Influenza virus sialidase substrates

Influenza Virus Sialidase Substrate Binding and Active Site 298... 

Fig. 4. A selection of substrate mimetics evaluated as inhibitors of influenza virus sialidase.

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. 

In the challenge to develop potent influenza virus sialidase inhibitors, a large amount of research has been dedicated to the manipulation of every position on 11 except C3. Structure-activity relationship (SAR) studies carried out on compounds derived from 11 before and during the development of zanamivir (reviewed in [101-103]) revealed structural requirements to conserve the main interactions between the substrate inhibitor and the active site of NA, particularly with regards to the carboxylate, C4-guanidino, and C5-acetamido moieties. 

A number of researchers have reported and demonstrated that maintaining the appropriate position of the substituents on a cyclic scaffold to interact with the established conserved amino acid residues involved in substrate binding can lead to development of new classes of influenza virus sialidase inhibitors [117]. Two drugs based on five-membered ring scaffolds have been developed as potent sialidase inhibitors. Cyclopentane derivative 24 (BCX-1812, peramivir) [117, 118] and pyrrolidine derivative 25 (ABT-675) [119] show nanomolar levels of inhibition of both influenza A and B viral sialidases (Fig. 17.13). 

The first substrate-based influenza virus sialidase inhibitor described was Neu5Ac2en (6) [71, 72], It is proposed that the olefin in (6) mimics, to a certain extent, the conformation of the proposed sialosyl cation transition-state intermediate (4) [62,68,69], Neu5Ac2en shows reasonably potent inhibition of influenza virus sialidase (with a A j in the range of 10 5 to 10 6 M) in vitro... 

Initial studies with sialidases demonstrated that some enzymes need divalent cation cofactors, Ca " being the most effective (Drzeniek 1972, 1973). For instance, V. cholerae, Diplococcus pneumoniae and some influenza virus sialidases require Ca " ions for activity and are consequently inhibited by the addition of EDTA. In the case of V. cholerae sialidase it was concluded that the Ca " " ions are required for stabilization of the enzyme rather than for direct enzyme-substrate binding. Mammalian enzymes are more complex and some examples of activation due to mono- and divalent cations were found to be concentration-dependent and related to the nature of the substrate (gangliosides), thus suggesting a substrate-enzyme interaction (Schengrund and Nelson 1975). 

The internal, sialyl residue of GM, is not completely resistant to some sialidases, so long as the oligosaccharide chain is bound to the ceramide part of GM, it is slowly cleaved by C. perfringens sialidase in the presence of bile salts.77 57 3511 Surprisingly, it is a relatively good substrate for the A. ureafaciens sialidase.360 Rapid hydrolysis of GM, sialic acid has also been observed with Sendai virus sialidase, in contrast to the enzymes from NDV or influenza viruses.361 Susceptibility towards mammalian sialidases has also been reported.362 However, it... 

The influence of the solvent in a conjugate reduction can be exemplified by the synthesis of sialic add derivatives which are potent inhibitors of the influenza virus surface enzyme sialidase. " A group of potential sialidase inhibitors were obtained by the 1,4-reduction of a galacto-enonate derivative (Scheme 2). The catalytic hydrogenation of the enone substrate with 10% Pd/C in methanol afforded the 2-Ha,dai penta-acetylated... 

M. (1995) Effect of substrate aglycon on enzyme mechanism in the reaction of sialidase from influenza virus. FEBS Letters, 372, 148-150. 

The initial demonstration of the a-anomeric specificity was made with IPNeu5AcLac (Kuhn and Brossmer 1958) with influenza and mumps virus sialidases. The preparation of synthetic p-glycosides of sialic acid allowed this specificity to be tested and verified for several viral and bacterial sialidases (Meindl and Tuppy 1965, Faillard et al 1966, Kuhn et al 1966). A further analysis was carried out on synthetic a-N- and a-S-glycosides of Neu5Ac, which proved not to be substrates for viral and bacterial sialidases (Khorlin et al 1970). Subsequent studies with V. cholerae sialidase and 4-nitrophenyl-a-Neu5Ac, however, showed low but significant release by the enzyme (Eschenfelder 1979). 

Munoz-Barroso, I., Garcia-Sastre, A., Villar, E., Manuguerra, J. C., Hannoun, C., and Cabezas, J. A., 1992, Increased influenza A virus sialidase activity with N-acetyl-9-O-acetylneuraminic acid-containing substrates resulting from influenza C virus O-acetylesterase action. Virus Res. 25 145-153. 

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