2. Academic Validation
  3. Workflow for E3 Ligase Ligand Validation for PROTAC Development

Workflow for E3 Ligase Ligand Validation for PROTAC Development

  • ACS Chem Biol. 2025 Feb 21;20(2):507-521. doi: 10.1021/acschembio.4c00812.
Nebojša Miletić 1 2,  Janik Weckesser 1 2,  Thorsten Mosler 3,  Rajeshwari Rathore 3,  Marina E Hoffmann 3,  Paul Gehrtz 4,  Sarah Schlesiger 4,  Ingo V Hartung 4,  Nicola Berner 5 6,  Stephanie Wilhelm 5,  Juliane Müller 7,  Bikash Adhikari 7,  Václav Němec 1 2,  Saran Aswathaman Sivashanmugam 1 2,  Lewis Elson 1 2,  Hanna Holzmann 1 2,  Martin P Schwalm 1 2,  Lasse Hoffmann 1 2,  Kamal Rayees Abdul Azeez 1 2,  Susanne Müller 1 2,  Bernhard Kuster 5 6,  Elmar Wolf 7,  Ivan Đikić 3,  Stefan Knapp 1 2 8
Affiliations

Affiliations

  • 1 Institute of Pharmaceutical Chemistry, Goethe University, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany.
  • 2 Structural Genomics Consortium (SGC), Buchmann Institute for Life Sciences, Max-von-Laue-Str. 15, 60438 Frankfurt am Main, Germany.
  • 3 Institute of Biochemistry II, School of Medicine, Goethe University Frankfurt, Frankfurt am Main 60590, Germany.
  • 4 Medicinal Chemistry, Global Research & Development, Merck Healthcare KGaA, 64293 Darmstadt, Germany.
  • 5 Chair of Proteomics and Bioanalytics, Technical University of Munich, Emil-Erlenmeyer-Forum 5, 85354 Freising, Germany.
  • 6 German Cancer Consortium (DKTK), partner site Munich, a partnership between DKFZ and University Center Technical University of Munich, Frankfurt am Main 60590, Germany.
  • 7 Institute of Biochemistry, University of Kiel, Rudolf-Höber-Str. 1, 24118 Kiel, Germany.
  • 8 German Cancer Consortium (DKTK) site Frankfurt/Mainz, Frankfurt am Main 60590, Germany.
PMID: 39932098 DOI: 10.1021/acschembio.4c00812
Abstract

Proteolysis targeting chimeras (PROTACs) have gained considerable attention as a new modality in drug discovery. The development of PROTACs has been mainly focused on using CRBN (Cereblon) and VHL (Von Hippel-Lindau Ligase) E3 Ligase ligands. However, the considerable size of the human E3 Ligase family, newly developed E3 Ligase ligands, and the favorable druggability of some E3 Ligase families hold the promise that novel degraders with unique pharmacological properties will be designed in the future using this large E3 Ligase space. Here, we developed a workflow aiming to improve and streamline the evaluation of E3 Ligase ligand efficiency for PROTAC development and the assessment of the corresponding "degradable" target space using broad-spectrum kinase inhibitors and the well-established VHL ligand VH032 as a validation system. Our study revealed VH032 linker attachment points that are highly efficient for kinase degradation as well as some of the pitfalls when using protein degradation as a readout. For instance, cytotoxicity was identified as a major mechanism leading to PROTAC- and VHL-independent kinase degradation. The combination of E3 Ligase ligand negative controls, competition by kinase parent compounds, and neddylation and Proteasome inhibitors was essential to distinguish between VHL-dependent and -independent kinase degradation events. We share here the findings and limitations of our study and hope that this study will provide guidance for future evaluations of new E3 Ligase ligand systems for degrader development.

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