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Neuraminidase-1 (NEU1) is the predominant sialidase expressed in human airway epithelia and lung microvascular endothelia where it mediates multiple biological processes. We tested whether the NEU1-selective sialidase inhibitor, C9-butyl-amide-2-deoxy-2,3-dehydro-N-acetylneuraminic acid (C9-BA-DANA), inhibits one or more established NEU1-mediated bioactivities in human lung cells. We established the IC50 values of C9-BA-DANA for total sialidase activity in human airway epithelia, lung microvascular endothelia and lung fibroblasts to be 3.74 µM, 13.0 µM and 4.82 µM, respectively. In human airway epithelia, C9-BA-DANA dose-dependently inhibited flagellin-induced, NEU1-mediated mucin-1 ectodomain desialylation, adhesiveness for Pseudomonas aeruginosa and shedding. In lung microvascular endothelia, C9-BA-DANA reversed NEU1-driven restraint of cell migration into a wound and disruption of capillary-like tube formation. NEU1 and its chaperone/transport protein, protective protein/cathepsin A (PPCA), were differentially expressed in these same cells. Normalized NEU1 protein expression correlated with total sialidase activity whereas PPCA expression did not. In contrast to eukaryotic sialidases, C9-BA-DANA exerted far less inhibitory activity for three selected bacterial neuraminidases (IC50 > 800 µM). Structural modeling of the four human sialidases and three bacterial neuraminidases revealed a loop between the seventh and eighth strands of the β-propeller fold, that in NEU1, was substantially shorter than that seen in the six other enzymes. Predicted steric hindrance between this loop and C9-BA-DANA could explain its selectivity for NEU1. Finally, pretreatment of mice with C9-BA-DANA completely protected against flagellin-induced increases in lung sialidase activity. Our combined data indicate that C9-BA-DANA inhibits endogenous and ectopically expressed sialidase activity and established NEU1-mediated bioactivities in human airway epithelia, lung microvascular endothelia, and fibroblasts in vitro and murine lungs in vivo., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

People throughout the world continue to be at risk for death from influenza A virus, which is always creating a new variant. Here we present a new effective and specific anti-influenza viral neuraminidase (viNA) inhibitor, 9-cyclopropylcarbonylamino-4-guanidino-Neu5Ac2en (cPro-GUN). Like zanamivir, it is highly effective against N1-N9 avian and N1-N2 human viNAs, including H274Y oseltamivir-resistant N1 viNA, due to its C-6 portion still being anchored in the active site, different from the disruption of oseltamivir's C-6 anchoring by H274Y mutation. Unlike zanamivir, no sialidase inhibitory activity has been observed for cPro-GUN against huNeu1-huNeu4 enzymes. Broad efficacy of cPro-GUN against avian and human influenza viruses in cell cultures comparable to its sialidase inhibitory activities makes cPro-GUN ideal for further development for safe therapeutic or prophylactic use against both seasonal and pandemic influenza.

We here report the design and synthesis of selective human lysosomal sialidase (NEU1) inhibitors. A series of amide-linked C9 modified DANA (2-deoxy-2,3-dehydro-N-acetylneuraminic acid) analogues were synthesized and their inhibitory activities against all four human sialidases (NEU1-NEU4) were determined. Structure-based approach was used to investigate the basis of selectivity of the compounds with experimentally observed activity. Results from the present study are found to be informative in a qualitative manner for the further design of isoform selective human sialidase inhibitors for therapeutic value.

Four types of human sialidases have been cloned and characterized at the molecular level. They are classified according to their major intracellular location as intralysomal (NEU1), cytosolic (NEU2), plasma membrane (NEU3) and lysosomal or mitochondrial membrane (NEU4) associated sialidases. These human isoforms are distinct from each other in their enzymatic properties as well as their substrate specificity. Altered expression of sialidases has been correlated with malignant transformation of cells and different sialidases have been known to behave differently during carcinogenesis. Particularly, increased expression of NEU3 has been implicated in the survival of various cancer cells and also in the development of insulin resistance. In the present study, we have modeled three-dimensional structures of NEU1, NEU3 and NEU4 based on the crystal structure of NEU2 using the homology modeling program MODELER. The best model in each enzyme case was chosen on the basis of various standard protein analysis programs. Predicted structures and the experimental protein-ligand complex of NEU2 were compared to identify similarities and differences among the active sites. The molecular electrostatic potential (MEP) was calculated for the predicted models to identify the differences in charge distribution around the active site and its vicinity. The primary objective of the present work is to identify the structural differences between the different isoforms of human sialidases, namely NEU1, NEU2, NEU3 and NEU4, thus providing a better insight into the differences in the active sites of these enzymes. This can in turn guide us in the better understanding and rationale of the differential substrate recognition and activity, thereby aiding in the structure-based design of selective NEU3 inhibitors.