Preclinical Evaluation of Protein Disulfide Isomerase Inhibitors for the Treatment of Glioblastoma

Cancer cells require increased rates of protein synthesis to sustain rapid cell growth and proliferation. Increased secretory and membrane protein synthesis relies on an upregulation of the translational and protein folding machinery in the endoplasmic reticulum to aid tumor growth. For example, many critical cancer signaling kinases, such as EGFR (epithelial growth factor receptor), function as membrane proteins. Protein disulfide isomerase (PDI) is the major enzyme responsible for disulfide bond formation in the endoplasmic reticulum, and knockdown of PDI halts tumor progression. Thus, the goal of this dissertation project was to identify novel PDI inhibitors and provide an extensive preclinical evaluation of their activity for the treatment of cancer, specifically glioblastoma. Through a phenotypic screening approach, we identified the pyrimidotriazinedione 35G8 as a potent cytotoxic agent that inhibited PDI. Because of its known pan-assay interference (PAINS) properties, we first validated that the activity of 35G8 was not due to its redox cycling characteristics and used a variety of assays to confirm PDI inhibition. 35G8 destabilized PDI in cell-based target-engagement assays and had a transcriptomic profile similar to PDI knockdown. These results demonstrated the ability of 35G8 to inhibit PDI and potently kill cancer cells. The chalcone BAP2 was also identified through a phenotypic screening approach, and an initial structure-activity relationship (SAR) campaign with 67 analogues revealed important binding characteristics that allowed us to hypothesize that the compounds were binding in the b’ domain of PDI. Mutation of His256 to Ala abolished BAP2 activity and confirmed the binding hypothesis. Furthermore, BAP2 and analogues inhibit glioblastoma cell growth, induce ER stress, increase expression of G2M checkpoint proteins, and reduce expression of DNA repair proteins. BAP2 and analogues also sensitized glioblastoma (GBM) cells to radiation. These results establish the BAP2 series as PDI inhibitors and support their further study as a novel strategy to treat glioblastoma. Finally, a manual biochemical screen of over 1,000 compounds in the PDI reductase assay produced a benzyl-benzodioxole, AS15, as a potent hit with an IC50 value under 1 μM. SAR analysis was performed with over 100 analogues of AS15. The SAR indicated that the compounds were binding PDI via a retro-Michael addition onto the cysteines, and protein mass spectrometry confirmed covalent binding. Cytotoxicity of the AS15 analogues was improved when combined with glutathione synthesis inhibitor buthionine sulfoximine (BSO), which confirmed that PDI competed with glutathione for binding the AS15 series in the cells. Thus, this study provides an excellent foundation to build analogues that are less sensitive to glutathione and more selective for PDI in the cells. The work as a whole provides an extensive characterization of PDI inhibition and its role in cancer. We were able to provide extensive preclinical evaluation of lead PDI inhibitors identified from medium throughput screens. This work provides the foundation for a guided optimization of the PDI inhibitors discovered to further improve the potency and selectivity of the compounds and design a PDI inhibitor for testing in clinical trials.PHDMedicinal ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/155125/1/ashergal_1.pd

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.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141828/1/cmdc201700629.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/141828/2/cmdc201700629-sup-0001-misc_information.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/141828/3/cmdc201700629_am.pd