Manno-epi-cyclophellitols Enable Activity-Based Protein Profiling of Human α-Mannosidases and Discovery of New Golgi Mannosidase II Inhibitors

Abstract

Golgi mannosidase II (GMII) catalyzes the sequential hydrolysis of two mannosyl residues from GlcNAc-Man 5 GlcNAc 2 to produce GlcNAcMan 3 GlcNAc 2 , the precursor for all complex N-glycans, including the branched N-glycans associated with cancer. Inhibitors of GMII are potential cancer therapeutics, but their usefulness is limited by off-target effects, which produce α-mannosidosis-like symptoms. Despite many structural and mechanistic studies of GMII, we still lack a potent and selective inhibitor of this enzyme. Here, we synthesized manno-epicyclophellitol epoxide and aziridines and demonstrate their covalent modification and time-dependent inhibition of GMII. Application of fluorescent manno-epi-cyclophellitol aziridine derivatives enabled activity-based protein profiling of α-mannosidases from both human cell lysate and mouse tissue extracts. Synthesized probes also facilitated a fluorescence polarization-based screen for dGMII inhibitors. We identified seven previously unknown inhibitors of GMII from a library of over 350 iminosugars and investigated their binding modalities through X-ray crystallography. Our results reveal previously unobserved inhibitor binding modes and promising scaffolds for the generation of selective GMII inhibitors.

Enzymatic Assemblies of Thiophosphopeptides Instantly Target Golgi Apparatus and Selectively Kill Cancer Cells**

“…Although Golgi is emerging as an important target for cancer therapy, there are, however, few approaches for targeting Golgi [3] . While Golgi mannosidase II inhibitors are able to inhibit cancer cells, the selectivity [4] or efficacy [3a] of the inhibitors remains to be improved. In addition, several studies reported the imaging of Golgi, including the commercial dyes for staining Golgi, [5] a smart “off‐on” fluorescence probe for imaging the Golgi in cancer cells, [6] and carbon quantum dots localizing at Golgi [7] .…”

Chemistry for the Identification of Therapeutic Targets: Recent Advances and Future Directions

“…In addition, these ABPs ( Figure 4A) have made possible the throughput profiling of α-mannosidases from both human cell lysates and mouse tissue extracts. [20] As a part of glycoside hydrolase family, α-Lfucosidase catalyzes the hydrolytic removal of L-fucose residues from glycoconjugates. The lack or diminished activity of this enzyme is related to several pathologies, ranging from cystic fibrosis to genetic disorders.…”

“…The development of manno‐epi‐cyclophellitol epoxide and aziridines covalent ABPs has enabled, for the first time, the identification of several promising scaffolds for the generation of selective GMII inhibitors. In addition, these ABPs (Figure 4A) have made possible the throughput profiling of α‐mannosidases from both human cell lysates and mouse tissue extracts [20] . As a part of glycoside hydrolase family, α‐L‐fucosidase catalyzes the hydrolytic removal of L‐fucose residues from glycoconjugates.…”

N-Alkylated Iminosugar Based Ligands: Synthesis and Inhibition of Human Lysosomal β-Glucocerebrosidase

“…In general, representatives of this substance class are known as ligands for carbohydrate-processing enzymes (CPEs), as they interact with the active site of the respective protein as mimics of the natural substrate [ 7 , 8 , 9 , 10 ]. Iminosugar-based structures are versatile probes and have been used in several different applications as inhibitors, [ 11 , 12 ] therapeutics [ 8 , 9 , 10 ] and probes for activity-based protein profiling [ 13 , 14 ]. In particular, these structures have proved interesting for the therapy of CPE- associated diseases such as cancer [ 15 ], bacterial infections [ 16 ], HIV [ 17 ], HPV [ 18 ], influenza [ 19 ], hepatitis [ 20 ], the dengue virus [ 21 ], malaria [ 22 ] and fungal infections [ 23 ].…”