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 ..
BioActive Compounds: 56
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.
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.
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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)