Functionalized High Mannose‐Specific Lectins for the Discovery of Type I Mannosidase Inhibitors

Abstract

An engineered cyanovirin‐N homologue that exhibits specificity for high mannose N‐glycans has been constructed to aid type I α 1,2‐mannosidase inhibitor discovery and development. Engineering the lectins C‐terminus permitted facile functionalization with fluorophores via a sortase and click strategy. The resulting lectin constructs exhibit specificity for cells presenting high mannose N‐glycans. Importantly, these lectin constructs can also be applied to specifically assess changes in cell surface glycosylation induced by type I mannosidase inhibitors. Testing the utility of these lectin constructs led to the discovery of type I mannosidase inhibitors with nanomolar potency. Cumulatively, these findings reveal the specificity and utility of the functionalized cyanovirin‐N homologue constructs, and highlight their potential in analytical contexts that require high mannose‐specific lectins.

Lectin Drug Conjugates Targeting High Mannose N‐Glycans

“…To determine the ability of Cyt-CVNH to bind to high mannose presenting cancer cells, we utilized a fluorophore labelled Cyt-CVNH derivative (i. e., Cyt-CVNH MB 488) that we have previously described and applied in assays to demonstrate the presence of high mannose N-glycans on cells. [10] Here, Cyt-CVNH MB 488 was applied to assess the ability of Cyt-CVNH to bind to triple negative breast cancer (TNBC) MDA-MB-231 cells, lung adenocarcinoma A549 cells, and adenocarcinoma HT-29 cells, which have been previously described as high mannose presenting cancer cell lines by Matoba and co-workers. [9] Using flow cytometry, we observed that Cyt-CVNH MB 488 readily bound to MDA-MB-231, A549, and HT-29 cells (Figure 3 A).…”

“…[19] To test this hypoth- ChemBioChem esis, we utilized TNBC MDA-MB-468 cells, as this cell line presents low levels of high mannose N-glycans, and the kifunensine analogue JDW-II-010 (5) to inhibit type I mannosidase (MAN I) enzymes. [10] When MDA-MB-468 cells were treated with 5 a significant increase in the binding of Cyt-CVNH MB 488 to the cells was observed by flow cytometry (Figure 4A), indicating that 5 increased the abundance of high mannose Nglycans. Next, we determined if treating MDA-MB-468 cells with 5 altered the sensitivity of these cells to Cyt-CVNH-GGG (4) and the LDCs 3 a and 3 b (Figure 4B-C).…”

“…Previously, we engineered a high-mannose N-glycan specific cyanovirin-N homologue from the cyanobacterium Cyano-thece7424 (1, Cyt-CVNH). [10] The engineered lectin (1) demonstrates specificity for cells presenting high-mannose N-glycans and can be functionalized at the C-terminus via a sortase ligation without disrupting the carbohydrate binding properties of the lectin. As such, we reasoned that this lectin would be an ideal candidate to enable the delivery of cytotoxic agents to high mannose N-glycan presenting malignant cells.…”

“…Previously, we engineered a high‐mannose N‐glycan specific cyanovirin‐N homologue from the cyanobacterium Cyanothece7424 ( 1 , Cyt‐CVNH) [10] . The engineered lectin ( 1 ) demonstrates specificity for cells presenting high‐mannose N‐glycans and can be functionalized at the C‐terminus via a sortase ligation without disrupting the carbohydrate binding properties of the lectin.…”

More Is Always Better Than One: The N-Terminal Domain of the Spike Protein as Another Emerging Target for Hampering the SARS-CoV-2 Attachment to Host Cells

“…NMR analysis allowed the characterization of the specific glycan epitopes recognized by each lectin, and paves the basis to unveil the roles played by glycosylation patterns in the interaction with receptors during infection. Another paper suggests that RBD can be put into relation with lectins, as a red-alga-derived lectin, griffithsin (GRFT), inhibits SARS-CoV-2 infection by targeting the glycosylation sites in the RBD of the SARS-CoV-2 S protein [ 23 , 24 ]. Although of interest, all these studies border their attention on the RBD of the S protein, despite the evidence that its extracellular portion is also constituted by the N-terminal domain ( Figure 1 —S1-NTD) encompassing residues 20–286.…”