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Fluorescence-Based Detection of FEN1 Nuclease Activity and Screening of Small-Molecule Inhibitors
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Fluorescence-Based Detection of FEN1 Nuclease Activity and Screening of Small-Molecule Inhibitors

Published on: June 27, 2025

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Cellularly active N-hydroxyurea FEN1 inhibitors block substrate entry to the active site

Jack C Exell1, Mark J Thompson1, L David Finger1

  • 1Centre for Chemical Biology, Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield, UK.

Nature Chemical Biology
|August 16, 2016
PubMed

Insights

Researchers crystallized human flap endonuclease-1 (hFEN1) with inhibitors, revealing how they block DNA repair. While inhibitors engaged hFEN1 in cells, their effectiveness was lower than expected, impacting its drug target potential.

Area of Science:

  • Biochemistry
  • Structural Biology
  • Molecular Oncology

Background:

  • Human flap endonuclease-1 (hFEN1) is a structure-specific nuclease crucial for DNA replication and repair.
  • hFEN1 is a potential oncology target due to its role in maintaining genomic stability.

Purpose of the Study:

  • To elucidate the structural basis of hFEN1 inhibition by cyclic N-hydroxyurea compounds.
  • To investigate the mechanism of action and cellular efficacy of hFEN1 inhibitors.

Main Methods:

  • X-ray crystallography was used to determine the structure of inhibitor-bound hFEN1.
  • In vitro enzyme assays and cellular thermal shift assays (CETSA) were performed.
  • DNA damage response activation was assessed upon inhibitor treatment.

Main Results:

  • The crystal structure revealed cyclic N-hydroxyurea inhibitors coordinated to magnesium ions in the hFEN1 active site.
  • Inhibitors exhibited distinct modes of action, either blocking catalytic metal ions or competing with the DNA substrate.
  • Both inhibitor types engaged hFEN1 in cells, activating the DNA damage response.
  • Cellular efficacy (EC50) was significantly lower than in vitro inhibition (IC50).

Conclusions:

  • Structural insights into hFEN1 inhibition provide a basis for drug design.
  • The discrepancy between in vitro and cellular activity suggests challenges in targeting hFEN1 for cancer therapy.
  • Further research is needed to optimize hFEN1 inhibitors for clinical applications.

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