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剖析聚糖功能和潜在候选药物的宝贵工具。糖基转移酶抑制剂的筛选主要通过体外酶测定进行，但难以将候选物转移到细胞和动物中。在这里，我们通过使用表达定制报告糖蛋白的糖工程细胞进行基于细胞的筛选测定来规避这一点。我们关注 GalNAc 型 O 糖基化，并选择选择性糖基化内吞低密度脂蛋白受体 (LDLR) 相关蛋白的 GalNAc-T11 同工酶作为靶标。我们对有限小分子化合物库的筛选并未鉴定出 GalNAc-T11 的选择性抑制剂，但是，我们鉴定了两种广泛抑制高尔基体定位糖基化过程的化合物。这些化合物介导高尔基体系统的可逆断裂而不影响分泌。我们展示了如何使用这些抑制剂来操纵细胞中的糖基化，以诱导截短的 O-聚糖的表达并增强癌症特异性 Tn 糖蛋白抗体的结合，并抑制硫酸乙酰肝素的表达以及 SARS-CoV-2 的结合和感染。