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Research Overview



Development of Neuraminidase Inhibitors as Anti-Influenza Virus Drugs

Joseph N. Varghese*

Biomolecular Research Institute, Parkville, Victoria, Australia


Strategy, Management and Health Policy


Venture Capital


Preclinical Development Toxicology, Formulation Drug Delivery, Pharmacokinetics

Clinical Development






Phases I-III

Regulatory, Quality,



Phase IV

ABSTRACT Structure-based design and synthesis of potent influenza virus neuraminidase inhibitors are now being evaluated in human trials as anti-influenza virus drugs. The first drug of this class, Relenza™ (Zanamivir/GG167), is now awaiting pharmaceutical evaluation and registration in Australia, Europe, and North America for both treatment and prophylaxis of influenza. The target for the drug is the active site of neuraminidase, which is a pocket that has been totally conserved in both Type A and B influenza in all known subtypes of influenza (animal and human). Mutations in residues that surround this conserved pocket allow the virus to escape binding to circulating antibodies that recognise the molecular surface

around the active site of the wild-type virus. High-affinity neuraminidase inhibitors have been designed that interact only with the conserved active site residues. 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 in- fluenza neuraminidase, and have been shown to inhibit influenza virus replication in both cell culture and animal models. The development of drugs against a rapidly mutating organism like influenza has to address to the possibility of emerging drug resistance. This is examined in the light of drug resistant mutants se- lected after in vitro passaging of virus in the presence of neuraminidase inhibitors. Drug Dev. Res. 46:176–

196, 1999.

© 1999 Wiley-Liss, Inc.

Key words: sialidase; Zanamivir; Relenza™


Influenza, a viral infection of the upper respiratory tract in humans, has plagued mankind since the dawn of history. The disease in modern times continues to affect a significant proportion of the population irrespective of age or previous infection history. These periodic epidem- ics that reinfect otherwise healthy people have devas- tated communities worldwide. Some pandemics like the 1917–1919 “Spanish flu” were responsible for the deaths of tens of millions of people throughout the world. The origins, spread, and severity of influenza epidemics have been a puzzle that has only in the last two decades been adequately addressed. The virus is spread by aerosols produced by infected animals, and the continual produc- tion of new strains of the virus results in reinfection [re- viewed by Kilbourne, 1987]. There are three types of influenza virus classified

© 1999 Wiley-Liss, Inc.

on their serological cross-reactivity with viral matrix pro- teins and soluble nucleoprotein (A, B, and C). Only type

A and B are known to cause severe human disease. Type

B is only found in humans, while type A has a natural

reservoir in birds and some mammals like pigs and horses [Webster et al., 1993]. Influenza, an orthomyxovirus, is a 100 nm lipid-enveloped virus containing an eight-seg- ment negative single-stranded genome [Lamb, 1989]. Two of the segments code for the surfaces glycoproteins he- magglutinin 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 Re- search Institute, 343 Royal Parade, Parkville, Victoria 3052, Austra- lia. E-mail:



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 depen- dent on interferon induction [Ennis and Meager, 1981] 48 h postinfection, which attenuates viral replication until the cell-mediated 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 effec- tive treatment is available at present to halt the progres- sion of the disease. Death can result for susceptible populations (neonate and elderly), primarily as a result of secondary infections [Sprenger et al., 1993]. In this article, a structural basis for the continual emergence of new influenza strains is discussed, and the reasons why current vaccines against influenza fail to protect against all strains of influenza. 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 po- tent NA inhibitors. The therapeutic use of these inhibi- tors as anti-viral drugs against influenza virus infections shall be examined and the possibility of drug resistance to NA inhibitors shall be addressed.

Antigenic Variation

The plethora of different strains of the virus that are responsible for continued reinfection of the virus in humans is primarily related to mutations in the viral genes of two surface glycoproteins, HA and NA [Smith and Palase, 1989]. The current paradigm for this genetic varia- tion [Skehel, 1974; Webster and Laver, 1975] is that these mutations arise primarily from incremental changes in the amino acid sequences of these glycoproteins by se- lection pressure of the immune system of the infected

host. This mechanism, termed “Antigenic Drift,” accounts for most of the strain variation within a particular sub- type of influenza. However, infrequently a mutation arises by genetic re-assortment of viruses from different animal hosts (“An- tigenic Shift”), whereby an entirely new gene for one of the surface glycoproteins is generated which is signifi- cantly different (~50%) in amino acid sequence of the parent virus. This is the mechanism by which new sub- types of influenza arise which are primarily responsible for the major pandemics that occur. Such a great change in the antigenic surface of these glycoproteins results in little or no cross-protection by circulating antibodies to previous influenza infections, leading to the global spread of severe influenza and the high mortality rate such pandemics inflict. Strains of influenza virus are classified by type (A, B, or C), geographic location, date of original isolation, and the subtype of the HA and NA antigens. There exist nine known subtypes (N1 to N9) of NA and 13 known subtypes (H1 to H13) of HA from influenza A in all ani- mal populations. Two NA (N1 and N2) and three HA (H1, H2, and H3) subtypes of influenza A have occurred in strains that have infected humans since 1933, when iso- lates were first characterised [Smith et al., 1933]. Prior to 1933 there is indirect evidence of antigenic shift oc- curring in human populations [Beveridge, 1977]. The N1 subtype was associated with virus isolated between 1933 and 1957, after which time the N2 subtype appeared in the Asian influenza. No major change in the structure of NA has occurred since, although the HA subtype has changed from H2 to H3 in 1968 in the Hong Kong pan- demic, and H1N1 reappeared in 1978 as the Russian pandemic. Recently (1998) an avian strain, H5N1, caused a brief outbreak in humans in Hong Kong that was quickly contained, as it appears that infections were only trans- mitted from avian to human and not human to human [Yeun et al., 1998; Subbaroa et al., 1998; Claas et al., 1998]. Influenza B, which infects only human hosts, has only one subtype, but like type A undergoes continual anti- genic drift.

Current Therapeutics and Vaccines

Amantadine and rimantadine are the only class of drugs that have been approved for therapy [Douglas, 1990; Wintermeyer and Nahata, 1995]. At high concen- tration (>50 g/mL), amantadine is thought to buffer the pH of the endosome and prevent the conformational change of the HA necessary for fusion. Drug-resistant mutants arise where the hemagglutinin trimers are thought to be less stable than the wild type [Daniels et al., 1985]. At low concentrations (<1 g/mL), amanta- dine blocks the activity of the viral M2 ion channel pro- tein, which plays a role in virus uncoating and



glycoprotein maturation [Hay et al., 1993]. Amantadine prevents fusion by altering the ability of the virus to change the ion balance [Pinto et al., 1992] within the en- dosome and the trans-Golgi network. However, amanta- dine rapidly gives rise to drug-resistant strains by mutations in the HA and the M2 protein that circumvent or block the activity of the drug. Amantidine and rimantadine can cause adverse CNS complaints and other complications [Srange et al., 1991; Monto et al., 1995], although rimantadine can be taken at lower doses, in part related to lower plasma levels, and much less dependence on renal secretion for elimination than amantidine. Rimantadine has been shown to lead to drug resistance in humans 2 days posttreatment [Hayden, 1993]. Influenza virus vaccines [Tyrrell, 1980; Murphy and Webster, 1990] prepared from killed (formalin inactivated) virus are used currently worldwide as the only prophy- lactic treatment available against the disease. Killed vac- cines contain whole virus or fractionated subunits. These vaccines attempt to incorporate antigens from influenza strains that are predicted to circulate in the community during the next season. They also confer immunity to vi- ral strains that are closely related to the strains incorpo- rated into the vaccine. However, the susceptibility of the virus to neutralisation by antibodies raised by immuni- sation is reduced by antigenic drift. For example, com- mercial vaccines containing inactivated A/Beijing/353/89 H3N2 strain circulating among humans from 1990 to 1993 provided significantly less protection [Chakraverty et al., 1993] against the antigenic drift [Both et al., 1983] vari- ant A/Georgia/03/93. A more radical vaccination treatment involving the direct injection into muscle of plasmid DNA encoding HA, nuclear protein, and M1 influenza viral proteins has suc- cessfully been attempted in animal models [Donnelly et al., 1995]. This is thought to involve the incorporation of the plasmid into muscle cells, and the production of the viral proteins cell-mediated immune (CMI) response elic- ited offers cross-protection due to reactivity with the more conserved epitopes of the HA and nuclear proteins, thus overcoming antigenic drift in the virus. The success of the method arises from a CMI response to segments of con- served amino acids in these proteins. Problems arising from possible autoimmune response to DNA, and in ensuring the exogenous DNA is not integrated into the cell genome or sensitive cell lines elsewhere in the host, have precluded testing in humans at present. Furthermore, mutations in the nuclear proteins of influenza (which have been rela- tively stable to date) arising from CMI selection pressure could undermine the efficacy of this technique.


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 glyco- proteins 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 Neu5Ac- containing receptors on the surface of susceptible cells [Rogers and Paulson, 1983]. NA, which also removes ter- minal 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 mu- cosal secretions in the upper respiratory tract. By remov- ing 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 sur- face. It has been shown that neuraminidase-deficient vi- rus [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, replica- tion, assembly, or budding in mice. It has an important role in facilitating the spread of the infection by prevent- ing aggregation at the cell surface and possible immobilisation in the mucin by HA. During virus repli- cation, the freshly synthesised viral glycoproteins have to be desialylated to prevent self-aggregation at the in- fected host cell surface by HA binding to terminal sialic acid on these glycoproteins. Finally, on elution of prog- eny 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- ing the rate of viral attachment and subsequent release of progeny virons. This would allow the host immune system to eliminate the virus.

Molecular Structure of Neuraminidase

There are between 50 to 100 NA spikes per virion [Bucher and Palese, 1975], which are approximately 10% of the visible spikes projecting out of the surface of the virion [White, 1974]. These spikes can be removed from the virus by treatment with detergent [Laver, 1963]. Elec- tron microscopic images of the NA spikes [Laver and Valentine, 1969] reveal a mushroom-shaped molecule made up of a box-like head of about 80 80 40 A, with



a narrow centrally attached stalk (15 A wide and 100 A long) which terminates in a hydrophobic knob anchored in the viral envelope. The detergent-released spikes can be digested by pronase to release the NA “heads,” which retain full antigenic and enzyme activity [Drzenick et al., 1968; Laver, 1978]. The molecule was found to be a tet- ramer of molecular weight 240,000, reducing to 200,000 when treated with pronase [Blok et al., 1982]. The X-ray 3-dimensional molecular structure of NA heads was determined [Varghese et al., 1983] for two N2 subtypes, A/Tokyo/3/67 and A/RI/5+/57. The structure of A/Tokyo/3/67 N2 has since been refined [Varghese and Colman, 1991] to higher resolution (2.2 A), as have the structures of two avian N9 subtypes [Baker et al., 1987; Tulip et al., 1992a] and influenza type B [Burmeister et al., 1991]. They were found to have an identical protein fold with 60 residues (including 16 conserved cysteine residues) being invariant. Bacterial sialidases from Sal- monella [Crennell et al., 1993] and cholera [Crennell et al., 1994] have homologous structures to influenza NA, but only a few of the residues in the active site are struc- turally invariant. The protein fold consists of a symmetric arrange- ment of six four-stranded antiparallel -sheets arranged as the blades of a propeller, the propeller axis being ap- proximately parallel to but tilted away from the circular 4-fold axis of the tetramer. This tilt angle varies between the known subtypes. There is a calcium-binding site connected to the active site via conserved residues. The functional role of calcium in the structure is unknown, although calcium has been shown to be necessary for NA activity [Baker and Gandhi, 1976] and it is essential for the thermostablity of the molecule [Burmeister et al., 1994].

Carbohydrate Structure

Carbohydrates at four N-linked glycosylation sites were observed in N2 NA at residues 86, 146, 200, and 234 in the X-ray structure. Two N-actylglucosamines were resolved at Asp86 and Asp234, both at the bottom sur- face of the monomer. The carbohydrate at Asp200 con- sists of at least eight sugar residues with linkages consistent with known mannose-rich simple N-linked car- bohydrate [Wagh and Bahl, 1981]. This oligosaccharide emerges from the side of the monomer and covers a neighbouring subunit (see Fig. 1). The oligosaccharide site at Asn146 is the most con- served of all neuraminidase glycosylation sites, except that of the neurovirulent virus A/WSN/33 (H1N1) [Francis and Moore, 1940; Hitte and Nayak, 1982]. The absence of this glycosylation site in A/WSN/33 has been shown to confer neurovirulence in mice [Li et al., 1993]. It has recently been shown [Goto and Kawaoka, 1998] that the NA of A/WSN/33 (a descendent of the virus responsible

for the 1918 pandemic, selected by serial passaging by mouse brain) binds and sequesters plasminogen. This leads to higher local concentrations of this protease pre- cursor and, thus, increasing cleavage of HA. This is a possible explanation for the pantropism of this strain [Kunkel et al., 1987]. The structural basis for this unusual function of NA appears to be the presence of a carboxyl- terminus lysine and the absence of the carbohydrate at Asn146. Goto and Kawkaoka [1998] suggest that this car- bohydrate obstructs the binding of plasminogen to the terminal lysine, suppressing HA cleavage in cells other than its usual targets. This oligosaccaride is a complex sugar containing N-acetylgalactosamine [Ward et al., 1983] that is not found in any other of the known oligosaccarides of influenza virus glycoproteins, and is the only glycopeptide antigenically related to chick em- bryo “host antigen” [Ward et al., 1982]. The oligosaccha- ride appears as a spike emanating from the top of the monomer, forming a crystal contact with a neighbouring tetramer in crystals of A/Tokyo/3/67 NA. The four carbo- hydrate spikes of a tetramer form an open “barrel” struc- ture of eight carbohydrate chains with the neighbouring tetramer, with no apparent inter-carbohydrate contacts [Varghese and Colman 1991]. This oligosaccharide may play an important but as yet unidentified role in NA struc- ture or activity.

A Second Sialic Acid Binding Site

Hemagglutination activity has been reported for the N9 subtype of NA of influenza type A virus [Laver et al., 1984] at 4C, which was not related to aberrant neuraminidase activity, but was associated with a second Neu5Ac binding site on the surface of NA away from the active site [Webster et al., 1987]. The residues on the surface of NA responsible for the haemabsorbing (HB) activity have been identified by monoclonal variants which lost the capacity to bind red blood cells [Webster et al., 1987], and the activity was successfully transferred to the N2 subtype of NA [Nuss and Air, 1991] by site- directed mutagenesis. Furthermore, it was shown that N9 NA activity did not remove the putative sialic acid- related moiety that bound red blood cells to this HB site [Air and Laver, 1995], as the agglutination was restored on cooling to 4C. An HB site has also been discovered on the NA of A/FPV/Rostock/34 H7N1 [Hausmann et al., 1995], which appears to have the same location on the NA surface as in N9 NA. Recently, Varghese and co-workers [1997] located the HB site on N9 by X-ray diffraction (Fig. 2). They have shown that six residues on three separate loops of N9 NA interact directly with the sialic acid in the second sialic acid binding site. These are the three serine resi- dues (367, 370, and 372) in the loop containing residues 367-SIASRS-372, Asn400, and Trp403 in the loop con-



180 VARGHESE Fig. 1. A CPK atomic model of the upper surface of an influenza virus

Fig. 1. A CPK atomic model of the upper surface of an influenza virus neuraminidase N2 tetrameric head [Varghese and Colman, 1991]. The long spike-like structures emerging from the face of the tetramer towards the right are carbohydrate moieties attached to Asn146. The dark spheres represent those residues that have been totally conserved in all known

taining 400-NTSW-403, and Lys432 in a third loop. The sequence variation at residues 400 and 432 in NAs with known HB activity indicates that these residues are not essential for HB activity, and the triple serine SxxSxS loop of 367 to 372 and Trp403 are a minimal signature for HB binding in NA.

influenza viruses, and they cluster near the corners of the molecule where the active site of the enzyme is situated. It is this site to which the neuramini- dase inhibitors are targeted. Residues that have mutated since 1933 are represented by lighter shaded spheres, and they surround the conserved active site.

All known strains from N1 to N9 which carry the signature are avian (or equine), with the exception of the two human N2 strains, RI5+/57 and Leningrad/134/57 [Varghese et al., 1997]. All avian sequences appear to carry this signature, and in general human and swine NAs do not, indicating that the HB site has a functional role in



ANTI-VIRAL DRUGS FOR INFLUENZA 181 Fig. 2. A surface rendered image [Nicholls et al., 1991] of

Fig. 2. A surface rendered image [Nicholls et al., 1991] of a subunit of influenza neuraminidase N9. Shown in darker grey are residues conserved in all influenza viruses that interact with sialic acid (twist-boat) in the

avian influenza. It has been shown [Air and Laver, 1995] that the HB site on A/tern/Australia/G70C/75 NA binds a moiety on red cells that is not cleavable by A/tern/Aus- tralia/G70C/75 NA. This is a consequence of the moiety either being Neu5Ac in a noncleavable linkage, or some- thing other than Neu5Ac. The biological significance of this HB site has not been determined, but the above re-

catalytic site. In dark grey are also residues that are conserved in all avian strains that interact with sialic acid (chair) in haemabsorption (HB) site.

sults would indicate that it may function as a lectin to some avian cell receptor, which is unaffected by influ- enza NA activity. Influenza is normally an asymptomatic infection in birds [Alexander, 1986], but replicates pref- erentially in the cells lining the intestinal tract of water- fowl [Slemons et al., 1974; Hinshaw et al., 1980], where all of the different subtypes of influenza A have been iso-



lated. This has led to the proposition that waterfowl are the primary vectors for the global spread of the disease [Webster et al., 1992] through faecal droppings. This avirulent adaptation of the virus to avian species enables it to survive and persist in a vast global reservoir. If this HB signature relates to some as-yet unidenti- fied role in avian influenza, then it could be inferred that the first human 1957 pandemic N2 strains carrying the HB signature (RI5+/57 and Leningrad/134/57) were most prob- ably derived from a genetic reassortment event with an avian strain [Skehel, 1974]. This HB signature has since disap- peared under antigenic drift in post-1957 human and swine stains, indicating that it serves no biological function in pathogenesis of influenza in humans and pigs.

Antigenic Variation in Neuraminidase Structures

Comparison of all known sequences of approximately 390 residues of the NA globular head [Varghese and Colman, 1991], indicates that only 54 (excluding 16 conserved cys- teine residues) are invariant (Fig. 1). Apart from 21 resi- dues involved in preserving the structural integrity of the molecule [Varghese and Colman, 1991], the main cluster- ing of these invariant residues is within the enzyme active site (Fig. 1), where 17 are in the active site and 16 neighbouring the active site. This is a cavity on the upper surface of the molecule into which sialic acid has been ob- served to bind [Colman et al., 1983; Varghese et al., 1992; Burmeister et al., 1991]. Excluding the active site pocket, strain variation occurs over the entire surface of the NA heads. The active site was found to be in a pocket of totally conserved (over all animal subtypes) residues [Colman et al., 1983]. In this way the enzyme active site pocket is sur- rounded by highly variable surface residues that prevent immune recognition by antibody molecules of the active site [Colman, 1994]. X-ray diffraction studies of NA–anti- body complexes have shown that the footprint of an anti- body in the complex is larger than the exposed surface of the conserved region of the active site [Colman et al., 1987, 1989; Malby et al., 1994]. These structural results indicate that antibodies are unable to exert mutational pressure on the conserved active site because they cannot bind there without engaging strain variable residues as part of the bind- ing surface [Colman, 1997]. However, since antibodies bind to strain variable elements of the structure, the virus can overcome host immune pressure by point mutations of these residues that do not have a catalytic or structural role [Varghese, 1988; Tulip et al., 1992b], but are able to disrupt the antigen–antibody binding interface. The rapid emer- gence of these escape mutants explains the failure to pro- duce a universal vaccine for influenza.

Enzyme Active Site

The structures of N-acetyl neuraminic acid (sialic acid Neu5Ac) and the 2-deoxy-2,3-dehydro-N-acetyl

neuraminic acid (Neu5Ac2en) inhibitor complexed with N2 NA [Varghese et al., 1992] revealed the na-

ture of the interactions of the molecules in the active site pocket (Fig. 3). Sialic acid binds in the active site in the -anomer, and in a distorted half-chair confor- mation through the same face as used in its interac- tion with HA [Weis et al., 1998]. The carboxylate group of the sugar interacts with three guanidinium groups of arginine residues 118, 292, and 371 and has an equa- torial conformation with respect to the sugar ring (this group, axial toward the floor in the undistorted struc- ture, is probably held equatorial by interactions with these arginine residues). The NH group of the 5-N- Acetyl side chain interacts with the floor of the active site cavity, via a bound water molecule. The oxygen of the 5-N-Acetyl side chain is hydrogen bonded to the

N of Arg152, while the methyl group lies in a hydro-

phobic pocket near Ile222 and Trp178. The last two hydroxyl groups of the 6-glycerol sidechain are hy- drogen bonded to carboxylate oxygens of Glu276 and

the 4-hydroxyl is directed to a carboxylate oxygen of Glu119, and the glycosidic oxygen O2 interacts with

a carboxylate oxygen of Asp151. Similar binding of

sialic acid in the active site was observed in type B [Burmeister et al., 1991]. Comparison of active sites of N2, N9, and type B NA [Varghese et al., 1995] show there are no significant differences between active site orientations, except for some minor displacements of Arg224 and Glu276, where the major interactions with the 6-glycerol group of sialic acid occur. However, there are some differences in the water structure in the active sites of the different sub- types. Comparison of the active sites of influenza neuraminidases and bacterial sialidases [Crennell et al., 1993, 1994] indicates that there is considerable conser- vation of the catalytic site at the carboxylate-binding end. The residues Asp151, Arg118, Glu277, Arg292, Val or Ile349, Arg371, Try406, and Glu425 are conserved over all known viral and bacterial strains. The argininyl resi- dues 118, 292, and 371 position the 2-carboxylate group and the Val(or Ile)349, Glu425 and Glu 277 are impor- tant in positioning the triarginyl cluster. Asp151 and Tyr406 are presumably important in bond cleavage, but the precise mechanism is still unclear. These eight resi- dues (Fig. 4) are thus most likely to be conserved in all neuraminidases. Differences between viral, bacterial, and mammalian NA structures may correspond with the dif- ferent role these enzymes have in vivo. These differences are likely to be in the interactions of the 6-glycerol, 5-N- acetyl, and 4-OH groups of silaic acid. In influenza, the turnover rate must be balanced against the requirement to maintain sufficient sialic acid at the cell surface to en- able attachment via the HA. This balance may require some configuration of residues in the active site not di-



ANTI-VIRAL DRUGS FOR INFLUENZA 183 Fig. 3. A ball and stick representation [Kraulis, 1991] of sialic

Fig. 3. A ball and stick representation [Kraulis, 1991] of sialic acid (Neu5Ac, in the centre of the diagram) bound in the active site of neuraminidase N2 [Varghese et al., 1992]. The tube represents the back-

rectly responsible for catalysis, but only involved in the binding and release of sialic acid [Varghese et al., 1995].


Earlier screening programs [Edmond et al., 1966] failed to identify potent inhibitors of viral NA. Neu5Ac2en (Fig. 5) was the first inhibitor synthesised [Meindl and Tuppy, 1969] whose K i was in the micromolar range. This was based on the proposed transition state of the reac- tion, where the anomeric carbon (C 2 ) bound to the ketosidic oxygen has a trigonal state. Several analogues of Neu5Ac2en were synthesised soon after, and the most potent of these, a trifluoracetyl derivative, had a K i of only 0.8 M [Meindl et al., 1974]. While this compound

bone of the protein, and black and grey spheres represent oxygen and nitrogen atoms. Thin lines represent bonding interactions between sialic acid and the conserved amino acid side-chains of the active site.

showed that in cell culture it retarded virus shedding [Palase and Compans, 1976; Palese et al., 1974], it failed as an effective antiviral agent in animals [Palese and Schulman, 1977]. The X-ray structure of Neu5Ac2en/neuraminidase complexes have been determined for N2 [Varghese et al., 1992], type B [Burmeister et al., 1993], and N9 [Bossart- Whitaker et al., 1993]. Neu5Ac2en binds in the active site of NA with the carboxylate oxygen atoms placed in the same location as the carboxylate of sialic acid. The 5- N-Acetyl, 4-hydroxy, and 6 Neu5Ac2en-glycerol are po- sitioned isosterically in the two molecules. An alternative approach to developing sialidase inhibitors has been made using synthetic thioglyco-



184 VARGHESE Fig. 4. A ball-and-stick representation [Kraulis, 1991] of Zanamivir/ GG167 (centre in light grey)

Fig. 4. A ball-and-stick representation [Kraulis, 1991] of Zanamivir/ GG167 (centre in light grey) in the active site of neuraminidase, show- ing “catalytic” residues (black) that are conserved over both bacte-

side-analogues of gangliosides such as Neu5Ac (2-S- 6)Glc (1-1)Ceramide [Suzuki et al., 1990], which in- hibit different subtypes of human and animal influenza virus with a K i of up to 2.8 M. These metabolically stable ganglioside analogues contain a thioglycosidic linkage to the terminal neuraminic acid that resists cleavage by the enzyme. Several flavonoid neuramini- dase inhibitors have been isolated from plant extracts [Nagai et al., 1990], one of which, 5,7,4 -trihydroxy-8- methoxyflavone was a more potent inhibitor than

rial and viral neuraminidases, and “recognition” residues (grey) con- served only in influenza virus neuraminidase. The tube is a repre- sentation of the protein backbone.

Neu5Ac2en. Recently, in vivo anti-influenza virus activ- ity of a Kampo (Japanese herbal medicine) preparation has shown promising results in inhibiting influenza vi- rus replication in mice [Nagai and Yamada, 1994], but the mode of action of these compounds is unclear.

Enzyme Mechanism

The similar positioning of the carboxylate oxygens and ring of Neu5Ac2en and sialic acid suggests that Neu5Ac2en is probably a transition state analogue. As



ANTI-VIRAL DRUGS FOR INFLUENZA 185 Fig. 5. Influenza neuraminidase inhibitors: 1 ) Sialic Acid, N- Acetylneuraminic

Fig. 5. Influenza neuraminidase inhibitors: 1) Sialic Acid, N- Acetylneuraminic acid (Neu5Ac); 2) 2-deoxy-2,3-dehydro-N- acetylneuraminic acid (Neu5Ac2en); 3) 4-amino-Neu5Ac2en; 4) Zanamivir, 4-guanidino-Neu5Ac2en; 5) 5-N-acetyl-4-guanidino-6- methyl(propyl) carboxamide-4,5-dihydro-2H-pyran-2-carboxylic acid; 6) 5-N-acetyl-4-amino-6-diethyl carboxamide-4,5-dihydro-2H-pyran-2-car- boxylic acid; 7) GS4071, 4-N-acetyl-5-amino-3-(1-ethylpropoxy)-1- cyclohexene-1-carboxylic acid.

Neu5Ac2en has a higher affinity for NA than sialic acid, a mechanism of catalysis was proposed [Miller et al., 1978] that involves the distortion of the substrate by the forma- tion of a oxycarbonium ion intermediate, which has a simi- lar structure to Neu5Ac2en. However, the structural results from sialic acid/NA complexes suggest that the tighter binding of Neu5Ac2en arises more likely from the relaxation of the conformational strain arising from the transition from chair to boat of the pyranose ring of sialic acid in the active site [Varghese et al., 1992]. Evidence for a sialyl cation transition state by iso-

topic effects [Chong et al., 1992] support the existence of

a oxycarbonium ion intermediate. However, the struc-

tural basis for NA activity is still unclear. It has been sug-

gested [Varghese et al., 1992] that the tyrosyl oxygen of Tyr406 assisted by the sialic acid carboxylate itself could stabilise the developing charge on the oxycarbonium ion intermediate. The reaction is completed by the activa- tion of a water molecule by a deprotonated Asp151, and its attack on the carbonium, resulting in the formation of the anomer of sialic acid [Friebolin et al., 1980]. How- ever, the pH activity profile of neuraminidase [Lentz et

al., 1987; Chong et al., 1991; Burmeister et al., 1993] sug- gest a bell-shaped profile, indicating normal activity from

a pH range of about 4.5 to 9, which would indicate that

the role of Asp151 as the acid group in the catalysis is unclear. A nonspecific proton donor has been proposed [Taylor and von Itzstein, 1994], probably a water mol- ecule as the acid group, with a deprotonated Tyr406

stabilising the oxycarbonium ion, and a proton transferred from water to the departing aglycon group. It has also been postulated that a proton elimination at C3 leads to the transformation of the oxycarbonium ion into Neu5Ac2en, which is produced irreversibly at low levels from sialic acid by the enzyme [Burmeister et al., 1993].

A S N 1-type mechanism has been suggested [Taylor and

von Itzstein, 1994] that is facilitated by an activated wa-

ter molecule that can be expelled upon inhibitor bind- ing. The catalytic mechanism could possibly proceed without an acid group, by the electrostatic potential of the enzyme lowering the barrier preventing the break- ing of the ketosidic bond, and the solvent protonating the aglycon after release [Janakiraman et al., 1994]. Clearly, details of the enzyme mechanism have yet to be elucidated definitively, as structural considerations can only indicate that Tyr406 (and possibly Glu277) and the triarginyl cluster are essential in the enzyme mechanism, and that Asp151 could be implicated in it.

Inhibitor Design Principles

All the nearest neighbour interactions with sialic acid and Neu5Ac2en and the protein are with totally con- served amino acids. Thus, an inhibitor designed to bind only to the conserved active site residues of NA would inhibit NA activity across all strains of influenza. This would enable the development of an anti-viral drug that would affect the spread of viral replication potentially in three ways, i.e., transport through the protective mucosal layer, desialyation of freshly synthesised viral glycopro- teins, and elution of progeny virions from infected cells. The development of potent inhibitors was based on the structural information of the N2 NA conserved ac- tive site and its complex with sialic acid and Neu5Ac2en. There are no reports of de novo molecules designed to fit into the cavity, and the most useful approach was to



consider molecules that were structurally related to Neu5Ac2en. This involved the design of molecules that would bind isosterically to Neu5Ac2en, but which were modified to increase the number of favourable interac- tions with the protein. The method of Goodford [1985] enabled the calcu- lation of favourable binding sites for a variety of chemi- cal probes. The validity of this method was indicated by its method’s ability to identify the positions of the car- boxylate binding site of sialic acid as an energy minima for a carboxylate probe, and the successful prediction of known bound water sites in the active sites by a water probe. Utilising this methodology, predictions of ener- getically favourable substitutions to Neu5Ac2en were examined [von Itzstein et al., 1993]. A replacement of the hydroxyl at the 4-position of the pyranose ring of Neu5Ac2en by an amino group was identified by this procedure as an energetically favourable substitution. A protonated primary amine probe identified a favourable binding site of -16 kcal mol 1 at this location, and in a pocket in the active site near two conserved glutamate residues, Glu119 and Glu227. This suggested that the substitution of the 4-hydroxyl group by an amino group would increase the overall binding interactions by form- ing a salt link with Glu119. Furthermore, the substitu- tion of the 4-hydroxyl group with a much more bulky guanidinyl group would lead to even tighter binding as a result of lateral interactions of the terminal nitrogens of the guanidinyl group and the carboxylate groups of Glu119 and Glu277. This led to the design and syntheses of 3 and 4 (4- amino-Neu5Ac2en and 4-guanidino-Neu5Ac2en in Fig. 5) [von Itzstein et al., 1993] (4 is GG167, Zanamivir, Relenza™), which bound to A/Tokyo/3/67 with a K i of 50 nM and 0.2 nM, respectively. These compounds were later shown by X-ray studies of 3 and 4 complexed with A/Tokyo/3/67 NA to bind close to that predicted by the design studies. However, details of the interactions of the guanidinyl group of 4 with the glutamic acid groups (Glu119 and Glu277) in the floor of the active site were slightly different. This was confirmed on a higher reso- lution X-ray study (Fig. 4) of 4 complexed with Tern N9 NA [Varghese et al., 1995]. One of the primary guanidinyl nitrogens of 4 is hydrogen bonded to the main chain oxy- gen at residue 178, a carbxylate oxygen of Glu227, and a water molecule. The other primary guanidinyl nitrogen interacts with the main chain oxygen of residues 178 and 151. The secondary guanidinyl nitrogen interacts with the carboxylate of Glu119 and Asp 151. The interactions with Glu119 lack the hydrogen bonding geometry (pos- tulated in the design study), as the carboxylate group of Glu119 stacks parallel to the guanidinyl group, with its interactions being electrostatic and van der Waals in char- acter. Furthermore, theoretical energy-minimised struc-

tures of the complex using AMBER [Pearlman et al., 1990] converged to the X-ray structure only if the protein nonhydrogen atom were kept rigid in the X-ray struc- ture [Varghese et al., 1995]. Otherwise, this resulted in active site residues showing large distortions in their con- formation. This is an example of the difficulty in correctly modelling even modest changes in the interactions of an inhibitor/active site complex. Zanamivir shows potent inhibition of NA activity in all known wild strains of influenza. Furthermore, it is very specific to influenza, as it shows weak inhibition to bacterial, para influenza, and mammalian neuraminidases [von Itzstein et al., 1993; Woods et al., 1993]. This is pos- sibly due to the specific interactions of the 4-guanidino group within the subpocket of the active site of influenza NA that is not conserved in other neuraminidases. In bacterial neuraminidases [Crennell et al., 1993, 1994], this pocket is much smaller, and would prevent the bind- ing of the 4-guanidino group in this region of the active site. This is consistent with the proposition that the in- teractions of sialic acid with the active site of NA are func- tion-specific at the C 4 ,C 5 , and C 6 position of sialic acid, and that modification at these positions confers specific- ity to the target enzyme [Varghese et al., 1995]. Recently, a new class of potent NA inhibitors, ex- emplified by 5 and 6 (Fig. 5) has been reported in which hydrophobic substituents have replaced the glycerol moiety at the 6-position [Sollis et al., 1996; Smith et al., 1996; Taylor et al., 1998]. A small conformational change in the active site of NA occurs to enable these inhibitors to be accommodated (Fig. 6). Glu276 changes its posi- tion to form a salt link with Arg224, and thereby creates the necessary hydrophobic pocket for the carboxamide substituents. It has been proposed that the unexpected strong binding of these inhibitors is a result of the burial of hydrophobic surface area and salt-bridge formation in an environment of low dielectric. Taylor and co-workers [1998] have shown that there is a greater degree of dis- tortion of the active site residues in influenza B NA than influenza A NA on binding with these carboxamide de- rivatives. This correlates with the decreased affinity for influenza B NA when compared with influenza A NA on binding to ligand. They have also shown by molecular dynamics calculations that the tendency for salt-bridge formation is greater in influenza A NA than influenza B NA, and this is a useful descriptor for the prediction of inhibitor potency. A carbocyclic analogue of sialic acid (GS4071, Ro640796) [Kim et al., 1997] (7 in Fig. 5), which has a hydrophobic group attached to the 6 position via an ether link, has also been shown to inhibit NA and virus repli- cation in vitro. This inhibitor binds in a similar mode as the 6-carboximide derivatives of Neu5Ac2en [Varghese et al., 1998], by forming a hydrophobic pocket created



ANTI-VIRAL DRUGS FOR INFLUENZA 187 Fig. 6. A surface rendered image [Nicholls et al., 1991] of

Fig. 6. A surface rendered image [Nicholls et al., 1991] of neuramini- dase N9 with a ball and stick representation of (a) Zanamivir [Varghese et al., 1993] and (b) a carboxamide derivative of Neu5Ac2en in the active site [Taylor et al., 1998]. The change in conformation of Glu276 (grey

by the salt link of Glu276 and Arg224. It could be in-

ferred, as with the carboxamide derivatives, that this com- pound would have a differential binding to influenza A

and B neuraminidases. GS4071 has a similar low bio-

availablity compared to GG167 [Li et al., 1998]; however, an ethyl ester linked to one of the carboxylate oxygens of GS4071 improves the oral bioavailablity following rapid conversion to the active form during gastrointestinal ab- sorption. This prodrug, GS4104, when administered orally results in high and sustained systemic absorption

in animal tests [Li et al., 1998] with a half-life of 5 h in most tissues. Whole body autoradiography in rats has shown a twofold higher concentration in the lungs com- pared to plasma 6 h postdose, and a 30-fold higher con- centration at 24 h postdose [Eisenberg et al., 1997]. Furthermore, distribution in brain tissue was minimal.

This would indicate that the GS4104 inhibitor is orally

active and the inhibitor is currently undergoing human

clinical trials for use as an anti-influenza therapeutic. Other approaches [Luo et al., 1995] to structure- based design of inhibitors have to date produced only


inhibition of neuraminidase activity.


In vitro inhibition of viral replication in tissue cul-


was demonstrated [Palese and Compans, 1976] for

the tri-fluro derivative of Neu5Ac2en, but its antiviral

patch at the upper centre of diagram) from H-bond interactions with the glycerol group of GG167 to formation of a salt-link with Arg224 is shown by the creation of a hydrophic binding pocket in (b). Black spheres repre- sent bound water molecules.

activity in vivo was not demonstrated [Palese and Schulman, 1977]. As a consequence of this, efforts were directed towards HA, which was then considered a bet- ter target for anti-influenza drugs. The interest in neuraminidase inhibitors as anti-influenza drugs has only been revived with the success of 4-guanidino-Neu5Ac2en and its analogues in attenuating viral titres in mice when administered directly into the lungs [von Itzstein et al., 1993; Ryan et al., 1994].

Inhibition In Vitro

von Izstein and co-workers [1993] have shown that the 4-amino- and 4-guanidino-Neu5Ac2en inhibit influ- enza strains A/Singapore/1/57 and B/Victoria/102/95 in MDCK cells with IC 50 values (the concentration required to inhibit plaque formation in MDCK cells by 50%) of 1.5 M and 0.065 M (4-amino) and 0.014 M and 0.005 M (4-guanidino), respectively. These IC 50 values, in particular for the 4-guanidino compound, are well below those found for amantadine, ribavirin, and Neu5Ac2en. Furthermore, in comparison to Neu5Ac2en the 4- guanidino-Neu5Ac2en inhibitor was 100-fold less active against human lysosomal sialidase and over 1,000-fold more active against a wide range of clinical isolates of influenza A and B, including amantidine- and riman- tadine-resistant variants [Woods et al., 1993]. The inhibition of replication of virus in MDCK cells has been confirmed [Thomas et al., 1994] and Hayden



and co-workers [1994] extended the inhibition studies to human respiratory epithelium cells in vitro, indicating high antiviral activity for strains of A(H1N1) and A(N3N2) isolates. They also found that delayed administration of the drug after viral replication was well established was associated with inhibition of virus replication. However, the viral titre was higher (1.3 log 10 compared to 4.0 log 10 at 10 g/ml concentration of the drug) for the delayed administration compared with the viral titre when the drug was present throughout the period of viral expo- sure. The clinical significance of this in the treatment of established infections has been explored in detail in clini- cal trails.

Administration and Inhibition In Vivo

The earlier work of Palese and Schulman [1977] indicated that the failure in inhibiting viral replication after intranasal and subcutaneous treatment by Neu5a- C2en in mice would produce similar results for Neu5Ac2en analogues. This failure of Neu5Ac2en as an anti-viral treatment in animal models can now be ascribed to the rapid excretion of the compound [Nohle et al., 1982], thereby not delivering sufficient concentration of the inhibitor to the infected tissue. It has been shown [von Itzstein et al., 1993] that when Zanamivir is admin- istrated intranasally in mice it is considerably more ef- fective than when the drug is injected intra-peritoneally. This can be attributed to the localisation of sufficiently high concentrations of inhibitor in the lining of the nasal and respiratory epithelia, where influenza virus replica- tion is believed to occur. Animal trials with ferrets challenged with influenza virus have shown that Zanamivir is effective in studies involving prophylactic administration of the drug [von Itzstein et al., 1993]. When the drug is administrated in- tranasally, 50 g/kg, twice daily, 1 day before infection with the virus and the succeeding 6 days, it substantially reduced virus titre in nasal washings and abolished fever that usually appears 3 days after infection. Results from a double-blind, randomised, placebo- controlled trial using this compound have been successful both for early treatment and prophylaxis of experimental inoculation of 166 adult human volunteers [Hayden et al., 1996] with 10 5 TCID 50 of influenza A/Texas/91 (H1N1). For all dose groups combined, Zanamivir was 82% effective in preventing laboratory evidence of infection, and 95% ef- fective in preventing febrile illness. These results indicate that NA is important for viral replication in humans. In separate randomised, double-blind studies in 38 centres in North America and 32 centres in Europe dur- ing the 1994–1995 influenza season, a total of 417 adults with influenza-like illness of less than 2 days duration were randomly assigned to the following groups: 6.4 mg of Zanamivir by intranasal spray plus 10 mg by inhala-

tion; 10 mg of Zanamivir plus placebo spray; or placebo by both routes [Hayden et al., 1997]. Treatments were self-administrated for 5 days. This study showed that in adult infections of both influenza A or B, Zanamivir di- rectly administered to the respiratory tract was safe and reduced symptoms if begun early. Administration of Zanamivir by both groups reduced from 7 to 4 days the median time to the alleviation of major symptoms when compared with the placebo group, if treatment com- menced within 30 h after onset of symptoms. Similar re- sults were obtained in a double-blind randomised, placebo-controlled study completed in the southern hemisphere winter of 1997, on healthy and “high risk” patients, showing Zanamivir was well tolerated and alle- viated symptoms of influenza 1.5 to 2.5 days earlier than placebo, and “high risk” patients had fewer complica- tions and a reduction in the use of antibiotics with these complications [Silagy et al., 1998]. In a double-blind pla- cebo controlled prophylaxis trail, Zanamivir (10 mg in- haled daily) was shown to be 67% (P < 0.001) effective in

preventing laboratory-confirmed clinical influenza illness [Monto et al., 1998], and when confirmed illness was re- stricted to those with fever, the efficacy rose to 84% (P = 0.001). Efficacy in prevention of total influenza infection was 31% (P = 0.034), indicating subclinical infection, which allows immunity to develop in subsequent infec- tions. Reduced single daily dosage of 6.4mg inhaled Zanamivir has also been shown to be protective against experimental infections in humans [Calfee et al., 1998]. Similar results have been obtained with the orally active prodrug GS4104. The parent drug, GS4071, has low bioavailability like GG167 (2–4%) compared to the high bioavailability of GS4104 [Li et al., 1998]. GS4104 undergoes rapid enzymatic conversion to GS4101 follow- ing gastrointestinal absorption. The oral administration of GS4104 in mice produced dose-dependent protective effects against the neurotropic influenza strain A/NWS/

33 (H1N1), where 1 mg/kg/day afforded 100% protec-

tion [Mendel et al., 1998]. Increasing the dose to 10 mg/ kg/day afforded similar protection against A/Victoria/3/

75 (H3N2) and B/HongKong/5/72. Similar results were

obtained with the ferret model [Oxford et al., 1998]. No significant drug-related toxicity was observed after the administration of oral dosages of GS4104 of up to 800 mg/kg/day for 14 days in nonclinical toxicology studies with rats [Mendel et al., 1998]. In human clinical trials in the US, following oral treatment (75 mg or 150 mg) of GS4104 for 5 days, after febrile respiratory naturally acquired illness of 36 h or less, in a placebo-controlled, double-blind study of healthy non-immunized adults ages 18–65 have shown the efficacy of the drug [Treanor et al., 1998]. The results showed a 30% reduction in both 75 mg (P = 0.0001) and 150 mg (P = 0.001) groups in duration of symptoms, 40%



reduction (both 75 mg, P = 0.0001; and 150 mg, P = 0.001) in severity of symptoms, and a 50% reduction (P = 0.017) of secondary complications. Similar results were obtained for treatment of naturally acquired influenza during the 1997–1998 influenza season in Canada, Eu- rope, and China [Aoki et al., 1998], and no serious ad- verse events were reported. A study of the long-term prophylaxis of natural influenza [Hayden et al., 1998] showed an overall protective efficacy of 74% against in- fluenza illness when 75 mg was taken once or twice daily during the period of local influenza activity. Long-term administration of once daily GS4104 was found to be safe and well tolerated.


Although the active site of influenza virus has been conserved in all known field strains of the virus, the pos- sibility of drug resistance needs to be addressed. NA in- hibitors, if used extensively as anti-influenza drugs, will apply selection pressure on the active site residues for the first time in its evolutionary history. Experience with influenza and other viruses, in particular HIV, have shown [Kimberlin et al., 1995] that drug-resistant mutants arise very rapidly, resulting in the effectiveness of antiviral drugs being short-lived. One attempted solution to the problem is the use of several drugs during therapy [Madren et al., 1995], making it more difficult for the vi- rus to develop resistance. In the case of influenza virus, to date there have been no reports of drug resistance to Zanamivir from field strains [Osterhaus et al., 1998], except in an immunocompromised host [Gubereva et al., 1998]. However, it has recently been reported [McKimm- Breschkin et al., 1994; Gubareva et al., 1995] that 4- guanidino-Neu5Ac2en-resistant mutants can arise from multiple serial passages of virus in MDCK cells in the presence of the inhibitor. It has been demon- strated [McKimm-Breschkin et al., 1996] that almost all of the mutations arise in the HA receptor binding site and not on the NA. These altered HA variants, which have weaker binding to HA receptors, appear to arise as a result of increased inhibition of the NA by the drug. This is consistent with the earlier proposition that the rate of desialylation of receptor is critically related to the rate of attachment to receptor for the virus infection and elution [Varghese et al., 1995]. The decreased activity of the NA by the drug selects HA mutants with decreased binding to receptors.

E119G Neuraminidase Variant

A Zanamivir-resistant NA mutation has been iso- lated [Blick et al., 1995; Gubareva et al., 1995] that re- sults in a single active-site residue mutation, with apparently unaltered activity, of glutamic acid 119 to gly-

cine in Tern N9. The crystal structure of this mutant and its complex (Fig. 7) with 4-guanidino-Neu5Ac2en has been determined [Blick et al., 1995]. The structures sug- gest that the decrease in inhibitor binding arises from the loss of stabilising interaction of the 4-guanidino group of Zanamivir [Varghese et al., 1995] and alterations in the solvent structure of the active site. This alteration arises from a water molecule that binds near the location of one of the carboxylate oxygens of the glutamic acid in the wild-type molecule. The location of the 4-guanidino- Neu5Ac2en drug in the complex with the mutant enzyme is isosteric compared to the drug/wild-type complex [Varghese et al., 1995], the only differences being the in- teractions with residue 119. This variant is, however, inherently thermally un- stable, as measured both by enzyme activity and NC10 monoclonal antibody reactivity [Sahasrabudhe et al., 1998]. Substrate, inhibitor, or monoclonal antibodies stabilised the NA against all methods of denaturation. These results suggests that the instability of the variant is primarily due to the level of polypeptide chain folding rather than the level of association of monomers and tet- ramers. Furthermore, the presence of high level of sub- strate, either cell- or virus-associated, may be sufficient to stabilise the NA during virus replication.

R276K Neuraminidase Variant

Resistance to the carboxamide inhibitor 5 (Fig. 5) has been investigated in type A influenza with the N9 subtype NA, and an active site mutant, R292K, has been isolated [McKimm-Breschkin et al., 1998]. The specific activity of the R292K variant is only 20% of that of wild- type, and virus bearing this mutant NA shows decreased sensitivity to all NA inhibitors [McKimm-Breschkin et al., 1998]. The R292K mutant has also been reported to arise in an avian N2 background after passaging in Zanamivir [Gubareva et al., 1997]. Varghese and co-workers [1998] have shown by X- ray diffraction that the mutation of Arg292 to Lys results in a very local change of structure when compared to the wild-type enzyme. In the wild-type NA Arg292, together with Arg118 and Arg371, engages the 2-carboxylate group of sialic acid in the active site and is partly responsible for distorting the pyranose ring from a chair to a boat conformation [Varghese et al., 1992]. The distal primary guanidinyl amide of Arg292 interacts with Glu277 and the tyrosyl oxygen of Tyr406 (Fig. 2), and the other two guanidinyl nitrogens interact with Asn294. The guani- dinyl group of Arg292 is parallel with the peptide plane of mainchain atoms of residues 348–349. Two water mol- ecules in the active site cavity have H-bond interactions with the primary amides of the Arg292 prior to the entry of substrate into the active site. The R292K variant of influenza NA affects the bind-



190 VARGHESE Fig. 7. A ball-and-stick representation of the complex of Zanamivir in the active site

Fig. 7. A ball-and-stick representation of the complex of Zanamivir in the active site of wild-type N9 neuraminidase and the Glu199 to Gly mutation superimposed on each other. Spheres represent oxygen, nitro- gen, and carbon atoms. A black sphere (Wat) represents a solvent mol- ecule that occupies the position of the carboxylate group of Glu119 when

ing of substrate by modification of the interaction with the substrate carboxylate. This may be one of the struc- tural correlates of the reduced enzyme activity of the variant. Inhibitors which have replacements for the glyc- erol at the 6-position are further affected in the R292K variant because of structural changes in the binding site which apparently raise the energy barrier for the confor- mational change in the enzyme required to accommo- date such inhibitors. It has been shown [Varghese et al., 1998] that there

it mutates to a Gly. Thin lines represent H-bond interactions of the guanidinium group of Zanamivir and the enzyme. The loss of the interac- tions with Glu119 in the mutant is thought to be responsible for the re- duction of binding of Zanamivir to the mutant. The tubes represent the backbone of the protein.

is a correlation between the effect of the Arg292 to Lys substitution on the structure of complexes with inhibi- tors and on the binding properties of the inhibitors. There is also a correlation between the degree of resistance of the variant over wild-type and the degree of structural dissimilarity of the inhibitor to the transition state ana- logue 2. The largest structural consequences occur with the inhibitors whose binding is most severely compro- mised, i.e., 7 and then 6 and 5. While 6 and 5 are able to form the hydrophobic pocket to accommodate the sub-



stitutions in the 6-position of the ring, 7 is unable to achieve this in the Arg292 to Lys mutation (Fig. 8). Smaller structural alterations are associated with inhibi- tors that retain the glycerol sidechain, and these com- pounds are only marginally impaired as inhibitors as a consequence of the Arg292 replacement by lysine. The R292K variant has retained only 20% of wild- type enzyme activity, but its high resistance to 7, 6, and 5 suggests that this mutant will be more successful against

these inhibitors than against those which retain a glyc- erol moiety at C6. Varghese and co-workers [1998] con- cluded that both the structural and binding effects of the Arg292 to Lys mutation are most pronounced on inhibi- tors which least resemble the natural ligand (for example, compound 7). They suggested that inhibitor design, at least for targets that have high mutation frequency, should aim to retain as many features as practicable of the natu- ral ligand. The principle behind such a design strategy is

ral ligand. The principle behind such a design strategy is Fig. 8. A ball-and-stick representation of

Fig. 8. A ball-and-stick representation of the complex of GS4071 in the active site of wild-type N9 neuraminidase and the Arg292 to Lys muta- tion superimposed on each other. Spheres represent oxygen, nitrogen, and carbon atoms, and the tube is a representation of the protein back-

bone. GS4071 fails to induce the formation of a salt-link of Glu276 with Arg224 and binds in a different conformation to that of the wild-type enzyme. The thin lines represent the H-bond interaction of GS4071 with the mutant enzyme.



simple — variants in the natural ligand binding site must retain some binding to that ligand to preserve the pro- tein function.


It has now been 15 years since the structure of in- fluenza 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 deter- mined to sufficient accuracy to permit modelling of po- tential inhibitors [Varghese et al., 1992]. The development was a multidisciplinary collaboration of biochemists, crys- tallographers, molecular modellers, and synthetic chem- ists, 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. Preliminary data on the efficacy of potent neuraminidase inhibitors on humans indicate its effectiveness in both prophylaxis and treatment of the disease [Hayden, 1998]. Zanamivir has completed stage III clinical trials in Aus- tralia, Europe, and North America, and awaits clearance as a pharmaceutical by regulatory authorities for use as a pharmaceutical both for prophylaxis and treatment of all influenza A and B infections. GS4104 is still undergoing clinical trials and offers an alternative oral therapy for influenza virus infections. While drug resistance of the virus has been observed in vitro, the question of whether this translates into resistance in field strains (as has oc- curred in the amantidine class of drugs) infecting human populations will be a cause for concern in the long-term viability of this class of antivirals. It has recently been reported that the Arg292 to Lys mutation in neuramini- dase has emerged in two immunocompetent patients in clinical trials of GS4104 [Hayden, 1998]. The significance of these isolates in terms of infectivity and spread of re- sistant variants is yet to be determined. The structural studies on drug-resistant variants of neuraminidase have suggested a minimalist approach [Varghese et al., 1998] to drug design against shifting targets like influenza vi- rus proteins. The further removed the drug is from its natural ligand the greater the potential of the organism developing resistance. This is one of the first structure-based design com- pounds to be a useful anti-viral drug and portends well for this methodology to be used as an additional weapon in controlling the many pathogens that have plagued humanity for so long.


The author would like to acknowledge Peter Colman for crystallographic and drug design strategies,

Jenny McKimm-Breschkin for drug resistance, Brian Smith for enzyme mechanisms.


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