qHTS Assay for the Inhibitors of FEN1: Confirmatory Assay for Cherry-picked Compounds
The major human flap endonuclease FEN1 is an efficient structure-specific enzyme that recognizes and cleaves a 5'-unannealed DNA flap. It belongs to the RAD2 family nucleases, which are involved in DNA metabolism and are highly conserved in prokaryotes and eukaryotes. FEN1 is a key enzyme in DNA replication, repair and maintenance of genomic stability. 5'-flap removal by FEN1 is critical for more ..
BioActive Compounds: 692
The major human flap endonuclease FEN1 is an efficient structure-specific enzyme that recognizes and cleaves a 5'-unannealed DNA flap. It belongs to the RAD2 family nucleases, which are involved in DNA metabolism and are highly conserved in prokaryotes and eukaryotes. FEN1 is a key enzyme in DNA replication, repair and maintenance of genomic stability. 5'-flap removal by FEN1 is critical for Okazaki fragment processing during lagging strand DNA synthesis, long-patch base excision repair (LP BER), and regulating or facilitating recombination events. The importance of FEN1 in maintaining genomic stability is demonstrated by the abnormal phenotypes of cells deficient for the protein. Mice carrying a homozygous null genotype (fen1-/-) are not viable and exhibit an early embryonic lethality, indicating that FEN1 is essential for normal embryonic development in mammals. Cells from fen1-/- blastocysts show increased apoptotic cell death after ionizing radiation treatment, and chicken cells lacking the fen1 gene are hypersensitive to DNA alkylating agents, e.g. methylmethane sulfonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), as well as hydrogen peroxide. FEN1 is found to be increased in many human cancers, including lung, brain cancer, gastric cancer, prostate cancer, pancreatic cancer and breast cancer indicating that it may be involved in tumor progression and development. FEN1 gene expression has been shown to be induced during cell proliferation and down-regulated during cell differentiation. Elevation of FEN1 in hormone refractory human prostate cancer cells also correlates with resistant to various anticancer agents including ionizing radiation, doxorubicin, paclitaxel and vinblastine. Conversely, down-regulation of FEN1 has been shown to sensitize human glioblastoma cells to MMS, and the clinical drugs temozolomide (TMZ) and cisplatin. These studies suggest that the levels of FEN1 expression influence cancer cell function as it relates to proliferation potential, survival and apoptosis.
This assay is used to confirm the activity of selected compounds against the human Fen1. Inhibition is the desired outcome for this screen.
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]
MLPCN Grant: MH092154
Assay Submitter (PI): David Wilson, National Institute of Aging
Three uL of reagents (buffer in column 3 and 4 as negative control and 10 nM FEN1 in columns 1, 2, and 5-48) were dispensed into 1,536-well black solid-bottomed plate. Compounds (23 nL) were transferred via Kalypsys pin tool equipped with 1536-pin array (10 nL slotted pins, V&P Scientific, San Diego, CA). The plates were then incubated for 15 min at room temperature, and 1 uL substrate (50 nM final concentration) were added to start the reaction read twice at 0 min read and 15 min on ViewLux reader equipped with 525 nm excitation and 598 nm emission filters. Throughout the screen, reagent bottle and all liquid lines were chilled and made light-tight to minimize reagent degradation.
FEN1 Substrate. All oligodeoxynucleotides were purchased from Biosearch Technologies, Inc., (Novato, CA). Double-stranded DNA substrate containing double flap strands for fluorogenic assay was prepared from three oligodeoxynucleotides: quencher (5'-TCA CCC TCG TAC GAC TCA TCC AAA AAA GC - BHQ2), flap (5'-Tamra-GC AAA AAA TTT ATT CAT CAA CTG ACA TCT CCT AC) and template (5'-GT AGG AGA TGT CAG TTG ATG AAT TGA GTC GTA CGA GGG TGA-3') strand by annealing. The 5' end of the downstream flap strand was labeled with 6-carboxytetramethyl rhodamine (6-TAMRA) as the fluorophore donor, whereas the 3' end of the quencher flap strand was labeled with a Black Hole Quencher-2 (BHQ-2) as the matching quencher.
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)