Characterization of Aminobenzylphenols as Protein Disulfide Isomerase Inhibitors in Glioblastoma Cell Lines

Abstract

Disulfide bond formation is a critical post-translational modification of newly synthesized polypeptides in the oxidizing environment of the endoplasmic reticulum and is mediated by protein disulfide isomerase (PDIA1). In this study, we report a series of α-aminobenzylphenol analogues as potent PDI inhibitors. The lead compound, AS15, is a covalent nanomolar inhibitor of PDI, and the combination of AS15 analogues with glutathione synthesis inhibitor buthionine sulfoximine (BSO) leads to synergistic cell growth inhibition. Using nascent RNA sequencing, we show that an AS15 analogue triggers the unfolded protein response in glioblastoma cells. A BODIPY-labeled analogue binds proteins including PDIA1, suggesting that the compounds are cell-permeable and reach the intended target. Taken together, these findings demonstrate an extensive biochemical characterization of a novel series of highly potent reactive small molecules that covalently bind to PDI.

Thiol Isomerases Orchestrate Thrombosis and Hemostasis

“…In parallel to PDI inhibitors identified after screening for neurodegenerative or cancer diseases, several groups have conducted high throughput screening in which the primary screen consisted of PDI reductase assay. This is the case for AS15, an aminobenzylphenol compound which covalently binds and inhibits PDI at nanomolar concentrations and decreases cell proliferation of Glioblastoma cell lines 123 . In a similar approach, this same group has identified 35G8, which is another nanomolar inhibitor of PDI that also inhibits proliferation of Glioblastoma cell lines 124 .…”

“…119 , 35G8124 , AS15123 , BAP1 and BAP2128 , CCF642121, E64FC26 78 , KSC-34 129 , LOC14 120 , Origamicin 130 , Securinine 131 , SK053 132 and STK076545 133 . Recent data from our lab suggests that LOC14 exerts anti-platelet effects, while CCF642 and 16F16 do not alter platelet function (data not shown).…”

Glioblastoma: Current Status, Emerging Targets, and Recent Advances

“…In 2020, Shergalis et al published some aminobenzophenol-based scaffolds as PDI inhibitors to treat GBM. 72 First, the authors screened approximately 1000 compounds from the National Cancer Institute and found 94 and 95 as potent lead compounds (PDI reductase assay), with IC 50 = 300 and 90 nM, respectively. The leads were also evaluated against U-87MG GBM cell lines, in which both compounds inhibited cell growth, with IC 50 = 18.3 μM for 94 and 10.6 μM for 95 .…”

Roles of Protein Disulfide Isomerase in Breast Cancer

“…Most PDI inhibitors are known to or likely to bind to catalytic sites of the a or a' domain of PDI: they include PACMA31 [114], P1 [115], 16F16 [116], AS15 [117], CCF642 [118], S-CW3554 [119], Origamicin [120], (±)-dMtcyDTDO [121], Ga-1 [122], 35G8 [123], Copper (II) complex 1 [124], and SK053 [125]. PACMA31 belongs to the class of propynoic acid carbamoyl methyl amides (PACMAs), and it covalently binds to the Cys400 of the active site of PDI, and it shows cytotoxicity on a broad range of human cancer cells [81].…”

“…16F16 is another small-molecule irreversible PDI inhibitor that binds to the cysteines in the active site via a chloroacetamide electrophile, and it suppresses apoptosis in cell and brain slice models of Huntington disease [116]. AS15 was recently identified as a covalent nanomolar PDI inhibitor that shows synergistic growth inhibition of glioblastoma cells when treated with the glutathione synthesis inhibitor buthionine sulfoximine (BSO) [117]. CCF642, S-CW3554, and Origamicin are also irreversible PDI inhibitors, showing cytotoxicity in multiple myeloma cells (CCF642 and S-CW3554) [118,119] and in neuroblastoma cells (Origamicin) [120].…”