Neuraminidase inhibitors inhibit enzymatic activity of the enzyme neuraminidase (sialidase). These types of inhibitors have been introduced as anti-influenza drugs as they prevent the virus from exiting infected cells and thus stop further spreading of the virus. Neuraminidase inhibitors for human neuraminidase (hNEU) have the potential to be useful drugs as the enzyme plays a role in several signaling pathways in cells and is implicated in diseases such as diabetes and cancer.Christopher W. Cairo. (2014) Inhibitors of the human neuraminidase enzymes. Med. Chem. Commun., 2014, 5, 1067.DOI: 10.1039/c4md00089g
History
The first neuraminidase Inhibitors (NAIs) were synthesized in the 1960s by Edmond et al.,Edmond, J. D., Johnston, R. G., Kidd, D., Rylance, H. J. and Sommerville, R. G. (1966) The Inhibition Of Neuraminidase And Antiviral Action. Br. J. Pharmacol. Chemother., 1966, 27: 415–426. doi: 10.1111/j.1476-5381.1966.tb01673.x through an attempt to understand the catalytic mechanism of the neuraminidase enzyme. They discovered that N-substituted oxamic acids had enzyme inhibitory properties. Then it was found that the synthetic compound 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en or DANA) which is an analogue of N-acetylneuraminic acid (Neu5Ac), inhibits the release of virus progeny in tissue culture but no antiviral activity in animals was detected.Kim, Choung U, Xiaowu Chen, and Dirk B Mendel. Neuraminidase inhibitors as anti-influenza virus agents. Antiviral chemistry & chemotherapy 10.4 (1999): 41-154von Itzstein, Mark. The war against influenza: discovery and development of sialidase inhibitors. Nature reviews Drug discovery 6.12 (2007): 967-974 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.
Influenza virus
The Influenza virus is an RNA virus that is divided into three serological types: A, B and C. Hemagglutinin (HA) and neuraminidase (NA) are two important glycoproteins on influenza virus membranes. The hemagglutinin is a sialic acid receptor-binding molecule and mediates entry of the virus into the host cell, while neuraminidase cleaves cellular-receptor sialic acid to form new particles. Neuraminidase is an exoglycosidase that destroys the hemagglutinin receptor by cleaving the α(2,6)- or α(2,3)-ketosidic linkage that exists between a terminal sialic acid and a sugar residue of the Neu5Ac containing receptor on the surface of host cells.Air, Gillian M, and W Graeme Laver. The neuraminidase of influenza virus. Proteins: Structure, Function, and Bioinformatics 6.4 (1989): 341-356 This helps the spread of the infection by preventing self-aggregation of new viruses at the cell surface and possible immobilisation in the mucin by hemagglutinin (HA) during virus replication. The virus will then be released from the host cells and will subsequently infect other cells.Varghese, J. N. (1999). Development of neuraminidase inhibitors as anti-influenza virus drugs. Drug Development Research, 46(3-4), 176-196 Neuraminidase also helps the invasion of the virus in the upper respiratory tract, possibly by cleaving sialic acid molecules on mucin of epithelial cells. Neuraminidase is found in influenza viruses of types A and B.Du, Juan, Timothy A Cross, and Huan-Xiang Zhou. Recent progress in structure-based anti-influenza drug design. Drug discovery today 17.19 (2012): 1111-1120 Neuraminidase has roles in the infection, replication and delivery of Influenza virus A and B. Type C Influenza virus expresses the enzyme esterase instead of neuraminidase.Wagaman PC, Spence HA, and O’Callaghan RJ. 1989. Detection of Influenza C Virus by Using an In Situ Esterase Assay Journal of Clinical Microbiology. 1:832-836.
The substrate
N-acetylneuraminic acid (Neu5Ac) is one of the two most common sialic acid in mammals.Varki, N. M., & Varki, A. (2007). Diversity in cell surface sialic acid presentations: implications for biology and disease. Lab Invest, 87(9), 851-857. doi: 10.1038/labinvest.3700656 It is a monosaccharide with a backbone of 9 carbons. It is usually attached to glycoproteins or gangliosides on a terminal end via α(2,3), α(2,6), and α(2,8) linkage.Varki, Ajit; Roland Schauer (2008). in Essentials of Glycobiology. Cold Spring Harbor Press. pp. Ch. 14 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.Racaniello, Vincent. (2009). Influenza virus attachment to cells. Retrieved October 2014, from http://www.virology.ws/2009/05/04/influenza-virus-attachment-to-cells/
Mechanism of action
Mechanism of enzyme catalysis
The mechanism of NA has been shown to proceed with the retention of configuration which means it preserves the absolute configuration on the atom in the stereocenter.Taylor, N. R., & von Itzstein, M. (1994). Molecular modeling studies on ligand binding to sialidase from influenza virus and the mechanism of catalysis. Journal of medicinal chemistry, 37(5), 616-624 There are four steps of catalytic pathways. In the first step, the binding step, the carboxylate group changes from the axial position into the pseudo-equatorial position. The second step is the proton donation from water molecule and formation of the endocyclic sialosyl cation transition-state intermediate. Step three involves nucleophilic attack of tyrosine on the sialosyl cation. The fourth step is the formation and release of Neu5Ac. A similar mechanism has been proposed by Janakiraman et al.Janakiraman, M. N., White, C. L., Laver, W. G., Air, G. M., & Luo, M. (1994). Structure of Influenza Virus Neuraminidase B/Lee/40 Complexed with Sialic Acid and a Dehydro Analog at 1.8-. ANG. Resolution: Implications for the Catalytic Mechanism. Biochemistry, 33(27), 8172-8179 where the double bond of Neu5Ac2en forces the pyranose of sugar ring into a planar structure were resembled the transition-state structure.
Mechanism of inhibition
There are two types of neuraminidase inhibitors commonly available for treatment and prophylaxis of influenza infections: Zanamivir and Oseltamivir. They interfere with the release of progeny virions from infected host cells, prevent infection process of new host cells and stop spread of infection in respiratory tract by mimicking natural substrate and fitting into active site of neuraminidase enzyme. They interrupt the detachment of progeny virions. Viral replication rate is then reduced and that allows human immune system to destroy the remaining viruses.Moscona, A. (2005). Neuraminidase Inhibitors for Influenza. New England Journal of Medicine, 353(13), 1363-1373. doi: doi:10.1056/NEJMra050740
Development
Binding site
Viral neuraminidase
Influenza virus neuraminidase (vNEU) consists of 4 co-planar roughly spherical subunits predominantly made of β-sheets, characterized as a 6-fold β-propeller and a hydrophobic region embedded in the virus’ membrane.Colman, P.M. (1994) Influenza virus neuraminidase: Structure, antibodies, and inhibitors.Protein Science. 3: 1687–1696. doi: 10.1002/pro.5560031007 The active site is located near the middle of the pseudo-symmetric sphere. Influenza virus neuraminidase only cleaves terminal Neu5Ac residues. X-ray crystallography has shown a distorted half-chair arrangement of the Neu5Ac substrate in the active site. 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 others highly conserved amino acid-residues that make direct contact to the substrate or its derivatives. Including a glutamic acid residue binding the C7 and C9 alcohol groups on the glycerol side-chain (at C6) with hydrogen bonds and several hydrophobic residues correlating with the methyl group on the C5 N-acetyl and the hydrophobic backbone of the glycerol.Mark von Itzstein (2007)The war against influenza: discovery and development of sialidase inhibitors. Nature publishing group. Volume 6. 967-974
Human Neuraminidase
Human neuraminidase (hNEU) shares many similar features with vNEU. 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.havas LMG, Tringali C, Fusi P, Venerando B, Tettamanti G, Kato R, Monti E, Wakatsuki S. Crystal structure of the human cytosolic sialidase Neu2 - Evidence for the dynamic nature of substrate recognition. J Biol Chem. 2005;280:469-475. The three arginine residues that bind the C1 acid-group with salt bridges in vNEU are also present in hNEU. 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. In hNEU the glycerol hydroxyl-groups are bound via several tyrosine residues but in vNEU the main interaction is with a glutamic acid residue. These overall similarities have called for concerns over potential side effects from drugs targeting vNEU. Nevertheless, most of the well studied vNEU inhibitors have shown very little affinity for hNEU except for the influenza drug Zanamivir which is an effective inhibitor for hNEU2.
Viral neuraminidase inhibitors
2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en) is a pan-selective inhibitor for neuraminidase. Neu5Ac2en is a dehydrogenated Neu5Ac and can be synthesized by the hNEU enzyme if Neu5Ac is in high enough concentration. Neu5Ac is also a mild inhibitor for the enzyme but as Neu5Ac2en is a transition-state analogue it is a much better inhibitor.
Zanamivir