doi:10.1038/s41467-023-36598-7engMadriz Sørensen, D.Büll, C.Madsen, T. D.Lira-Navarrete, E.Clausen, T. M.Clark, A. E.Garretson, A. F.Karlsson, R.Pijnenborg, J. F. A.Yin, X.Miller, R. L.Chanda, S. K.Boltje, T. J.Schjoldager, K. T.Vakhrushev, S. Y.Halim, A.Esko, J. D.Carlin, A. F.Hurtado-Guerrero, R.Weigert, R.Clausen, H.Narimatsu, Y.Identification of global inhibitors of cellular glycosylationART-2023-132938Small 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-2.2023http://zaguan.unizar.es/record/12530010.1038/s41467-023-36598-7http://zaguan.unizar.es/record/125300oai:zaguan.unizar.es:125300info:eu-repo/grantAgreement/ES/DGA-FEDER/E34-R17info:eu-repo/grantAgreement/ES/DGA/LMP58-18info:eu-repo/grantAgreement/EC/FP7/609409/EU/Supporting transnational mobility within the European life sciences by cofunding of the EMBO Fellowship Programme/LTFCOFUND2013info:eu-repo/grantAgreement/EC/H2020/787684/EU/Generation of a Cell-Based Sialoglycan Array to Decipher Biological Interactions and Functions of the Human Sialome/Sialoglycan ArrayThis project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No H2020 787684-Sialoglycan Arrayinfo:eu-repo/grantAgreement/ES/MICINN/BFU2016-75633-Pinfo:eu-repo/grantAgreement/ES/MICINN/PID2019-105451GB-I00Nature communications 14 (2023), 948 [19 pp.]byhttp://creativecommons.org/licenses/by/3.0/es/info:eu-repo/semantics/openAccess