inc., cat. no. AB561)
  • Phosphate‐buffered saline (PBS; see recipe) containing 0.05% (v/v) Tween 20
  • Anti‐IgG horseradish peroxidase (Zymed, cat. no. 81‐6100)
  • Anti‐β‐actin antibody (Sigma‐Aldrich, cat. no. A2066)
  • Amersham ECL Western Blotting Detection Reagents (GE Healthcare Life Sciences, cat. no. RPN2209)
  • 24‐well tissue culture plates
  • Refrigerated centrifuge
  • Heat block
  • Platform rotator
  • Immuno‐Blot PVDF Membrane (BioRad, cat. no. 162‐0177)
  • Kodak BioMax XAR films (Sigma‐Aldrich, cat. no. F5513)
  • Additional reagents and equipment for SDS‐PAGE (Gallagher, ) electrotransfer of proteins from gels to membranes (Ursitti et al., )

Alternate Protocol 1: Validation of PROTAC's Mode of Action Using Cell Lines

  • 786‐O and 786‐O/VHL cell lines (Baba et al., )
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Figures

Videos

Literature Cited

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