Active Site

The crystal structure of NA complexed with sialic acid, which is itself a weak inhibitor of influenza NA with a Ki« 1 mM, revealed sialic acid in a strained conformation with its hexose ring in a half-chair rather than a chair conformation (Fig. 3) [22]. Neu5Ac2en represents a transition-state analog, and its interactions with the active site are shown in Fig. 4a. A trifluoroacetyl derivative of Neu5Ac2en, FANA (Fig. 1, structure 2b), was the best inhibitor of the influenza NA for some years, with a Ki=0.8 |M [9]. Comparison of the several influenza NA structures now available reveal a relatively rigid active site, and so the challenge in inhibitor design has been to exploit the largely immobile features of this site [19,22-24]. The most effective inhibitors that have been developed to date include Relenza® (SKB) [25], Tamiflu® (Roche), BCX-1812 (Biocryst Pharmaceuticals) [26], and A-315675 (Abbott) [27]. Compound A-192558 from Abbott has been less successful [28], as have derivatives of benzoic acid [29]. Table 1 lists the Ki and IC50 of several ligands.

The sialic-acid-binding active site is a deep pocket, mainly acidic in nature, but with a basic side to it (Fig. 3). The four characteristic features of the site are:

1. A basic pocket formed by an arginine triad (Arg118, Arg292, Arg371) that interacts with the carboxylic acid of the ligand; this feature is a key determinant of the

Figure 2 The influenza virus NA. (Top) Schematic drawing of the influenza virus NA tetramer, as if looking down onto the virus surface. Coloring is from blue at the N terminus to red at the C terminus. (Bottom) Surface representation of the same tetramer, showing the location of Neu5Ac2en bound in the active site. The yellow coloring shows amino acids that vary within the N8 subtype, revealing the antigenic drift that the virus undergoes.

Figure 2 The influenza virus NA. (Top) Schematic drawing of the influenza virus NA tetramer, as if looking down onto the virus surface. Coloring is from blue at the N terminus to red at the C terminus. (Bottom) Surface representation of the same tetramer, showing the location of Neu5Ac2en bound in the active site. The yellow coloring shows amino acids that vary within the N8 subtype, revealing the antigenic drift that the virus undergoes.

binding, and all inhibitors developed to date preserve the carboxyl moiety.

2. An acidic pocket formed by glutamates (Glu276, Glu277); Glu277 forms a strong H-bond with Tyr406, and together these residues are thought to stabilize an oxocarbonium ion intermediate in the reaction [30,31]. Glu276 interacts with O8 and O9 of the glycerol moiety of sialic acid and Neu5Ac2en, and Relenza preserves this interaction. Other inhibitors have placed a hydrophobic moiety at this position to improve the lipophilicity of the compounds, which

Figure 3 Stereo views of the active site with sialic acid bound (tern N9 influenza neuraminidase, PDB code 1MWE). (Top) Colored by electrostatic potential: blue positive, red negative. (Bottom) Hydrogen bonding interactions shown as dotted green lines; W denotes a water molecule.

Table I Inhibition Parameters for Ligands of Influenza Virus Neuraminidase

Influenza A Virus Influenza B Virus

K IC50 K IC50 Refs.

Relenza 0.06-1.3 nM 0.3-2.3 nM 0.09-0.27 nM 1.5-17 nM 46

Tamiflu 0.10-1.3 nM 0.01-2.2 nM 1.1-2.1 nM 5.0-10.4 nM 49

BCX-1812 0.014-1.1 nM 0.1-1.4 nM 0.21-0.96 nM 0.6-11 nM 46

A-315675 0.024-0.21 nM 0.4-5.9 nM 0.14-0.31 nM 6.7-14.1 nM 49

subsequent crystal complexes have shown are accommodated by a movement of Glu276 to extend a hydrophobic pocket.

3. A hydrophobic pocket formed by Trp178, Ile222, and the methylene elements of the side chains of Arg152 and Arg224, all residues that are conserved across influenza viruses. This pocket accommodates the methyl group of the acetamido moiety of sialic acid and Neu5Ac2en. The oxygen of the acetamido group hyrodgen bonds to the guanidinium group of Arg152, and the acetamido nitrogen hydrogen bonds to a buried water molecule, which in turn interacts with Glu276 and Glu227. Most inhibitors have preserved the acetamido group or a triflouroacetamido group, such as in FANA and the Abbott A-192558 compound.

4. An acidic pocket formed from Glu119, Glu227, and Asp151, the latter residue being most likely to be involved in hydrolysis via a water molecule [30]. The two glutamates are conserved, yet play no obvious role in substrate binding or hydrolysis. The O4 hydroxyl of sialic acid and Neu5Ac2en does not hydrogen bond to any of these residues as there is a large cavity around this position. Most successful inhibitors have an amino or guanadino group substituted at this position, except for the Abbott A-315675 compound.

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