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Some GPCRs transmit signals through adenylyl cyclase (AC), a transmembrane enzyme. AC helps synthesize second messenger cyclic adenosine monophosphate (cAMP). AC catalyzes cyclization reaction and converts ATP to cAMP by releasing a pyrophosphate. The pyrophosphate is further hydrolyzed to phosphate by the enzyme pyrophosphatase, which drives cAMP synthesis to completion. However, cAMP is rapidly degraded to 5′ AMP by the enzymes phosphodiesterase (PDE), preventing overstimulation of...
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On-site reaction for PPARγ modification using a specific bifunctional ligand.

Hiroyuki Kojima1, Toshimasa Itoh1, Keiko Yamamoto1

  • 1Laboratory of Drug Design and Medicinal Chemistry, Showa Pharmaceutical University, 3-3165 Higashi-Tamagawagakuen, Machida, Tokyo 194-8543, Japan.

Bioorganic & Medicinal Chemistry
|November 4, 2017
PubMed
Summary

We developed a novel "on-site reaction" strategy for site-specific protein labeling using a bifunctional ligand. This method enables the conversion of covalent ligands into chemical tools for analyzing protein function and identifying drug targets.

Keywords:
Bioorthogonal reactionClick reaction on crystalConjugate additionCovalent ligandCovalent modifierProtein labelingProtein mass spectrometryReactivity of SPAAC

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Area of Science:

  • Chemical Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Site-specific labeling is crucial for understanding protein function.
  • Existing methods may have limitations in efficiency or scope.
  • Developing versatile labeling strategies is essential for biological research.

Purpose of the Study:

  • To introduce a novel "on-site reaction" strategy for site-specific chemical labeling of proteins.
  • To demonstrate the utility of a bifunctional ligand with enone and azide moieties for sequential reactions.
  • To apply this methodology for functional analysis of peroxisome proliferator activated receptor γ (PPARγ).

Main Methods:

  • Design and synthesis of a bifunctional ligand containing an enone and an azide.
  • Application of the "on-site reaction" involving conjugate addition followed by Huisgen cycloaddition.
  • Utilizing fluorescein as a probe and PPARγ as the target protein.
  • Analysis of reaction products using ESI-mass spectrometry.
  • Determination of binding sites and modes via X-ray crystallography.

Main Results:

  • Successful site-specific labeling of the target protein PPARγ was achieved.
  • The bifunctional ligand effectively underwent sequential reactions on the protein.
  • Fluorescein labeling allowed for visualization and analysis of protein interactions.
  • ESI-mass analysis confirmed the labeling events.
  • X-ray crystallography elucidated the precise binding site and interaction mode.

Conclusions:

  • The "on-site reaction" strategy provides a versatile approach for site-specific protein labeling.
  • This method facilitates the conversion of covalent ligands into valuable chemical tools.
  • The methodology aids in protein functional analysis and drug target identification.