Neu5Ac structure

The discovery of the potent in vitro sialidase inhibitory activity and in vivo efficacy of zanamivir 12, and the increasing availability of 3D structural data for influenza virus sialidases in the 1990s, particularly with Neu5Ac and various inhibitors bound into the active site, provided a platform for further drug discovery efforts targeting... 

Chan T-H, Xin Y-C, von Itzstein M (1997) Synthesis of phosphonic add analogs of siaUc acids (Neu5Ac and KDN) as potential sialidase inhibitors. J Org Chem 62 3500-3504 Chand P, Kotian PL, Dehghani A, El-Kattan Y, Lin T-H, Hutchison TL, Babu YS, Bantia S, Elliott AJ, Montgomery JA (2001) Systematic structure-based design and stereoselective synthesis of novel multisubstituted cyclopentane derivatives with potent antiinfluenza activity. J Med Chem 44 4379 392... 

In an interesting extension of this work, the Neu5Ac aldolase from E. coli was subjected to directed evolution to expand its catalytic activity for enantiomeric forms of the usual substrates to include A -acetyl-L-mannosamine and L-arabinose with formation of the synthetically important products L-sialic add and L-3-deoxy-L-manno-oct-2-ulosonic add (l-KDO) (163). The evolved Neu5Ac aldolases were characterized by sequence analysis, kinetics, stereoselectivity, and in one case even by an X-ray structure analysis. Again, remote mutations were identified. It is significant... 

The structures of TV-acetyl neuraminic acid (sialic acid Neu5Ac) and the 2-deoxy-2,3-dehydro-/V-acetyl neuraminic acid (Neu5Ac2en) inhibitor complexed with N2 neuraminidase [66] revealed the nature of the interactions of the... 

The X-ray crystal structure of virus sialidase was first solved in early 1980s.24,25 While the active site could be identified, resolution was insufficient to determine the orientation of the bound ligand (Neu5Ac) within the catalytic center. Subsequent efforts in protein crystallography allowed further refinement... 

The discovery of Zanamivir as a potent and selective inhibitor of influenza virus sialidase prompted several researchers to investigate the synthesis and structure-activity relationship studies of Neu5Ac2en-based compounds as potential sialidase inhibitors. Exploration of these SAR studies were undertaken to optimize inhibitory activity and to improve the physicochemical properties of the sialic acid-based influenza virus sialidase inhibitor. A few in vitro assays are commonly employed to measure the effectiveness of influenza virus sialidase inhibitors. The first involves a fluorometric assay that measures release of a synthetic fluorophore following its cleavage from Neu5Ac by sialidase. Dye-uptake assay, such as the Neutral Red uptake assay, measures the uptake of a vital stain, Neutral Red in cell culture. The process requires intact membranes and active metabolism in the cell, and is expressed as percent protective rate against virus infection. The plaque-reduction assay is used to measure sialidase inhibition indirectly in cell culture, and provides some measure of the inhibitor s effect on the viability of the influenza virus. In vitro and in vivo systems for analysis of inhibitors of influenza virus enzymes have been reviewed.71... 

Fig. 6. Structures and inhibitory activity of C-4 modified Neu5Ac derivatives against influenza A virus sialidase. values are normalized against that of Neu5Ac2en (A = 1.0).

X-ray crystal structure studies of influenza A/N2, A/N9, and B sialidases bound with a-Neu5Ac [66] show that the active site contains 18 invariant amino acid residues that either interact with the bound a-Neu5Ac or support these residues. These residues are conserved in all strains of influenza A and B viruses, suggesting their involvement in the enzymatic activity [65,66], The residues helped define the topology of the active site [66, 67], Of those conserved amino acids interacting with the substrate, many are polar, but there are also a number of nonpolar residues... 

FIGURE 17.6 X-ray structures of influenza virus sialidases. (a) Structure of influenza B virus sialidase tetramer (PDB 1 a4g) viewed from the top [62]. (b) Structure of influenza A virus sialidase N9 monomer (PDB lmwe) with a-Neu5Ac (shown as spheres) bound in the active site [63]. 

The mechanism by which influenza virus sialidases cleaves the Neu5Ac(a2 3)Gal or Neu5Ac(a2- 6)Gal linkage has been a topic of much interest for many years (e.g., see [71, 72]). Recently, it has been shown by structural analysis [73] that it involves a covalent enzyme-substrate intermediate as has been reported for other sialidases. A proposed mechanism is depicted in Scheme 17.1. 

X-ray crystal structures of a-Neu5Ac and 11 in complex with influenza virus sialidases in the 1980s and early 1990s led to the opportunity for structure-based design and development of influenza virus sialidase inhibitors [66, 85],... 

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