Abstract 7148: Small molecule inhibitor of FEN1 nuclease utilizing a novel metal-binding pharmacophore synergizes with inhibitors of USP1, PARP, PARG and ATR
Authors: Munguia Jason , Agarwalla Sanjay , Guo Elaine , Martin Dave , Fan Junhua , Lonergan Dave , Puerta David , Zimmerman Zachary , Taganov Konstantin 
Published: March 2024

Flap endonuclease 1 (FEN1) is a structure-specific metallonuclease that cleaves 5’ DNA flaps during replication and repair. FEN1 is an attractive target for development of anticancer therapeutics because it is overexpressed in many tumor types and has a large number of synthetic lethality partners including genes in Homologous Recombination (HR) pathway (Mengwasser et al., 2019; Guo et al., 2020).

Utilizing fragment-based drug discovery approach we identified a novel metal-binding pharmacophore that binds to the two magnesium ions in the FEN1 active site. Further elaboration using fragment growth strategies resulted in highly potent and selective inhibitors. The current lead (BSM-1516) is ~65-fold more potent against FEN1 than its related enzyme Exonuclease 1 (Exo1) in biochemical assays (IC50 of 7 nM and 460 nM, respectively), an improvement of more than an order of magnitude in selectivity compared to earlier efforts. FEN1 target engagement in live cells was validated by cellular thermal shift assay (CETSA EC50 of 24 nM). Inhibition of FEN1 led to its increased association with chromatin in S-phase cells and recruitment of PARP1 enzyme.

In clonogenic assay, BRCA2-deficient DLD1 cells were ~15-fold more sensitive to FEN1 inhibition than their isogenic BRCA2-wild-type counterparts (EC50 of 350 nM and 5 µM, respectively), confirming the increased susceptibility of HR deficient cancer cells to FEN1 inhibition. Treatment of BRCA2-deficient but not wild-type DLD1 cells with BSM-1516 resulted in cell cycle arrest and dose-dependent decrease of S-phase BrdU incorporation into DNA. Cell cycle arrest of BRCA2-deficient DLD1 cells was accompanied by DNA damage signaling and by accumulation of chromatin-bound RPA32, a marker of ssDNA.

To explore synthetic lethal interactions of FEN1 with other DNA repair genes we performed FEN1-inhibitor-anchored CRISPR screen. This analysis revealed that in addition to HR pathway inactivation, genomic perturbations in EXO1, USP1 and PARP1 genes sensitized cells to FEN1 inhibition. Synergistic relationships of BSM-1516 and its combination potential were further explored in viability studies with a panel of DDR inhibitors (n=25) in BRCA2-proficient and deficient cell lines. Strong synergy was identified with multiple drug classes that included inhibitors of USP1 (KSQ-4279), PARP (Olaparib, Niraparib, Talazoparib, AZD5305), PARG (PDD 00017273) and ATR (AZD6738, VE-822, Elimusertib).

In vitro ADME assays and in vivo PK studies showed that BSM-1516 has properties suitable for in vivo testing, either as a single agent or in combination with synergistic DDR inhibitors, an investigation that is currently underway.

Citation Format: Jason Munguia, Sanjay Agarwalla, Elaine Guo, Dave Martin, Junhua Fan, Dave Lonergan, David Puerta, Zachary Zimmerman, Konstantin Taganov. Small molecule inhibitor of FEN1 nuclease utilizing a novel metal-binding pharmacophore synergizes with inhibitors of USP1, PARP, PARG and ATR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7148.