title: Cellularly active N-hydroxyurea FEN1 inhibitors block substrate entry to the active site.
abstract: 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 IC50 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 EC50 values were significantly higher than in vitro inhibition constants, and the implications of this for exploitation of hFEN1 as a drug target are discussed.
authors.affiliation: Centre for Chemical Biology, Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield, UK.
authors.forename: Jack C
authors.initials: JC
authors.lastname: Exell
authors.affiliation: Centre for Chemical Biology, Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield, UK.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Thompson
authors.affiliation: Centre for Chemical Biology, Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield, UK.
authors.forename: L David
authors.identifier: 0000-0002-2342-9569
authors.initials: LD
authors.lastname: Finger
authors.affiliation: Centre for Chemical Biology, Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield, UK.
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Shaw
authors.affiliation: Discovery Sciences, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Cambridge, UK.
authors.forename: Judit
authors.initials: J
authors.lastname: Debreczeni
authors.affiliation: Bioscience, Oncology Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Alderley Park, Cheshire, UK.
authors.forename: Thomas A
authors.identifier: 0000-0003-0937-4554
authors.initials: TA
authors.lastname: Ward
authors.affiliation: Discovery Sciences, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Cambridge, UK.
authors.forename: Claire
authors.initials: C
authors.lastname: McWhirter
authors.affiliation: Pelago Bioscience AB, Solna, Sweden.
authors.forename: Catrine L B
authors.initials: CL
authors.lastname: Siöberg
authors.affiliation: Pelago Bioscience AB, Solna, Sweden.
authors.forename: Daniel
authors.initials: D
authors.lastname: Martinez Molina
authors.affiliation: Discovery Sciences, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Cambridge, UK.
authors.forename: W Mark
authors.initials: WM
authors.lastname: Abbott
authors.affiliation: Chemistry, Oncology Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Alderley Park, UK.
authors.forename: Clifford D
authors.initials: CD
authors.lastname: Jones
authors.affiliation: Chemistry, Oncology Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Cambridge, UK.
authors.forename: J Willem M
authors.identifier: 0000-0003-2572-9140
authors.initials: JW
authors.lastname: Nissink
authors.affiliation: Bioscience, Oncology Innovative Medicines and Early Development Biotech Unit, Cambridge, UK.
authors.forename: Stephen T
authors.initials: ST
authors.lastname: Durant
authors.affiliation: Centre for Chemical Biology, Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield, UK.|Bioscience, Oncology Innovative Medicines and Early Development Biotech Unit, Cambridge, UK.
authors.forename: Jane A
authors.initials: JA
authors.lastname: Grasby
mesh_terms: D020134:Catalytic Domain / Q000187:drug effects; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D045585:Flap Endonucleases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D015394:Molecular Structure; D013329:Structure-Activity Relationship; D013696:Temperature
chemical_list: D004791:Enzyme Inhibitors; D045585:Flap Endonucleases; C468317:FEN1 protein, human