Biophysical and computational characterization identifies FSTL1 as a novel binding target of doxorubicin

Mapping the protein-binding landscape of doxorubicin (DOX) is pivotal for optimizing therapeutic efficacy and overcoming resistance. In this study, we integrated data from prior protein microarray screenings that identified 363 DOX-binding proteins (with a signal-to-noise ratio (SNR) > 4) along with 224 Homo sapiens expression profiling series obtained from the Gene Expression Omnibus (GEO). Cross-analysis indicated that follistatin-like 1 (FSTL1) consistently demonstrated bidirectional expression across three GEO series (GSE202536, GSE13477, GSE155478), indicating sensitivity to DOX. Specifically, FSTL1 expression was suppressed in resistant cell lines (CAL-51/DOX, MCF-7/DOX) and restored after DOX treatment in their sensitive parental counterparts. Protein microarray analysis confirmed a strong interaction (SNR = 5.763), which was further corroborated by surface plasmon resonance (SPR) assays, indicating micromolar affinity. AlphaFold3 modeling combined with docking and 400 ns molecular dynamics simulations demonstrated that hydrophobic interactions and hydrogen bonding stabilize the FSTL1-DOX complex. TCGA-BRCA analysis revealed decreased FSTL1 expression in tumor tissues, modest diagnostic discrimination between tumor and normal samples (AUC = 0.76), and improved overall survival among patients with elevated FSTL1 expression. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses notably associated FSTL1 with extracellular matrix organization and the PI3K-Akt signaling pathway, supporting its potential relevance as a biomarker candidate and mechanistic hypothesis for anthracycline responsiveness.