You are on page 1of 17

Development of Tamiflu and Relenza INTRO VIRUS

The virus Influenza virus belongs to the orthomyxoviridae family, which is subdivided into three serologically distinct types: A, B and C. Only influenza virus A and B appear to be of concern as human pathogens as influenza C virus does not seem to cause significant disease12,13. Further classification of influenza virus is based on the antigenic properties of its surface glycoproteins haemagglutinin and sialidase14 (FIG. 1), both of which are essential for infection to proceed (FIG. 2). These surface glycoproteins are carbohydrate-recognizing proteins and in humans are known to recognize the sialic acid N acetylneuraminic acid (Neu5Ac; which has and forms when not conjugated; compound 1, FIG. 3), which is typically associated as the -linked terminal carbohydrate unit of upper respiratory tract and lung-associated glycoconjugates13,15. Haemagglutinin is made up of three identical subunits (FIG. 1a) and is anchored to the lipid membrane of the virus13. This glycoprotein seems to have two significant roles. The first is to provide an initial point of contact for the virus to the target host cell-surface glycoconjugates by ketosidically linked terminal Neu5Ac residues10,16,17. The second is to trigger the internalization process of the virus through fusion of the viral envelope with the host cell 10,18. A multitude of influenza virus haemagglutinin structures have been determined, as well as the structures of several haemagglutinin ligand complexes1922. Influenza virus sialidase is an enzyme made up of four identical subunits that is also anchored to the viral membrane23. The enzyme is an exoglycohydrolase and cleaves ketosidically linked Neu5Ac residues that cap the ends of various glycoconjugates11. The significance of the sialidase action is that it assists in the movement of virus particlesthrough the upper respiratory tract as well as in the release of virion progeny from infected cells24,25. Several influenza virus sialidase crystal structures, including a number of complexes of influenza virus sialidase with Neu5Ac (FIG. 1b) and its derivatives, have been determined2628.

Influenza, an orthomyxovirus, is a 100 nm lipid-enveloped virus containing an eight-segment negative single-stranded genome [Lamb, 1989]. Two of the segments code for the surfaces glycoproteins hemagglutinin HA (which binds to terminal sialic acid) and neuraminidase NA (which cleaves terminal sialic acid) which appear as spikes protruding out of the viral enve*Correspondence to: Joseph N. Varghese, Biomolecular Research Institute, 343 Royal Parade, Parkville, Victoria 3052, Australia. E-mail:

ANTI-VIRAL DRUGS FOR INFLUENZA 177 lope. The viral target in humans is the upper respiratory

tract epithelial cells. Replication begins with penetration of the virion through the mucin layer covering the epithelial surface, followed by attachment to the viral receptor by the HA. Penetration of the cell is achieved by endocytosis and the virion core is released after the fusion of the virion and vesicle membrane, mediated by the HA. Fusion is enabled by a conformational change in the HA, made possible by lowering the pH of the endosome by the M2 ion channel protein. Following replication, the progeny virions are released by budding off the cell membrane [Murphy and Bang, 1952; Compans and Dimmons, 1969]. The release of virons occurs 8 h postinfection and the onset of infection is sudden, resulting in pyroxia, muscular and joint pain, and a dry cough [Murphy and Webster, 1990]. Virus shedding continues for up to a week, when a rise in virus-specific antibody clears the virus from the host. The vulnerability of the host succumbing to viremea during this week of rising viral titre is dependent on interferon induction [Ennis and Meager, 1981] 48 h postinfection, which attenuates viral replication until the cellmediated immune response begins to clear the virus. The severity of the illness is thought to depend on the level of cross protection arising from antibodies raised from previous influenza infections [Fox et al., 1982]. The course of the illness can be dehabilitating, and no effective treatment is available at present to halt the progression of the disease. Death can result for susceptible populations (neonate and elderly), primarily as a result of secondary infections [Sprenger et al., 1993]. The discovery in the early 1980s of the molecular structure of influenza virus NA, the detailed atomic description of the active site of the molecule, and the exploitation of its structural conservation is discussed in terms of the design of potent NA inhibitors.


The majority of research efforts aimed at identifying suitable molecular targets for antiviral intervention are focused on the influenza neuraminidase, one of two major glycoproteins located on the influenza virus membrane envelope [2, 3]. This enzyme is responsible for the cleavage of terminal sialic acid residues from glycoconjugates and is essential for virus replication and infectivity [4-7]. Early efforts to identify influenza neuraminidase inhibitors were met with reasonable success. By utilizing rational drug design in conjunction with available high resolution X-ray crystal structures of sialic acid (3 ) and the transition state analog Neu5Ac2en (4 ) bound to influenza A and B neuraminidases [9-11], researchers were able to design potent inhibitors of the influenza neuraminidase enzyme. To date, this approach has led to the development of several potent and specific influenza neuraminidase inhibitors. Most notably, oseltamivir
Historically, the first drugs available for the treatment of influenza were the adamantane- based M2 ion channel protein inhibitors, rimantidine and amantadine4,5. These compounds have only been useful in the treatment of influenza A infection, because only the A strains of the virus have M2 ion channel proteins47. Although both drugs can be effective against influenza virus A infection, they have been reported to cause CNS side effects 5,6, and have given rise to the rapid emergence of drug-resistant viral strains7. Given such issues, there has been considerable effort worldwide to discover novel therapeutic agents against all types of influenza, and several valuable reviews concerned with aspects of influenza virus have been published (for example, REFS 811).

Amantadine and rimantadine are older antiviral agents that have been important adjuncts in the prevention and treatment of influenza A outbreaks. Zanamivir and oseltamivir are newer agents indicated for the treatment of both influenza A and B.
The threat of a major human influenza pandemic, in particular from highly aggressive strains such as avian H5N1, has emphasized the need for therapeutic strategies to combat these pathogens. At present, two inhibitors of sialidase (also known as neuraminidase), a viral enzyme that has a key role in the life cycle of influenza viruses, would be the mainstay of pharmacological strategies in the event of such a pandemic. This article provides a historical perspective on the discovery and development of these drugs zanamivir and oseltamivir and highlights the value of structure-based drug design in this process.
Here, after providing some background on the influenza virus and its key surface glycoproteins, this article describes the discovery and development of influenza virus sialidase (also known as neuraminidase/exo--sialidase; EC inhibitors, which are now at the forefront of defences against a flu pandemic. Focused efforts to develop such drugs using structural information began in the 1980s, and provide one of the earliest examples of the application of structurebased drug design


Enzyme activity on the surface of influenza virus was first detected by Hirst [1942], who observed that red blood cells once agglutinated by influenza virus could not again be agglutinated by either the eluted virus or fresh virus preparations. This activity is now attributed to NA, which is one of the two integral membrane glycoproteins of influenza virus [for review, see Colman, 1994; Varghese, 1997] Neuraminidase is an exoglycosidase which destroys the hemagglutinin receptor by cleaving the -ketosidic linkage of terminal sialic acid (N-actylneuraminic acid (Neu5Ac)) to an adjacent sugar [Klenk et al., 1955; Gottschalk, 1957]. Viral HA binds specifically to Neu5Accontaining receptors on the surface of susceptible cells [Rogers and Paulson, 1983]. NA, which also removes terminal sialic acid from a range of glycoconjugates, plays an important, but not completely understood, role in the viral replication cycle. Without NA activity, viruses [Burnett, 1947] were thought to be immobilised by mucosal secretions in the upper respiratory tract. By removing terminal sialic acid

from the sialic acid-rich mucous layer protecting target cells [Gottschalk, 1957, 1958], NA could facilitate penetration of the virus to the cell surface. It has been shown that neuraminidasedeficient virus [Liu and Air, 1993] can still replicate in vivo, albeit at a much reduced rate [Liu et al., 1995]. This shows that NA does not play an essential role in viral entry, replication, assembly, or budding in mice. It has an important role in facilitating the spread of the infection by preventing aggregation at the cell surface and possible immobilisation in the mucin by HA. During virus replication, the freshly synthesised viral glycoproteins have to be desialylated to prevent selfaggregation at the infected host cell surface by HA binding to terminal sialic acid on these glycoproteins. Finally, on elution of progeny virions from infected cells, NA activity is required to facilitate viral escape from the cell surface. Inactivation or inhibition of NA during budding has been observed to result in aggregation of virons on the cell surface [Palese et al., 1974; Palese and Compans, 1976; Griffin and Compans, 1979]. Inhibition of this glycohydrolase thus provides a means of controlling this disease, while the number of infected cells is low, by slow slowing the rate of viral attachment and subsequent release of progeny virons. This would allow the host immune system to eliminate the virus.


Detailed analysis of available X-ray crystal structures indicates several common critical interactions between neuraminidase and its inhibitors. For example, the negatively charged carboxylate group of sialic acid makes strong charge-charge interactions with the positively charged side chains of the Arg triad (Arg 118, 292, and in particular 371) of neuraminidase as shown in Fig. (1 ). Furthermore, the Nacetyl group of sialic acid makes both polar and nonpolar contacts with Arg152, Trp178, and Ile222. These two interactions help to anchor the scaffold of sialic acid in the active site of neuraminidase, thus laying a structural foundation for introducing additional interactions which could lead to more potent inhibitors. interactions with the glycerol moiety of sialic acid, novel nonpolar interactions have been discovered and prove to be the key in achieving high binding affinity with cyclohexene based neuraminidase inhibitors [12]. The X-ray crystal structure also reveals the presence of a well-formed hydrophobic pocket which is not utilized by sialic acid for binding. This pocket (Pocket 2) consists of the highly conserved amino acid residues Ile 222, Arg 224, and Ala 246. The cyclohexene series of inhibitors takes advantage of this pocket and forms favorable hydrophobic interactions with residues in this pocket [12]. The third binding pocket (Pocket 3) is large and contains both hydrophobic and hydrophilic residues (Glu 119, Asp 151, Arg 152, Trp 178, Ser179, Ile 222, and Glu 227). The C-4 hydroxyl and the C- 5 N-acetyl groups of sialic acid bind in this pocket but do not fully interact with all of the residues in the binding pocket. In particular, there is a cluster of negatively charged residues (Glu 119, Glu 227, and Asp 151) near the C-4 hydroxyl group of sialic acid The neuraminidase active site can be divided into three major binding pockets. The highly polar residues Glu 276, Glu 277, Arg 292, Asn 294, and hydrophobic Ala 246 form Pocket 1. Although this pocket appears to be highly polar in nature, as evidenced by its
Discovery and Development of GS 4104 (Oseltamivir) Current Medicinal Chemistry, 2000, Vol. 7, No. ? 665

which could be further explored for possible chargecharge interactions.

The Design of Influenza Neuraminidase Inhibitors

The X-ray crystal structure of the sialic acid/neuraminidase complex reveal that sialic acid binds to the enzyme in a considerably deformed conformation due to the strong ionic interactions of the sialic acid carboxyl moiety with the Arg 118, 292, and 371 triad. This binding mode is very similar to that found in

the X-ray crystal structure of Neu5Ac2en (4 ) complexed to neuraminidase. Namely, the doublebond of 4 constrains the pyranose ring into a planar structure around the ring oxygen. Based on structural information and biochemical mechanistic studies, it has been proposed that the catalytic mechanism for the neuraminidase cleavage of sialic acid from glycoconjugate 1 proceeds via transition-state 2 (Scheme 1). Efforts aimed towards mimicking transitionstate 2 have resulted in the design and synthesis of a variety of transition-state analogues based on a dihydropyran ring (4 -6 ), benzene ring (7 ) [14, 15], and cyclohexene ring (8 ). The majority of research efforts aimed at identifying suitable molecular targets for antiviral intervention are focused on the influenza neuraminidase, one of two major glycoproteins located on the influenza virus membrane envelope [2, 3]. This enzyme is responsible for the cleavage of terminal sialic acid residues from glycoconjugates and is essential for virus replication and infectivity [4-7].

High resolution crystal structures of influenza neuraminidase and its complex with sialic acid (3 ) (neuraminidases catalytic product) and other transition state analogues reveal several common structural features among all influenza neuraminidases. In particular, the neuraminidase active site contains several large well-defined pockets. All residues which make direct contact with the substrate are strictly conserved among both influenza A and B neuraminidases. These residues interact similarly with both substrate and inhibitor molecules, and, not suprisingly, the neuraminidase active site contains a rather large number of polar or charged residues since its carbohydrate substrates are quite polar.

The most successful structure-based anti-influenza drug discovery programme has arisen from targeting the sialidase function. This article describes aspects of the discovery of the first potent designed influenza virus sialidase inhibitor and now commercially available inhaled anti-influenza drug zanamivir (Relenza; GlaxoSmithKline)30, and the discovery of the subsequently approved orally bioavailable drug oseltamivir (Tamiflu; Gilead/Roche) Several high-resolution Xray crystal structures of sialidase complexed with various small-molecule inhibitors have been determined, including Neu5Ac (FIG. 4a). Most strikingly, the active site consists of a number of distinct adjoining pockets that are lined by eight highly conserved amino-acid residues that make direct contact with Neu5Ac and its derivatives27 (FIG. 4b). In addition, there are a further ten amino-acid residues invariant in all strains of influenza virus within the vicinity of the active site that appear to be important primarily in the stabilization of the architecture of the active site27,37. Upon binding, Neu5Ac-containing glycoconjugates are oriented in the active site through interaction with a cluster of three arginine residues and the Neu5Ac moiety makes a number of significant contacts with active-site residues38,39 (FIG. 4c). Specifically, further orientation of the Neu5Ac moiety is facilitated by several additional interactions within the active site, including hydrogen bonding of the C5 acetamido group carbonyl oxygen to Arg152 and its NH to a buried water molecule. Favourable hydrophobic contacts to residues Trp178 and Ile222 are also made by the methyl of the C5 acetamido group. Additional hydrogen-bond networks are formed by the C8 and C9 hydroxyl groups of the glycerol side chain to the carboxylate oxygens of residue Glu276, while the C4 hydroxyl group associates with the carboxylate oxygen of Glu119. All of the amino-acid residues mentioned above are fully conserved across the natural strains of influenza virus known so far23,37. an initial focus in the discovery of influenza virus sialidase inhibitors was on substrate-like Neu5Ac derivatives, particularly 2-deoxyd-Nacetylneuraminic acid (2-deoxy-Neu5Ac) derivatives (compound 3, FIG. 3). Based on an understanding of the enzymes catalytic mechanism33,40, it was thought that such compounds might not be rapidly metabolized and should be recognized by the enzyme as a result of the compounds substrate/productlike characteristics 41. the unsaturated Neu5Ac derivative 2deoxy2,3-didehydroN-acetylneuraminic acid (Neu5Ac2en; compound 2a, FIG. 3), a micromolar inhibitor of influenza virus sialidase, has provided the most potent inhibitor core template. Computational chemistry techniques were used to probe the active site of influenza virus sialidase in an attempt to design structurally modified Neu5Ac2en derivatives that might be more potent inhibitors30,39,46 Energetically favourable interactions between various functional groups and the residues within the bindingpocket were revealed through the application of GRID software47. Most importantly, the significance of the Neu5Ac2en C4 hydroxyl group-binding domain within the sialidase active site was realized. This realization, with further considerations, directed attention to the replacement of the Neu5Ac2en C4 hydroxyl group by a basic group such as, in the first instance, an amino group. The 4substituted Neu5Ac2en derivative, 4amino4-deoxy- Neu5Ac2en (compound 4, FIG. 3), was predicted39,46 to have higher affinity for the enzyme than the parent compound Neu5Ac2en as a result of salt-bridge formation with the conserved amino acid Glu119.

Importantly, with further analysis, it was found that the conserved Neu5Ac2en C4 hydroxyl group-binding domain could accommodate a larger basic functional group. This analysis, together with chemical intuition, led to the conclusion that incorporation of a larger, more basic functionality in place of the Neu5Ac2en C4 hydroxyl group was of value. Thus, substitution of the C4 hydroxyl group with a guanidinyl functionality to provide 4deoxy4-guanidinoNeu5Ac2en (compound 5, FIG. 3) was predicted to significantly improve affinity for the enzyme. The two target molecules 4amino 4-deoxy-Neu5Ac2en and 4deoxy4- guanidino-Neu5Ac2en (FIG. 3) were synthesized using the key C4 azide intermediate48 (compound 6, FIG. 3). Evaluation of these C4 derivatives as influenza virus sialidase inhibitors confirmed that both were competitive inhibitors30,49. Moreover, both compounds were found to be highly potent inhibitors of virus replication for all influenza A and B virus strains evaluated in vitro30,49,50 and in vivo30. In the case of influenza A (N2) virus sialidase, 4amino4- deoxy-Neu5Ac2en inhibited the enzyme by a factor of 100-times greater than the parent compound Neu5Ac2en (4amino4-deoxy- Neu5Ac2en: Ki 4 108 M; Neu5Ac2en: Ki 4 106 M)49. As expected, the derivative 4deoxy4-guanidino-Neu5Ac2en was found to be more potent, with an improved affinity as much as 10,000-fold compared with Neu5Ac2en (4deoxy4-guanidino- Neu5Ac2en: Ki 2 1010 M)30,49,50. X-ray crystallographic structure determination of influenza virus sialidaseinhibitor complexes for 4amino4-deoxy-Neu5Ac2en and 4deoxy4-guanidino-Neu5Ac2en confirmed, in general, that these inhibitors engaged the enzymes active site in the predicted binding modes30. Specifically, the 4amino group of 4amino4-deoxy- Neu5Ac2en was shown30 to establish a salt bridge with Glu119, and although with Glu119, and although 4deoxy4-guanidino-Neu5Ac2en displayed the predicted30,46 lateral binding between the terminal guanidinyl nitrogens and the carboxylate of Glu227, Glu119 was found to be slightly further removed30 than proposed and stacked parallel to the guanidinyl group38. However, Glu119 is still within a distance close enough for electrostatic interaction with the guanidinyl group30,38. Fortuitously, 4deoxy4-guanidino- Neu5Ac2en was found to be highly selective for influenza virus sialidase and displayed considerably lower affinity for other sialidases from different sources49. The selectivity of influenza virus sialidase inhibitors has become of increasing interest as a result of potential side effects in patients as a result of, in part, the possible inhibition of endogenous human sialidases51. The more potent inhibitor, 4deoxy4-m guanidino-Neu5Ac2en (compound 5, FIG. 3), was selected as the lead drug candidate by Glaxo under the generic name zanamivir. Owing to its limited oral bioavailability (due to its highly polar nature and rapid excretion of the compound), it was developed as an inhaled formulation. Following its success in clinical trials, zanamivir was approved in 1999 as the first sialidase-targeting anti-influenza drug, with the tradename Relenza.

RELENZA/ZANAMAVIR/ On 3 June 1993 a significant article entitled "Rational design of potent sialidase-based inhibitors of influenza virus replication" was published in the high-impact medical science journal, Nature. This publication is considered a milestone paper in the structure-based drug design of a drug to treat influenza infection and the work has been acclaimed worldwide for its contribution to medical science. This anti-influenza drug, Relenza, was designed, synthesised and biologically evaluated (in vitro) in Mark von Itzsteins laboratory. This discovery is considered to be the most significant outcome and flagship in glycotherapeutic drug development in the last century. Relenza has saved many lives and is one of Australias greatest discoveries. As new strains of influenza develop with epidemics

and pandemics, Relenza will be at the forefront for human treatment. Relenza (trade name) or Zanamivir (INN) is a neuraminidase inhibitor used in the treatment and prophylaxis of Influenzavirus A and Influenzavirus B. Zanamivir was the first neuraminidase inhibitor commercially developed. It is currently marketed by GlaxoSmithKline. Relenza ZANAMAVIR DANA Relenza works by inhibiting the life cycle of the flu virus. It blocks neuraminidase, a viral enzyme that allows the virus to multiply. Iteffectively prevents the virus escaping from the infected cells and spreading to healthy cells Zanamivir was discovered in 1989 by scientists led by Mark von Itzstein, at the Victorian College of Pharmacy, Monash University, in collaboration with the CSIRO and scientists at Glaxo, UK. Zanamivir was the first of the neuraminidase inhibitors. The discovery was funded initially by the Australian biotechnology company Biota and was part of Biota's ongoing program to develop antiviral agents through rational drug design. Its strategy relied on the availability of the structure of influenza neuraminidase, by X-ray crystallography. It was also known, as far back as 1974, that 2-deoxy-2,3-didehydro-''N''-acetylneuraminic acid (DANA), a sialic acid analogue, was an inhibitor of neuraminidase. Sialic acid (''N''-acetyl neuraminic acid, NANA), the substrate of neuraminidase, is itself a mild inhibitor of the enzyme, but the dehydrated derivative DANA, a transition-state analogue, is a better inhibitor. Computational chemistry techniques were used to probe the active site of the enzyme, in an attempt to design derivatives of DANA that would bind tightly to the aminoacid residues of the catalytic site, and so would be potent and specific inhibitors of the enzyme. The software GRID from Molecular Discovery was used to determine energetically favourable interactions between various functional groups and residues in the catalytic site canyon. This showed there was a negatively charged zone in the neuraminidase active site that aligned with the C4 hydroxyl group of DANA. This hydroxyl was therefore replaced with a positively charged amino group; the 4-amino DANA was 100 times better an inhibitor than DANA, owing to the formation of a salt bridge with a conserved glutamic acid (119) in the active site. It was also noticed that Glu 119 was at the bottom of a conserved pocket in the active site just big enough to accommodate a more basic functional positively charged group, such as a guanidino group, which was also larger than the amino group. Zanamivir, a transition-state analogue inhibitor of neuraminidase, was the result.

Rational drug design utilizing available X-ray crystal structures of sialic acid analogues bound to the active site of influenza virus neuraminidase has led to the discovery of a series of potent carbocyclic influenza neuraminidase inhibitors. From this series, GS 4104 (oseltamivir, TAMIFLU) has emerged as a promising antiviral for the treatment and prophylaxis of human influenza infection. By utilizing rational drug design in conjunction with available high resolution X-ray crystal structures of sialic acid (3 ) and the transition state analog Neu5Ac2en (4 ) bound to influenza A and B

neuraminidases [9-11], researchers were able to design potent inhibitors of the influenza neuraminidase enzyme. To date, this approach has led to the development of several potent and specific influenza neuraminidase inhibitors. Most notably, oseltamivir (9 ) (GS 4104, the oral prodrug of GS 4071 (8 )) [12, 13] and zanamivir (6 ) (GG167
The discovery of zanamivir provided a platform for further sialidase-targeted antiinfluenza drugs. Significant work has been undertaken in structureactivity relationship studies with Neu5Ac-based derivatives and uronic acid carbohydrate-based templates derived from Nacetylglucosamine (compound 7, FIG. 3)52. Moreover, substantial effort has been applied towards the development of influenza virus sialidase inhibitors that are based on non-carbohydrate templates (for a review, see REF. 11). For example, potent and selective inhibitors of influenza virus sialidase and of influenza virus infection in vivo have been developed based on a range of core templates including cyclohexenes such as oseltamivir carboxylate (compound 8, FIG. 3; originally known as GS 4071)31; cyclopentanes such as peramivir53 (compound 9, FIG. 3 ); and pyrrolidines such as A-315675 (compound 10, FIG. 3)54. Most noteworthy has been the development of the cyclohexene derivative GS 4071, and its prodrug, oseltamivir, the first orally active sialidase inhibitor to be approved for treating influenza chemistry based on the functionalized cyclohexene and cyclohexane shikimic acid and quinic acid core templates, respectively, were undertaken in the discovery of GS 4071. On the basis of interactions of the unsaturated Neu5Ac derivatives 4 and 5, three key concepts were used in the discovery of GS 4071 and oseltamivir. First, based on previous mechanistic studies33,36,39, positioning the double bond in the inhibitor to more closely mimic the putative transition state sialosyl cation (BOX 1) was investigated. Second, replacing the glycerol moiety of 4amino4-deoxy-Neu5Ac2en by a lipophilic group was explored on the basis that the hydrophobic backbone of the glycerol side chain makes contact with the protein, even though the C 8 and C9 hydroxyl groups make a bidentate interaction with Glu276 Xray crystallographic study of an influenza virus sialidaseGS 4071 complex clearly showed that the architecture of the active site had been altered on binding of GS 4071 (REFS 31,55). Specifically, Glu276 reorients outwards from the glycerol side-chain binding domain to interact with Arg224 and in doing so generates a considerable hydrophobic area within this domain. This rearrangement, which was not predictable, provided exciting new opportunities for further sialidase inhibitorbased anti-influenza drug discovery. Although GS 4071 binds in an identical fashion to zanamivir and other Neu5Ac2en derivatives, this induced fit is essential for the inhibitor to successfully engage the active site to provide the inhibitors potent efficacy (FIG. 5c). Third, although it had been hoped that GS 4071 (which is more lipophilic than zanamivir) might have had sufficient oral bioavailability, this was found not to be the case, and so a prodrug strategy was used. Oseltamivir (GS 4104; compound 11, FIG. 3), the ethyl ester prodrug of GS 4071, is readily converted to the active form in vivo by the action of endogenous esterases. Following its success in clinical trials as an orally administered treatment for influenza virus infection, oseltamivir (developed by Gilead) was approved in late 1999, and is now marketed by Roche under the trade name Tamiflu.

Oseltamivir INN /sltmvr/, marketed under the trade name Tamiflu, is an antiviral drug, which is licenced to prevent or slow the spread of influenza (flu) virus between cells in the body by stopping the virus from chemically cutting ties with its host cell. It is used to treat influenza A virus and influenza B virus. It was the first orally active neuraminidase inhibitor commercially developed] It was developed by C.U. Kim, W. Lew, and X. Chen of US-based Gilead Sciences,[2] and is marketed by Genentech There are concerns that oseltamivir may cause dangerous psychological, neuropsychiatric side effects including self-harm in some users. MECHANISM OF ACTION The prodrug oseltamivir is itself not virally effective; however, once in the liver it is hydrolysed to its active metabolite - the free carboxylate of oseltamivir (GS4071). Oseltamivir is a neuraminidase inhibitor, serving as a competitive inhibitor of the activity of the viral neuraminidase (NA) enzyme upon sialic acid, found on glycoproteins on the surface of normal host cells. By blocking the activity of the enzyme, oseltamivir prevents new viral particles from being released by infected cells

Sialic Acid-Based Influenza Neuraminidase Inhibitors

The design of these sialic acid analogues was based on the crystal structure of influenza virus neuraminidase and its complex with N-acetyl neuraminic acid (sialic acid) and 2-deoxy-2,3-dehydro-N-acetyl neuraminic acid. These novel inhibitors are highly specific for influenza neuraminidase, and have been shown to inhibit influenza virus replication in both cell culture and animal models.

Based on information derived from the available Xray crystal structures of sialic acid and its analogues. bound with neuraminidase, inhibitors 5 and 6 were rationally designed and synthesized [11, 16]. Compounds 5 and 6 exhibited potent neuraminidase inhibitory activity with Ki values of 10-8 M and 1010 M, respectively. Consistent with its potent in vitro inhibitory activity, 6 (zanamivir) exhibited potent antiviral activity against a variety of influenza A and B strains in cell culture and demonstrated in vivo efficacy in the influenza infected animal models via intranasal administration [11]. Currently zanamivir, marketed as Relenza, is approved for the treatment of influenza infection in the U.S., Europe and Australia and is administered via inhalation.

Discovery of GS 4071
As shown in Table 1, the length, substitution, and geometry of the C-3 alkyl side chain in 10 profoundly influenced neuraminidase inhibitory activity [12, 17]. A greater than 20-fold increase in inhibitory activity of npropyl analogue 14 compared to that of methyl analogue 12 implicated a significant hydrophobic interaction of the n-propyl group with either Pocket 1 or

Figure 8 seemed to be the best inhibitor because of the large chain that occupies some space in the active site.
Branching of the npropyl group adjacent to the ether oxygen led to a significant increase in potency, as demonstrated by the 3-pentyl analogue 8 (GS 4071). entirely unexpected since hydrophobic interactions play a vital role in inhibitor binding. A truly surprising discovery from this structure is the ability of the other ethyl group of the 3-pentyl side chain to bind in an apparently highly polar region (Pocket 1). Such binding becomes possible because the Glu 276 side chain can adopt two different conformations [12]. When sialic acid binds in the neuraminidase active site, the Glu 276 side chain adopts a conformation in which a bidentate hydrogen bonding interaction with the glycerol side chain of sialic acid is achieved; Whereas upon binding of 8 in the neuraminidase active site, the Glu 276 side chain rotates away from the center of pocket 1 and adopts an alternate conformation which is stabilized by a strong charge-charge interaction with the nearby guanidine group of Arg 224. Such a conformational change effectively enlarges Pocket 1 and creates a much less polar environment which can accommodate a hydrophobic binding group such as the ethyl group of the 3-pentyl side chain of 8 . The X-ray crystal structure of 8 bound to neuraminidase (Fig. (2 )) [12] indicates that the binding mode for this series of carbocyclic inhibitors is similar to that of 4-amino-Neu5Ac2en (5). Namely, the corresponding C-1 carboxylate, C-4 acetyl, and C-5 amino groups of 8 and 5 interact with the same active site amino acid residues upon binding in the neuraminidase active site. More importantly, the X-ray crystal structure of 8 reveals that the 3-pentyl side chain binds in an extended conformation and forms hydrophobic interactions previously not observed in the sialic acid/neuraminidase complex. One of the ethyl groups of the 3-pentyl side chain of 8 makes several favorable hydrophobic contacts with amino acid residues Ile 222, Arg 224, and Ala 246, resulting in a significant increase in binding affinity. This is not entirely unexpected since hydrophobic interactions play a vital role in inhibitor binding. A truly surprising discovery from this structure is the ability of the other ethyl group of the 3-pentyl side chain to bind in an apparently highly polar region (Pocket 1). Such binding becomes possible because the Glu 276 side chain can adopt two different conformations [12]. When sialic acid binds in the neuraminidase active site, the Glu 276 side chain adopts a conformation in which a bidentate

hydrogen bonding interaction with the glycerol side chain of sialic acid is achieved; Whereas upon binding of 8 in the neuraminidase active site, the Glu 276 side chain rotates away from the center of pocket 1 and adopts an alternate conformation which is stabilized by a strong charge-charge interaction with the nearby guanidine group of Arg 224. Such a conformational change effectively enlarges Pocket 1 and creates a much less polar environment which can accommodate a hydrophobic binding group such as the ethyl group of the 3-pentyl side chain of 8 . Animal Studies Due to the poor oral bioavailability of GS 4071, in vivo efficacy in animal models has largely been conducted using oral administration of oseltamivir. Oral administration of oseltamivir has been shown to provide protection against the lethal effects of influenza A and B virus infection in mice [23, 24]. In addition to the mouse animal model, ferrets provide a useful experimental animal model in which to measure symptomatic and virological responses to treatment with neuraminidase inhibitors. When infected with the influenza virus, ferrets develop a disease state of limited duration similar to that seen in humans. Oral administration of oseltamivir twice a day for 5 days beginning 4 hours after infection was found to be effective in the ferret influenza model [24].

A rational approach to drug design in conjunction with available X-ray crystal structures was utilized in the discovery of potent inhibitors targeting influenza neuramindase. In the past several years, influenza neuramindase has been validated as a target for the development of anti-influenza drugs as demonstrated by the efficacy of zanamivir and oseltamivir. Although it remains to be further investigated, the resistance development from neuraminidase inhibitors appears to not be clinically significant. Oseltamivir (TAMIFLU) was approved in October 1999 by the U.S. Food and Drug Administration for use as the first orally administered neuraminidase inhibitor for the treatment of influenza infection. OSELTAMVIR LOOK FOR REFERENCES

LIFE CYCLE OF INFLUENZA For influenza virus to be infelctive, its haemagglutinin must first bind to sialic acid glycoconjugatres, the putative receptors on the host cell for the virus. Binding of the HA allows the virus to penetrate the plasma membrane, uncoat and enter the cytoplasm. Viral RNA strands replicate in the nucleus and new virus particles are produced. Neuraminidase is a glycohydrolase enzyme that is responsible for the terminal sialic acid residues from carbohydrate moieties on the surface of host cells and in influenza virus envelope. Neuraminidase is responsible for the release of newly formed virus from the surface of infected cells and aids the motility of the virus through the mucous lining of the respiratory tract. Neuraminidase inhibitors such as zanamivir are sialic acid aanalogues. They work by preventing the enzyme from cleaving sialic acid residues on the surface of host cells and influenza viral envelope (Von Itzstein at al 1993; Whittington and Bethell 1995) viral heamagglutinin binds to uncleaved sialic acid residues, resultingin viral aggregation and a reduction in the amount of virus that is able to infct cells. A breakthrough at inhibiting influenza neuraminidase came when its structure was finally crystallised, thereby allowing the 3D structure of the active site to be elucidated. Various sialic acid analogues were developed aided by computer assisted modelling of the active site. This led chemists to identifying 4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid (zanamavir) as a potent inhibitor of influenza neuraminidase.

Neuraminidases (salidases) are found in a wide variety of organimsm and even in mammalian cells where they play important roles. In vitro studies have shown that zanamavir is a specific influenza neuraminidase inhibitor with only partial activity for mammalian, bacterial and parainfluenza sialidases (holzer et al. 1993; von Itzstein et al. 1993)

zanamivir, a drug derived from the naturally occurring sialic acid Neu5Ac with minimal additional functionalization, would not result in viable mutants as functionally important amino-acid residues had been targeted or unknown side effects. So far, over 8 years of clinical experience with zanamivir has not provided evidence to suggest otherwise, although this may simply be the result of the limited use of this drug. In the case of oseltamivir, which has been much more widely used, a viable resistant influenza virus mutant has emerged56. Interestingly, this mutation targets and effectively blocks the essential rearrangement of Glu276 within the sialidase active site and as a consequence the drug has significantly reduced affinity (FIG. 5b). This oseltamivirresistant virus remains sensitive to zanamivir 56. These experiences may provide some evidence that maintaining a strong resemblance to the natural substrate, Neu5Ac, mightreduce the prospect of the development of viable drug-resistant mutants

With regards to adverse effects of sialidase inhibitors in general, the only significant adverse effects that have been reported in the past 2 years have been some speculation about adverse neuropsychiatric effects of oseltamivir in certain age groups 57.

SUMMARY Zanamavir was the first neuraminidase inhibitor to be approved for the treatment of influenza in humans. It is delivered by inhalation to the respiratory tract, which is the site fo viral replication, in order to ensure immediate antiviral activity. FUTURE DEVELOPMENTS
the development of next-generation antiinfluenza drugs must be a high priority. To this end, the US FDA has established a fast-track programme for the development of such drugs. So far, the FDA has provided fast-track designation for the Biocryst injectable candidate peramivir (compound 9, FIG. 3). Biota and Sankyo have also announced their intention to develop a new influenza virus sialidase inhibitor that is a divalent zanamivir (compound 12, FIG. 3), which is currently in clinical trials. An injectable drug such as peramivir might be of great value to patients who cannot readily take tablets or have limited lung capacity. Also, a long-acting sialidase inhibitor, such as divalent zanamivir, that might reduce the number of treatments required to as little as once a week compared with the current twice-daily. requirement is appealing.
As a proven anti-influenza drug target, neuraminidase continues to be attractive for the development of new inhibitors. The crystal structure of H5N1 avian influenza neuraminidase (PDB code: 2HTY) provides the three-dimensional structural information and opportunity for finding new inhibitors in this regard, because the existing inhibitors, such as oseltamivir and zanamivir, were developed based on different structures of neuraminidase, such as subtypes N9, N2, and type B genus of influenza virus

It has now been 15 years since the structure of influenza virus neuraminidase was determined [Varghese et al., 1983; Colman et al., 1983]. The development of the 4-subtituted Neu5Ac2en analogue inhibitors dates from 1987, when the structure of sialic acid and Neu5Ac2en complexed with neuraminidase were determined to sufficient accuracy to permit modelling of potential inhibitors [Varghese et al., 1992]. The development was a multidisciplinary collaboration of biochemists, crystallographers,

molecular modellers, and synthetic chemists, and culminated in the synthesis [von Itzstein et al., 1994] and biological testing of the compounds [von Itzstein et al., 1993]. Similar results have been obtained by the orally active Gilead carbocyclic analogue GS4104.

Both references

references OSELTAMVIR not printed