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

Structural, enzymatic and cellular target engagement studies reveal the mechanism of action by N -hydroxyurea small molecule inhibitors of the DNA repair enzyme, human flap endonuclease-1 (FEN1) that prevent cleavage of DNA flaps in cancer cells. The structure-specific nuclease human flap endonuclea...

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Veröffentlicht in:Nature chemical biology Jg. 12; H. 10; S. 815 - 821
Hauptverfasser: Exell, Jack C, Thompson, Mark J, Finger, L David, Shaw, Steven J, Debreczeni, Judit, Ward, Thomas A, McWhirter, Claire, Siöberg, Catrine L B, Molina, Daniel Martinez, Abbott, W Mark, Jones, Clifford D, Nissink, J Willem M, Durant, Stephen T, Grasby, Jane A
Format: Journal Article
Sprache:Englisch
Veröffentlicht: New York Nature Publishing Group US 01.10.2016
Nature Publishing Group
Schlagworte:
631/337/1427
631/45/535/1266
631/92/173
631/92/613
Biochemical Engineering
Biochemistry
Bioorganic Chemistry
Catalytic Domain - drug effects
Cell Biology
Cell Line, Tumor
Chemistry
Chemistry/Food Science
Crystallography
Deoxyribonucleic acid
DNA
DNA damage
DNA repair
Dose-Response Relationship, Drug
Enzyme Inhibitors - chemistry
Enzyme Inhibitors - pharmacology
Enzymes
Exploitation
Flap Endonucleases - antagonists & inhibitors
Flap Endonucleases - chemistry
Flap Endonucleases - metabolism
Humans
Inhibitors
Magnesium
Metal ions
Mode of action
Models, Molecular
Molecular biology
Molecular Structure
Structure-Activity Relationship
Substrates
Temperature
ISSN:1552-4450, 1552-4469
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Zusammenfassung:Structural, enzymatic and cellular target engagement studies reveal the mechanism of action by N -hydroxyurea small molecule inhibitors of the DNA repair enzyme, human flap endonuclease-1 (FEN1) that prevent cleavage of DNA flaps in cancer cells. The structure-specific nuclease human flap endonuclease-1 (hFEN1) plays a key role in DNA replication and repair and may be of interest as an oncology target. We present the crystal structure of inhibitor-bound hFEN1, which shows a cyclic N -hydroxyurea bound in the active site coordinated to two magnesium ions. Three such compounds had similar IC 50 values but differed subtly in mode of action. One had comparable affinity for protein and protein–substrate complex and prevented reaction by binding to active site catalytic metal ions, blocking the necessary unpairing of substrate DNA. Other compounds were more competitive with substrate. Cellular thermal shift data showed that both inhibitor types engaged with hFEN1 in cells, and activation of the DNA damage response was evident upon treatment with inhibitors. However, cellular EC 50 values were significantly higher than in vitro inhibition constants, and the implications of this for exploitation of hFEN1 as a drug target are discussed.
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ISSN:1552-4450
1552-4469
DOI:10.1038/nchembio.2148