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Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis
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Time-dependent inhibition of PHD2

Isabelle Tcholakov1, Charles E Grimshaw2, Lihong Shi2

  • 1In Vitro Pharmacology, Immunology, Takeda California, Inc., 10410 Science Center Drive, San Diego, CA 92121, U.S.A.

Bioscience Reports
|June 9, 2017
PubMed

Insights

Small molecule inhibitors targeting prolyl hydroxylases (PHDs) show promise for anemia treatment. These compounds reversibly inhibit PHDs by competing with 2-oxoglutarate, acting as pan PHD inhibitors with long-lasting effects.

Area of Science:

  • Biochemistry
  • Pharmacology
  • Cellular Biology

Background:

  • Prolyl hydroxylases (PHDs) regulate hypoxia-inducible factors (HIFs) through proline residue hydroxylation, impacting cellular response to hypoxia.
  • PHD inhibitors are under investigation for anemia treatment, with several in clinical trials.

Purpose of the Study:

  • To perform detailed kinetic analysis of small molecule PHD inhibitors.
  • To elucidate the mechanism of inhibition for these compounds.

Main Methods:

  • Enzyme kinetics assays were performed to determine inhibition constants and mechanisms.
  • Analysis included determination of co-substrate competition and time-dependent inhibition (TDI).

Main Results:

  • The inhibitors were found to be reversible and competitive with 2-oxoglutarate (2-OG) at the enzyme active site.
  • Most compounds acted as pan PHD inhibitors, affecting multiple PHD isoforms.
  • A time-dependent inhibition (TDI) mechanism was observed, characterized by a slow dissociation rate constant (koff) and long residence time.

Conclusions:

  • Small molecule PHD inhibitors exhibit competitive inhibition with 2-OG.
  • The observed TDI mechanism suggests prolonged target engagement, potentially beneficial for therapeutic applications.
  • These findings provide valuable kinetic insights for the development of PHD inhibitors for anemia.
Keywords:
hypoxiahypoxia inducible factorliquid chromatography-tandem mass spectrometryprolyl hydroxylase 2residence timetime-dependent inhibition

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