Department of Biochemical Science "A. Rossi Fanelli", Faculty of Farmacy and Medicine, Sapienza University of Rome, Pl. A. Moro 5, 00185 Rome, Italy.
Department of Bioscience and Agro-Food and Environmental Technology, University of Teramo, Campus "Aurelio Saliceti", Via R. Balzarini 1, 64100 Teramo, Italy.
Int J Mol Sci. 2024 Sep 30;25(19):10531. doi: 10.3390/ijms251910531.
PDIA3 is a pleiotropic protein primarily located in the endoplasmic reticulum where it is involved in protein folding, catalyzing the formation, breakage, and rearrangement of disulfide bonds. PDIA3 is implicated in numerous pathologies such as cancer, inflammation, and neurodegeneration. Although punicalagin has been proven to be a highly promising PDIA3 inhibitor and can be used as target protein in glioblastoma, it does not have sufficient selectivity for PDIA3 and is a quite-large molecule. With the aim of finding punicalagin derivatives with a simplified structure, we selected punicalin, which lacks the hexahydroxy-diphenic acid moiety. Previous docking studies suggest that this part of the molecule is not involved in the binding with PDIA3. In this study we compared the ability of punicalin to bind and inhibit PDIA3 and PDIA1. Tryptophan fluorescence quenching and disulfide reductase activity (using both glutathione and insulin as substrates) were evaluated, demonstrating the ability of punicalin to bind and inhibit PDIA3 even to a lesser extent compared to punicalagin. On the other hand, punicalin showed a very low inhibition activity towards PDIA1, demonstrating a higher selectivity for PDIA3. Protein thermal shift assay evidenced that both proteins can be destabilized by punicalin as well as punicalagin, with PDIA3 much more sensitive. Additionally, punicalin showed a higher change in the thermal stability of PDIA3, with a shift up to 8 °C. This result could explain the presence of PDIA3 aggregates, evidenced by immunofluorescence analysis, that accumulate within treated cells and that are more evident in the presence of punicalin. The results here obtained show punicalin is able to bind both proteins but with a higher selectivity for PDIA3, suggesting the possibility of developing new molecules with a simplified structure that are still able to selectively bind and inhibit PDIA3.
PDIA3 是一种多功能蛋白，主要位于内质网中，参与蛋白质折叠，催化二硫键的形成、断裂和重排。PDIA3 与许多病理学有关，如癌症、炎症和神经退行性疾病。虽然鞣花酸已被证明是一种很有前途的 PDIA3 抑制剂，并可用作神经胶质瘤的靶蛋白，但它对 PDIA3 的选择性不够，而且是一个相当大的分子。为了寻找结构简化的鞣花酸衍生物，我们选择了缺乏六羟基二苯酸部分的鞣花单宁。以前的对接研究表明，该分子的这一部分不参与与 PDIA3 的结合。在这项研究中，我们比较了鞣花单宁结合和抑制 PDIA3 和 PDIA1 的能力。色氨酸荧光猝灭和二硫键还原酶活性（使用谷胱甘肽和胰岛素作为底物）进行了评估，结果表明鞣花单宁能够结合和抑制 PDIA3，即使与鞣花酸相比，其结合和抑制 PDIA3 的能力也有所降低。另一方面，鞣花单宁对 PDIA1 的抑制活性非常低，对 PDIA3 的选择性更高。蛋白质热转移测定表明，鞣花酸和鞣花酸都可以使两种蛋白质失稳，PDIA3 更为敏感。此外，鞣花单宁使 PDIA3 的热稳定性发生更高的变化，上升了 8°C。这一结果可以解释免疫荧光分析所证实的 PDIA3 聚集体的存在，这些聚集体在处理过的细胞内积累，在鞣花单宁存在下更为明显。这里获得的结果表明，鞣花单宁能够结合两种蛋白质，但对 PDIA3 的选择性更高，这表明有可能开发出具有简化结构的新分子，仍然能够选择性地结合和抑制 PDIA3。
