Discovery and development of neuraminidase inhibitors
[3][4] In the early 1990s, the determination of biological crystal structure of influenza virus surface protein led to the discovery of the active site and provided the opportunities to discover and design new and specific inhibitors.
[6] Neuraminidase also helps the invasion of the virus in the upper respiratory tract, possibly by cleaving sialic acid molecules on mucin of epithelial cells.
[10] Neuraminidase is an enzyme which hydrolyses that bond to produce a free neuraminic acid and a glycoprotein or a sugar chain.
Influenza virus will bind via the hemagglutinin protein on these sialic acid attached glycoproteins on the cell membrane.
The second step is the proton donation from water molecule and formation of the endocyclic sialosyl cation transition-state intermediate.
There are two types of neuraminidase inhibitors commonly available for treatment and prophylaxis of influenza infections: Zanamivir and Oseltamivir.
Viral replication rate is then reduced and that allows human immune system to destroy the remaining viruses.
This distorted structure forms a sialosyl cation after the release of the aglycon and is then trapped in the active site by a nucleophilic attack of the tyrosine residue.
The orientation of the substrate in the active site is facilitated mainly by three strain-preserved Arginine residues binding the C1 acid group with salt bridges.
Furthermore, the active site consists of eight other highly conserved amino acid-residues that make direct contact to the substrate or its derivatives.
The human genome has four different neuraminidase enzymes (NEU1, NEU2, NEU3, NEU4) and only one of them (NEU2) is not membrane-associated or in a membrane-complex and has been studied with X-ray crystallography.
Active site topology and interactions with the substrate are very similar with the exception of the glycerol side chain which offers some strategic options in designing inhibitors targeting either vNEU or hNEU.
These overall similarities have called for concerns over potential side effects from drugs targeting vNEU.
[1] In the beginning the X-ray crystallography did not have a very good resolution so the initial focus was on substrate derived inhibitors instead of structurally based.
With better X-ray crystal structure a number of important residues in the active site were identified, specifically C4 hydroxyl group.
4-amino-4-deoxy-Neu5Ac2en and 4-deoxy-4-guanidino-Neu5Ac2en were synthesized and proved to be competitive inhibitors for viral neuraminidase and significantly inhibited both A and B influenza replication in vitro and in vivo.
With focus on positioning the double bond in the inhibitor to more closely resemble the transition state of the substrate and replacing the glycerol side chain with a lipophilic group on the basis of the hydrophobic backbone of the glycerol interacting with the protein lead to the discovery of GS 4071.
Babu et al.[18] found that the addition of n-butyl side chain makes the compound fit better to the hydrophobic region of the enzyme.
[21] Since the compound processed similar binding interaction with active site of NA to zanamivir and due to the mutation in some zanamivir-resistance strains, the position of guanidino group was altered and the n-butyl group was replaced in order to change its active site interaction.
[23] New NA inhibitor analogues were synthesized, based on Zanamivir, Oseltamivir and Peramivir, with rational structure-based drug design and can be categorized into four groups.
[24] Laninamivir is designed by replacing C7 hydroxy moiety with small lipophilic group, -OCH3, which resulted in an excellent inhibitory activity.
The C8 and C9 diol play an important role in the binding affinity with neuraminidase, prolonging the effect.
The polymer scaffolds at the C7 position of zanamivir via an alkyl ether has gained more attention as this showed enhanced antiviral activity.
For example, the triazole-containing carbocycles by Von Itzstein and Pinto group and the phosphonate analogue of oseltamivir has been reported to show stronger activity resulted from a pertinent binding mode of phosphonate with three Arginine residues in the active site.
DANA is a pan-selective inhibitor for all hNEU isoenzymes, with a difference from 2 to 10 fold inhibition activity, most effective for hNEU3 and hNEU4.
These studies show that the glycerol side-chain pocket in the active site can potentially be manipulated in the design of isoenzyme selective inhibitors for hNEU.
[23] Laninamivir is approved for the treatment of influenza under the tradename Inavir in Japan but it is still in clinical trial in the USA.
The most common side effects of Oseltamivir are headache and nausea (in adults) and vomiting, cough and nasal congestion (in children).
[38] Oseltamivir has a large hydrophobic side chain and the NA must undergo rearrangement to form a pocket for drug binding by rotating aminoacid E276 and bond with R224.
