Manno-Configured Epi-Cyclophellitol Aziridine Based Fluorescence Polarization Activity-Based Protein Profiling Identifies New Golgi A-Mannosidase Inhibitors

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Monday, July 1, 2019 - 12:00 to 12:15

Zachary Armstrong1, Daniël Lahav2, Rachel Johnson1, Chi-Lin Kuo2, Thomas J. M. Beenakker2, Casper de Boer2, Chung-Sing Wong2, Marjoke F. Debets2, Mario van der Stelt2, Jeroen Codée2, Johannes M. F. G. Aerts2, Liang Wu1, Hermen Overkleeft2, Gideon Davies1

1University Of York, York, United Kingdom, 2Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands

As part of the N-glycan processing pathway, Golgi mannosidase II (GMII) catalyzes the sequential hydrolysis of two mannosyl residues from GlcNAcMan₅GlcNAc₂ . The product of this hydrolysis (GlcNAcMan₃GlcNAc₂) is the precursor for all complex N-glycans, including the branched N-glycans associated with cancer. Inhibitors of GMII reduce the production of complex N-glycans and are therefore potential cancer therapeutics. Despite many studies targeting the inhibition of GMII, potent and selective inhibitors GMII over other alpha-mannosidases remain elusive, and α-mannosidosis-like symptoms due to inhibition of lysosomal alpha-mannosidase are therefore a major concern. It is in this context that we sought to design a fluorescence polarization screen for new GMII inhibitors. We synthesized both manno-epi-cyclophellitol epoxide and aziridine and demonstrated covalent modification and time-dependent inhibition of Drosophila melanogaster GMII (dGMII) with these inhibitors. The manno-epi-cyclophellitol aziridine was then used as a scaffold to design a fluorescent α-mannosidase activity based probe, which was implemented in a fluorescence polarization based screen for dGMII inhibitors. We identified 7 previously unknown inhibitors of dGMII from a library of over 350 iminosugars and investigated their binding modalities through X-ray crystallography. This revealed previously unobserved inhibitor binding modes and promising scaffolds for the generation of selective inhibitors.