Complementary non-radioactive assays for investigation of human flap endonuclease 1 activity
Dorjbal Dorjsuren1, Daemyung Kim, David J Maloney
1NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892-3370, USA.
Abstract:
FEN1, a key participant in DNA replication and repair, is the major human flap endonuclease that recognizes and cleaves flap DNA structures. Deficiencies in FEN1 function or deletion of the fen1 gene have profound biological effects, including the suppression of repair of DNA damage incurred from the action of various genotoxic agents. Given the importance of FEN1 in resolving abnormal DNA structures, inhibitors of the enzyme carry a potential as enhancers of DNA-interactive anticancer drugs. To facilitate the studies of FEN1 activity and the search for novel inhibitors, we developed a pair of complementary-readout homogeneous assays utilizing fluorogenic donor/quencher and AlphaScreen chemiluminescence strategies. A previously reported FEN1 inhibitor 3-hydroxy-5-methyl-1-phenylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione displayed equal potency in the new assays, in agreement with its published IC(50). The assays were optimized to a low 4 µl volume and used to investigate a set of small molecules, leading to the identification of previously-unreported FEN1 inhibitors, among which aurintricarboxylic acid and NSC-13755 (an arylstibonic derivative) displayed submicromolar potency (average IC(50) of 0.59 and 0.93 µM, respectively). The availability of these simple complementary assays obviates the need for undesirable radiotracer-based assays and should facilitate efforts to develop novel inhibitors for this key biological target.
Insights
New assays were developed to identify inhibitors of flap endonuclease 1 (FEN1), a key enzyme in DNA repair. Aurintricarboxylic acid and NSC-13755 were found to be potent FEN1 inhibitors, aiding cancer drug development.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Discovery
Background:
- Flap endonuclease 1 (FEN1) is crucial for DNA replication and repair, cleaving flap DNA structures.
- FEN1 dysfunction or deficiency impairs DNA damage repair, impacting cellular integrity.
- FEN1's role in resolving aberrant DNA structures makes it a target for anticancer drug development.
Purpose of the Study:
- To develop homogeneous assays for studying FEN1 activity and identifying novel inhibitors.
- To validate the developed assays using a known FEN1 inhibitor.
- To screen small molecules for FEN1 inhibitory potential.
Main Methods:
- Development of complementary-readout homogeneous assays using fluorogenic donor/quencher and AlphaScreen chemiluminescence.
- Optimization of assays to a low 4 µl volume.
- Screening of small molecules to identify FEN1 inhibitors.
Main Results:
- The developed assays confirmed the potency of a previously reported FEN1 inhibitor.
- Aurintricarboxylic acid and NSC-13755 were identified as novel FEN1 inhibitors with submicromolar potency (IC50 values of 0.59 µM and 0.93 µM, respectively).
- The assays provide a sensitive and efficient alternative to radiotracer-based methods.
Conclusions:
- The developed homogeneous assays are effective for FEN1 activity studies and inhibitor screening.
- Novel FEN1 inhibitors, including aurintricarboxylic acid and NSC-13755, have been identified.
- These assays facilitate the development of FEN1-targeting anticancer drugs.
