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BioAssay: AID 488816

qHTS Validation Assay for the Inhibitors of Human Flap endonuclease 1 (FEN1)

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 ..
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 Tested Compounds
 Tested Compounds
All(1266)
 
 
Active(56)
 
 
Inactive(1183)
 
 
Inconclusive(27)
 
 
 Tested Substances
 Tested Substances
All(1280)
 
 
Active(56)
 
 
Inactive(1197)
 
 
Inconclusive(27)
 
 
 Related BioAssays
 Related BioAssays
AID: 488816
Data Source: NCGC (FEN147)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-10-18

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 56
Related Experiments
AIDNameTypeComment
488813Probe Development Summary for Inhibitors of Human Flap endonuclease 1 (FEN1)Summarydepositor-specified cross reference
587qHTS Assay for Spectroscopic Profiling in Texas Red Spectral RegionOthersame project related to Summary assay
588795qHTS Assay for the Inhibitors of Human Flap endonuclease 1 (FEN1).Confirmatorysame project related to Summary assay
720498qHTS Assay for the Inhibitors of FEN1: Confirmatory Assay for Cherry-picked CompoundsConfirmatorysame project related to Summary assay
Description:
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. Deletion of the yeast FEN1 gene, i.e. rad27, results in an increased frequency of short DNA (3-32 bp) repeats, micro- and mini-satellite formation, trinucleotide repeat expansion, spontaneous recombination events, and a severe growth defect in association with cell cycle arrest in late S/G2 phase. It is also shown that mice carrying a homozygous null genotype (fen1-/-) are not viable and exhibit an early embryonic lethality (E4.5), 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. These results indicate that FEN1 deficiency fails to repair DNA lesions generated by DNA alkylating and oxidizing compounds.

FEN1 is found to be increased in many human cancers, including lung, brain cancer, gastric cancer, prostate cancer, pancreatic cancer and breast cancer. These results suggest that FEN1 may, in part, be involved in tumor progression and development. In fact, FEN1 gene expression has been shown to be induced during cell proliferation and down-regulated during cell differentiation. In particular, elevation of FEN1 in hormone refractory human prostate cancer cells 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.
Protocol
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. Throughout the screen, reagent bottle and all liquid lines were chilled and made light-tight to minimize reagent degradation. All screening operations were performed on a fully integrated robotic system (Kalypsys, San Diego, CA) containing one RX-130 and two RX-90 anthropomorphic robotic arms (Staubli, Duncan, SC). Library plates were screened starting from the lowest and proceeding to the highest concentration, and a "double-dipping" step of the highest concentration was required to access higher concentrations of compounds. Vehicle-only plates, with DMSO being pin-transferred to the entire column 5-48 compound area, were inserted uniformly at the beginning and the end of each library in order to monitor for and record any shifts in the background, which can be affected by reagent dispensers or loss in enzyme activity overtime. Screening data was corrected, normalized, and concentration-effect relationships were derived by using publicly-available curve fitting algorithms developed in-house (http://ncgc.nih.gov/pub/openhts). A four parameter Hill equation was fitted to the concentration-response data by minimizing the residual error between the modeled and observed responses.
Comment
Compound Ranking:
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
From ChEMBL:
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PhenotypeIndicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive.String
2Potency*Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.FloatμM
3EfficacyMaximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.Float%
4Analysis CommentAnnotation/notes on a particular compound's data or its analysis.String
5Curve_DescriptionA description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control.String
6Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
7Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
8Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
9Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
10Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
11Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12Excluded_PointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
13Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14Activity at 0.00366 uM (0.00366μM**)% Activity at given concentration.Float%
15Activity at 0.018 uM (0.0183μM**)% Activity at given concentration.Float%
16Activity at 0.091 uM (0.0914μM**)% Activity at given concentration.Float%
17Activity at 0.457 uM (0.457μM**)% Activity at given concentration.Float%
18Activity at 2.290 uM (2.29μM**)% Activity at given concentration.Float%
19Activity at 11.40 uM (11.4μM**)% Activity at given concentration.Float%
20Activity at 57.10 uM (57.1μM**)% Activity at given concentration.Float%
21Compound QCSource of compound QCString

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH092154-01

Data Table (Concise)
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