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Related Concept Videos

Allosteric Regulation01:08

Allosteric Regulation

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Allosteric regulation of enzymes occurs when the binding of an effector molecule to a site that is different from the active site causes a change in the enzymatic activity. This alternate site is called an allosteric site, and an enzyme can contain more than one of these sites. Allosteric regulation can either be positive or negative, resulting in an increase or decrease in enzyme activity. Most enzymes that display allosteric regulation are metabolic enzymes involved in the degradation or...
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Potent Fluorescent Probe for Target-Engagement Studies of Allosteric Pyruvate Kinase Modulators.

Oscar Nilsson1, Anna P Valaka1, Liliana Håversen2

  • 1Department of Chemistry and Molecular Biology, University of Gothenburg, Medicinaregatan 7B, Gothenburg, SE-413 90, Sweden.

Angewandte Chemie (International Ed. in English)
|August 30, 2025
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Summary

Researchers developed LumiPK, a fluorescent tracer for monitoring allosteric binding to liver pyruvate kinase (PKL). This novel tool offers high affinity and enables intracellular target engagement assessment, advancing PKL research.

Keywords:
AllosterismBiological activityDrug discoveryFluorescent probesMedicinal chemistry

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Area of Science:

  • Biochemistry
  • Enzyme kinetics
  • Chemical biology

Background:

  • Pyruvate kinases (PKs) are crucial allosterically regulated enzymes in cellular metabolism.
  • PKs are significant therapeutic targets for cancer, metabolic diseases, and diabetes.
  • Limited methods exist for directly assessing allosteric ligand engagement with PK isoforms.

Purpose of the Study:

  • To develop a novel fluorescent tracer, LumiPK, for monitoring allosteric binding to the liver isoform of pyruvate kinase (PKL).
  • To establish a tool for assessing target engagement of PKL allosteric modulators in both in vitro and cellular settings.

Main Methods:

  • Development of LumiPK, an environment-sensitive fluorescent tracer based on a potent allosteric modulator scaffold.
  • Characterization of LumiPK's binding affinity to PKL using recombinant assays (KD = 37 ± 5 nM).
  • Utilizing NanoBRET assays with PKL-Nluc fusion proteins for intracellular target engagement studies.

Main Results:

  • LumiPK demonstrated high affinity for PKL, representing the most potent fluorescent PK reporter to date.
  • Intracellular assays confirmed LumiPK's high potency (EC50 = 18.4 nM) and enabled monitoring of unlabeled ligand engagement.
  • Competitive assays validated the binding of known PKL activators, showing consistent KD values across methods.

Conclusions:

  • LumiPK is a robust and sensitive tool for probing PKL allosteric modulation.
  • This tracer technology addresses a critical gap in target engagement assessment for PKL.
  • LumiPK facilitates further research and drug development targeting PKL.