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Updated: Jun 20, 2026
Fluorescence-Based Detection of FEN1 Nuclease Activity and Screening of Small-Molecule Inhibitors
Published on: June 27, 2025
Amino acid Asp181 of 5'-flap endonuclease 1 is a useful target for chemotherapeutic development
Harekrushna Panda1, Aruna S Jaiswal, Patrick E Corsino
1Department of Anatomy and Cell Biology and UF Shands Cancer Center, University of Florida, Gainesville, Florida 32610, USA.
Abstract:
DNA alkylation-induced damage is one of the most efficacious anticancer therapeutic strategies. Enhanced DNA alkylation and weakened DNA repair capacity in cancer cells are responsible for the effectiveness of DNA-alkylating therapies. 5'-Flap endonuclease 1 (Fen1) is an important enzyme involved in base excision repair (BER), specifically in long-patch BER (LP-BER). Using the site-directed mutagenesis approach, we have identified an important role for amino acid Asp181 of Fen1 in its endonuclease activity. Asp181 is thought to be involved in Mg(2+) binding in the active site. Using structure-based molecular docking of Fen1 targeted to its metal binding pocket M2 (Mg(2+) site), we have identified a potent low-molecular weight inhibitor (LMI, NSC-281680) that efficiently blocks LP-BER. In this study, we have demonstrated that the interaction of this LMI with Fen1 blocked its endonuclease activity, thereby blocking LP-BER and enhancing the cytotoxic effect of DNA-alkylating agent Temozolomide (TMZ) in mismatch repair (MMR)-deficient and MMR-proficient colon cancer cells. The results further suggest that blockade of LP-BER by NSC-281680 may bypass other drug resistance mechanisms such as mismatch repair (MMR) defects. Therefore, our findings provide groundwork for the development of highly specific and safer structure-based small molecular inhibitors targeting the BER pathway, which can be used along with existing chemotherapeutic agents, like TMZ, as combination therapy for the treatment of colorectal cancer.
Insights
Researchers identified a new inhibitor that blocks DNA repair in cancer cells. This approach enhances the effectiveness of chemotherapy drugs like Temozolomide (TMZ) and may overcome drug resistance in colorectal cancer treatment.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Therapeutics
Background:
- DNA alkylation is a key anticancer strategy, relying on cancer cells' heightened sensitivity to DNA damage and impaired repair.
- 5'-Flap endonuclease 1 (Fen1) is crucial for long-patch base excision repair (LP-BER), a pathway involved in DNA repair.
Purpose of the Study:
- To identify and characterize inhibitors of Fen1's endonuclease activity to enhance DNA-alkylating agent efficacy.
- To investigate the role of Asp181 in Fen1's Mg(2+) binding and endonuclease function.
- To evaluate the potential of a novel inhibitor (NSC-281680) in combination therapy for colorectal cancer.
Main Methods:
- Site-directed mutagenesis to probe Fen1's Asp181 residue.
- Structure-based molecular docking to identify low-molecular weight inhibitors (LMIs) targeting Fen1's active site.
- Assays to assess the effect of LMIs on Fen1 activity, LP-BER, and cytotoxicity of Temozolomide (TMZ) in colon cancer cells.
Main Results:
- Asp181 was identified as critical for Fen1's endonuclease activity, likely involved in Mg(2+) binding.
- A novel LMI, NSC-281680, was identified that potently inhibits Fen1 and blocks LP-BER.
- NSC-281680 enhanced the cytotoxic effects of TMZ in both mismatch repair (MMR)-deficient and MMR-proficient colon cancer cells.
- Inhibition of LP-BER by NSC-281680 showed potential to bypass MMR-mediated drug resistance.
Conclusions:
- Targeting Fen1 with specific inhibitors like NSC-281680 can enhance the efficacy of DNA-alkylating chemotherapy.
- Blocking LP-BER offers a promising strategy to overcome drug resistance mechanisms in cancer.
- This research supports the development of structure-based inhibitors for combination therapy in colorectal cancer.
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