Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, Copenhagen, Denmark.
Department of Biomolecular Chemistry, Institute for Molecules and Materials, Radboud University, 6525 AJ, Nijmegen, The Netherlands.
Nat Commun. 2023 Feb 20;14(1):948. doi: 10.1038/s41467-023-36598-7.
Small molecule inhibitors of glycosylation enzymes are valuable tools for dissecting glycan functions and potential drug candidates. Screening for inhibitors of glycosyltransferases are mainly performed by in vitro enzyme assays with difficulties moving candidates to cells and animals. Here, we circumvent this by employing a cell-based screening assay using glycoengineered cells expressing tailored reporter glycoproteins. We focused on GalNAc-type O-glycosylation and selected the GalNAc-T11 isoenzyme that selectively glycosylates endocytic low-density lipoprotein receptor (LDLR)-related proteins as targets. Our screen of a limited small molecule compound library did not identify selective inhibitors of GalNAc-T11, however, we identify two compounds that broadly inhibited Golgi-localized glycosylation processes. These compounds mediate the reversible fragmentation of the Golgi system without affecting secretion. We demonstrate how these inhibitors can be used to manipulate glycosylation in cells to induce expression of truncated O-glycans and augment binding of cancer-specific Tn-glycoprotein antibodies and to inhibit expression of heparan sulfate and binding and infection of SARS-CoV-2.
糖基化酶的小分子抑制剂是解析聚糖功能和潜在药物候选物的有价值的工具。糖基转移酶抑制剂的筛选主要通过体外酶测定进行，但将候选物转移到细胞和动物中存在困难。在这里，我们通过使用表达定制报告糖蛋白的糖基工程细胞的基于细胞的筛选测定法来避免这一问题。我们专注于 GalNAc 型 O-糖基化，并选择了 GalNAc-T11 同工酶作为靶点，该同工酶选择性糖基化内吞性低密度脂蛋白受体 (LDLR) 相关蛋白。我们对有限的小分子化合物文库进行了筛选，但没有鉴定出 GalNAc-T11 的选择性抑制剂，然而，我们鉴定出两种广泛抑制高尔基定位糖基化过程的化合物。这些化合物介导了高尔基体系统的可逆碎片化，而不影响分泌。我们展示了如何使用这些抑制剂在细胞中操纵糖基化，以诱导截断 O-聚糖的表达，并增强对癌症特异性 Tn-糖蛋白抗体的结合和 SARS-CoV-2 的结合和感染。
