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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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Many proteins’ biological role depends on their interactions with their ligands, small molecules that bind to specific locations on the protein known as ligand-binding sites. Ligand-binding sites are often conserved among homologous proteins as these sites are critical for protein function.
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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...
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Hit-Validation Methodologies for Ligands Isolated from DNA-Encoded Chemical Libraries.

Gunther Zimmermann1, Yizhou Li1, Ulrike Rieder2

  • 1Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology, ETH Zürich, Vladimir-Prelog-Weg 3, 8093, Zürich, Switzerland.

Chembiochem : a European Journal of Chemical Biology
|January 10, 2017
PubMed
Summary

DNA-encoded chemical libraries (DECLs) enable efficient screening of compounds. New methods using fluorescent DNA probes allow direct determination of binding affinity and kinetics, reducing the need for resynthesis.

Keywords:
DNA-encoded chemical librariesdrug discoveryfluorescence anisotropyhigh-throughput screeninghit validationkoff

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Drug Discovery

Background:

  • DNA-encoded chemical libraries (DECLs) are powerful tools for identifying small molecule binders to target proteins.
  • Traditional DECL screening requires hit resynthesis and affinity measurements for confirmation.

Purpose of the Study:

  • To develop novel methods for direct determination of binding affinity and kinetic constants from DECL screening data.
  • To validate these new methods using a known binder-target interaction.

Main Methods:

  • Utilized hybridization of oligonucleotide conjugates with fluorescently labeled complementary oligonucleotides.
  • Employed fluorescence polarization, Alphascreen, and microscale thermophoresis for binding event detection.
  • Demonstrated the methodology with acetazolamide binding to carbonic anhydrase IX.

Main Results:

  • Successfully determined affinity constants and kinetic dissociation constants using the novel hybridization-based methods.
  • Achieved nanomolar dissociation constant measurements for acetazolamide-carbonic anhydrase IX.
  • Showcased compatibility with multiple detection platforms.

Conclusions:

  • The presented methods enable direct affinity and kinetic characterization of DECL hits.
  • These techniques streamline the hit validation process in DECL-based drug discovery.
  • The approach offers a versatile platform for quantitative analysis of molecular interactions.