Influenza Virus Sialidase Active Site

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]. 

FIGURE 17.7 Some important direct interactions between conserved amino acid residues of the active site of influenza A virus sialidase and a-Neu5Ac. Ala, alanine Arg, arginine Asn, asparagine Asp, aspartic acid Glu, glutamic acid His, histidine lie, isoleucine Typ, tryptophan Tyr, tyrosine. 

To facilitate the description of the binding between a-Neu5Ac and NA, the influenza virus sialidase active site can be divided into five regions (Fig. 17.7) [31,69], Subsite 1 

SCHEME 17.1 Proposed enzymatic mechanism for the cleavage of an a-sialoside by the influenza virus sialidase. 

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Analysis of Potential Binding Interactions within the Influenza Virus Sialidase Active Site... 

An interesting feature of the influenza virus sialidase active site that offers the potential for developing inhibitors specific for N1 sialidases, including avian influenza A/H5N1 virus sialidase, has recently been revealed by X-ray crystallography. The... 

FIGURE 17.12 Interaction of oseltamivir carboxylate (20) with the influenza virus sialidase active site. 

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... 

Fig. 7 The influenza virus A sialidase active site showing the five potential inhibitor binding subsites (with S5 containing the hydrophobic pocket formed by reorientation of the Glu276 side-chain), with oseltamivir carboxylate 18 placed in the active site...

Viruses that contain amino acid substitutions in the sialidase that impart resistance to the developed inhibitors have been isolated from serial passage of virus in the presence of drug in cell culture and from the clinical setting (reviewed in McKimm-Breschkin 2000 Zambon and Hayden 2001 Cinatl et al. 2007a Reece 2007). In addition, influenza B virus variants with reduced drug sensitivity have been isolated from previously untreated patients (Hurt et al. 2006 Hatakeyama et al. 2007). The types of mutations that are observed are sub-type specific. The mutations present in variants isolated from clinical samples are shown in Table 1, and their locations within the sialidase active site are shown diagrammatically in Fig. 9. 

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

Fig. 3. A schematic representation of some key interactions of Neu5Ac2en 5 with conserved influenza virus A sialidase active site residues.

Although a large number of candidates were synthesized, none of the resulting compounds demonstrated significantly increased activity against influenza virus sialidase. Moreover, the interactions of individual substituents on the benzene ring with the active site were not found to be additive. The overall interaction of the molecules with the active site of the enzyme was dependent upon the electronic and steric interaction of each unique substituent, which made the design of inhibitors difficult.111 No compound of this family has proceeded to clinical trials. 

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. 

Functionalization of the C7 Position of Zanamivir X-ray studies of influenza virus sialidase in complex with a-Neu5Ac [66] and zanamivir [91] showed that the C7-OH is not involved in any direct interaction in the active site of influenza virus NA, and only the C8- and C9-hydroxy groups are involved in the binding interactions. Based on this knowledge, different manipulations have been attempted at Cl, for example, by a simple substitution of the C7-hydroxy group or alkylation... 

An X-ray crystal structure of peramivir (24) in complex with influenza A N9 showed interactions of the 3-pentyl group with S4 and S5 [117]. Due to the different stereochemistry of the guanidino moiety of 24 compared to zanamivir, a water molecule was displaced from S2 when 24 was bound. The different binding mode of the guanidine moiety within the active site provided the reason by which 24 showed inhibitory activity also for zanamivir-resistant influenza virus sialidase strains [117,120], Compound 24, which showed comparable or better efficacy in vivo than zanamivir and oseltamivir [121, 122], successfully completed animal studies and is in phase III clinical trials. 

Modeling studies carried out by Mark von Itzstein s group [ 137] on the 150-loop open structures of group 1 sialidase Nl revealed the possibility to access the predominantly hydrophobic 150-cavity by an appropriate substitution at C3 of 11. As shown in Figure 17.18, an opportune substitution at C3 of 11 would be accommodated in the 150-cavity in the more open form of influenza virus sialidase of group 1 and could also potentially bind to the active site with minimal distortion of the normal binding mode of 11. 

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