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Related Concept Videos

Ligand Binding Sites02:40

Ligand Binding Sites

Proteins are dynamic macromolecules that carry out a wide variety of essential processes; however, the activities of most proteins depend on their interactions with other molecules or ions, known as ligands.
Protein-ligand interactions are quite specific; even though numerous potential ligands surround a cellular protein at any given time, only a particular ligand can bind to that protein. Moreover, a ligand binds only to a dedicated area on the surface of the protein, known as the...
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Allosteric proteins have more than one ligand binding site; the binding of a ligand to any of these sites influences the binding of ligands to the other sites. When a protein is allosteric, its binding sites are called coupled or linked.  In the case of enzymes, the site that binds to the substrate is known as the active site and the other site is known as the regulatory site. When a ligand binds to the regulatory site, this leads to conformational changes in the protein that can influence the...
Ligand Binding and Linkage00:49

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Ligand Binding Sites02:40

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Proteins are dynamic macromolecules that carry out a wide variety of essential processes; however, the activities of most proteins depend on their interactions with other molecules or ions, known as ligands.
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Updated: Jun 12, 2026

In Vitro Analysis of E3 Ubiquitin Ligase Function
06:06

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Published on: May 14, 2021

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Workflow for E3 Ligase Ligand Validation for PROTAC Development.

Nebojša Miletić1,2, Janik Weckesser1,2, Thorsten Mosler3

  • 1Institute of Pharmaceutical Chemistry, Goethe University, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany.

ACS Chemical Biology
|February 11, 2025
PubMed
Summary
This summary is machine-generated.

This study introduces a streamlined workflow for evaluating E3 ligase ligands in proteolysis targeting chimeras (PROTACs) development. It highlights efficient linker attachment points for kinase degradation and identifies cytotoxicity as a pitfall for VHL-independent degradation.

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Area of Science:

  • Drug Discovery and Development
  • Molecular Biology
  • Biochemistry

Background:

  • Proteolysis targeting chimeras (PROTACs) represent a novel drug discovery modality.
  • Current PROTAC development primarily utilizes Cereblon (CRBN) and Von Hippel-Lindau (VHL) E3 ligase ligands.
  • The extensive human E3 ligase family offers potential for novel degraders with unique properties.

Purpose of the Study:

  • To develop and validate a streamlined workflow for assessing E3 ligase ligand efficiency in PROTAC development.
  • To evaluate the 'degradable' target space using kinase inhibitors and the VHL ligand VH032.
  • To identify potential pitfalls in protein degradation assays.

Main Methods:

  • Development of a workflow for evaluating E3 ligase ligand efficiency.
  • Utilized broad-spectrum kinase inhibitors and the VHL ligand VH032 as a validation system.
  • Employed E3 ligase ligand negative controls, competition assays, and inhibitors of neddylation and proteasome pathways.

Main Results:

  • Identified VH032 linker attachment points that are highly efficient for kinase degradation.
  • Revealed cytotoxicity as a significant mechanism causing PROTAC- and VHL-independent kinase degradation.
  • Established the necessity of controls and inhibitors to differentiate VHL-dependent from VHL-independent degradation.

Conclusions:

  • The developed workflow aids in evaluating E3 ligase ligand efficiency for PROTAC development.
  • Cytotoxicity can confound protein degradation readouts, necessitating careful experimental design.
  • This study provides guidance for future evaluations of novel E3 ligase ligand systems in degrader development.