Pyrazolone-based ERO1 inhibitors in ERO1-driven Triple-Negative Breast Cancer and SEPN1-Related Myopathy: Structure-activity relationship and therapeutic potential

: Endoplasmic reticulum oxidoreductin 1 alpha (ERO1A) is a disulfide oxidase that facilitates oxidative protein folding by reoxidizing protein disulfide isomerase (PDI), a process essential for maintaining endoplasmic reticulum (ER) homeostasis. Under ER stress, ERO1A expression is upregulated via the unfolded protein response (UPR), promoting cell survival. However, sustained ERO1A activity can impair proteostasis and contribute to disease. Notably, ERO1A is overexpressed in triple-negative breast cancer (TNBC), where it supports tumor growth and adaptation to hypoxia, and in SEPN1-related myopathy, a rare congenital muscle disorder linked to ER and oxidative stress. To investigate ERO1A as a therapeutic target, we conducted a structure-activity relationship (SAR) study of EN460-based pyrazolone inhibitors. Forty derivatives and three EN460 salts were synthesized to optimize potency and solubility. In vitro and cell-based assays revealed that effective inhibition required covalent binding to Cys397, interactions with Arg287 and Trp200, and distortion of the phenyl ring. While sulfonic acid substitution improved solubility, it abolished activity by disrupting key interactions. The most potent compound, I29, featuring a mono ortho-fluorine substitution, demonstrated improved inhibitory activity (IC₅₀ = 2.6µM) and efficacy in preclinical models of TNBC and SEPN1-related myopathy. These findings highlight ERO1A's pathological role in cancer and congenital muscle disease and support its inhibition as a promising therapeutic strategy for conditions characterized by chronic ER and oxidative stress.