Discovery and Mechanistic Elucidation of a Class of Protein Disulfide Isomerase Inhibitors for the Treatment of Glioblastoma

Protein disulfide isomerase (PDI) is overexpressed in glioblastoma, the most aggressive form of brain cancer, and folds nascent proteins responsible for the progression and spread of the disease. Herein we describe a novel nanomolar PDI inhibitor, pyrimidotriazinedione 35G8, that is toxic in a panel of human glioblastoma cell lines. We performed a medium‐throughput 20 000‐compound screen of a diverse subset of 1 000 000 compounds to identify cytotoxic small molecules. Cytotoxic compounds were screened for PDI inhibition, and, from the screen, 35G8 emerged as the most cytotoxic inhibitor of PDI. Bromouridine labeling and sequencing (Bru‐seq) of nascent RNA revealed that 35G8 induces nuclear factor‐like 2 (Nrf2) antioxidant response, endoplasmic reticulum (ER) stress response, and autophagy. Specifically, 35G8 upregulated heme oxygenase 1 and solute carrier family 7 member 11 (SLC7A11) transcription and protein expression and repressed PDI target genes such as thioredoxin‐interacting protein 1 (TXNIP) and early growth response 1 (EGR1). Interestingly, 35G8‐induced cell death did not proceed via apoptosis or necrosis, but by a mixture of autophagy and ferroptosis. Cumulatively, our data demonstrate a mechanism for a novel PDI inhibitor as a chemical probe to validate PDI as a target for brain cancer. Iron‐clad PDI inhibition: We describe a nanomolar, cytotoxic protein disulfide isomerase (PDI) inhibitor, 35G8, that is potent in a panel of human glioblastoma cell lines. Bromouridine‐labeling and sequencing of nascent RNA revealed that 35G8 induces Nrf2 antioxidant response, endoplasmic reticulum stress response, autophagy, and may induce cell death via ferroptosis.