Proteomic profiling of FEN1 inhibition by BSM-1516 reveals chromatin-associated biomarkers for preclinical pharmacodynamic evaluation

Flap endonuclease 1 (FEN1) is a structure-specific metallonuclease essential for Okazaki fragment maturation and DNA repair. We previously reported the discovery of BSM-1516, a potent and selective small-molecule FEN1 inhibitor that synergizes with PARP-targeted and other DNA damage response therapies and exhibits favorable in vivo pharmacokinetic properties. Pharmacologic inhibition of FEN1 increases its chromatin association, induces poly(ADP-Ribosyl)ation and ssDNA gaps, and is selectively cytotoxic to cells with homologous recombination deficiency. To characterize chromatin protein dynamics following FEN1 inhibition and identify potential pharmacodynamic (PD) biomarkers of target engagement, we employed isolation of Proteins On Nascent DNA (iPOND) coupled to mass spectrometry in proliferating cells treated with BSM-1516, alone or in combination with olaparib. FEN1 inhibition reproducibly enriched replication and DNA repair proteins, including FEN1, PARP1/2, LIG3, XRCC1, and CHD1L, reflecting PARP-dependent engagement of an alternative Okazaki fragment maturation pathway that was abrogated by co-treatment with olaparib. Orthogonal assays for chromatin-bound proteins confirmed selective enrichment of several iPOND-identified hits, establishing tractable PD biomarker candidates. Collectively, these findings delineate a proteomic signature of FEN1 inhibition at the replication forks and lay the groundwork for ongoing in vivo studies assessing these markers as indicators of target engagement in preclinical models.