Inhibitors of the human neuraminidase enzymes

The human neuraminidase enzymes (hNEU) are a family of four isoenzymes that hydrolyze sialosides, including gangliosides and glycoproteins. These enzymes are proposed to play roles in several important signaling pathways and are implicated in diseases such as diabetes and cancer. Despite their importance, only a limited number of studies have sought to identify potent inhibitors for these enzymes. This review summarizes the substrate speci ﬁ city and known inhibitors of the hNEU isoenzymes, as well as the emerging work on development of isoenzyme-speci ﬁ c inhibitors.


Introduction
The human neuraminidase enzymes (hNEU) are a family of glycosyl hydrolases (GH) that play critical roles in human health and disease.There are currently four known isoenzymes: NEU1, NEU2, NEU3, and NEU4.][3][4] NEU1 is part of a membrane complex, and is found primarily in the lysosome and plasma membrane. 5,6Murine NEU1 has four sites of N-link glycosylation, several of which are critical for enzyme function. 7NEU2 has been found to be localized to the cytosol, [8][9][10] while NEU3 and NEU4 are thought to be membraneassociated. 11,12The NEU4 isoenzyme is found in endoplasmic reticulum, 13,14 mitochondrial, 13,15 and lysosomal 4 membranes and occurs as both a long and truncated form, which affect its subcellular localization. 13,15he hNEU have been implicated in regulation of a variety of human pathologies.The identication of such a large number of hNEU isoenzymes may suggest that cells exploit these enzymes to remodel existing epitopes to rapidly generate modied glycan structures. 16,17Such a mechanism would avoid the time and energy required to biosynthesize a new glycan structure and may help explain the more rapid turnover of terminal carbohydrate residues in glycoproteins. 18,19Plasmamembrane associated forms of the enzyme can act on substrates found on other cells. 20The expression levels of hNEU isoenzymes are linked to malignancy. 21,22For example, NEU4 is overexpressed in neuroblastoma, 23 and NEU1 expression has been linked to metastasis through regulation of integrin adhesion. 24hNEU have been linked to regulation of lipid metabolism, which may be relevant in cardiovascular disease. 25NEU1 has recently been linked to signaling by the insulin receptor in a model of diabetes. 26][29][30][31][32] The family of hNEU are clearly important for a variety of biological mechanisms; however, few small molecule inhibitors of these enzymes have been reported.In contrast, a large body of research has been devoted to development of inhibitors for the viral neuraminidase enzymes (vNEU) as anti-inuenza therapeutics. 33Importantly, compounds which target vNEU have not been found to be as active in the human enzymes. 34hus, there remains a need for the development of compounds which target hNEU specically.When considering the design of hNEU inhibitors, one should remember that certain functions of hNEU are essential for homeostasis.Genetic deciency of the NEU1 isoenzyme results in sialidosis; 35,36 thus, potential therapeutics must show specicity to avoid off-target or non-specic inhibition of undesired isoenzymes.The high homology within the enzyme family presents a challenging problem for the medicinal chemist, not only must potential therapeutics be active against the desired isoenzymebut they must be highly selective.This review will summarize the current state of known small molecule inhibitors for the hNEU, as well as relevant molecular details regarding active-site structure and substrate specicity.Particular attention will be devoted to features and activities which are distinct among the isoenzymes.

Structure of hNEU
The hNEU are classied as family 33 glycosyl hydrolases (GH33) and cleave the terminal glycosidic linkage of sialic acid (exosialidases; EC 3.2.1.18). 37Alignment of the four human isoenzyme sequences shows the greatest similarity between NEU2 and NEU3.The NEU2 isoenzyme is the only hNEU studied by Xray crystallography.Chavas et al. have reported co-crystals of the enzyme with 2-deoxy-2,3-didehydro-N-acetyl-neuraminic acid (DANA; 15), 38 as well as viral neuraminidase enzyme (vNEU) inhibitors. 39The enzyme shows a 6-bladed b-propellar fold common to GH33 members which is conserved in many species. 40Unfortunately, there are currently limited structural data for the remaining hNEU members.This is likely due to the nature of the NEU1, NEU3, and NEU4 isoenzymeseach of these are either a component of a membrane complex or membrane-associated.The enzymes may contain post-translational modications including phosphorylation 4 and glycosylation 7 that may be important for activity.
In the absence of experimental protein structures, indirect methods of examining hNEU structure have been proposed.Magesh et al. rst introduced homology models of the three remaining isoenzymes based on the reported structure of NEU2. 41Although NEU3 has been proposed to contain a transmembrane domain, 2 further study has suggested that it is more likely a peripheral membrane-associated protein. 12,42A homology model of NEU3 has also been reported and validated using site-directed mutagenesis. 43A homology model of NEU4 was proposed to rationalize the binding of NEU4-selective inhibitors. 44The binding epitope of NEU3 has been examined using a trisaccharide analog of GM3. 45 Some insight into the structure of hNEU can be gained from examining mutations of the enzymes.Naturally occurring mutations on NEU1 have been characterized in relation to sialidosis diseases. 46Mutagenesis of NEU1 has also revealed that N-link glycosylation of the protein is essential for its activity. 7he NEU2 enzyme has been studied by mutagenesis, conrming the catalytic pair as Y334 and E218. 47,48Site-directed mutagenesis of NEU3 was used to identify key active site residues, including the catalytic pair (Y370 and E225) and the arginine triad (R25, R245, R340). 42,43Deletion of a proline-rich segment of NEU4 has been found to alter protein-protein interactions and to alter substrate specicity. 49here are several key structural features of the active site of neuraminidase enzymes necessary for recognition of inhibitors across species. 33,40,48The C1 carboxylate of sialosides is recognized by a triad of arginine residues (Fig. 1).The catalytic residues of GH33 are a Tyr-Glu pair, with the Glu residue acting as a general acid-base and the Tyr-OH acting as a nucleophile. 43,47,50inally an Asp residue is required as a general acid-base within the active site. 48The vNEU contain a exible recognition site for the glycerol sidechain of the sialoside, and this has been exploited in the design of inhibitors. 51The recognition of the glycerol sidechain in hNEU appears to be distinct and relies on several contacts to Tyr residues. 38,39

Substrate recognition in hNEU
The hNEU are exo-sialidases which cleave a-linked N-acetyl-neuraminic acid (Neu5Ac); among these, NEU2 and NEU3 have been conrmed as retaining GH. 43,52 Neu5Ac linkages in glycoproteins are typically a(2,6), though a(2,3) are possible, while a(2,3), a(2,6), and a(2,8) linkages are present in glycolipids (Fig. 2; 1-3). 53All four enzymes have acidic pH optima, typically found to be between pH 3.5 and 5.5. 4,42,54Reports differ on the activity of NEU2 in the presence of metal ions, although high concentrations of EDTA appear to reduce enzyme activity. 52,55The presence of Cu 2+ ions is known to be inhibitory for NEU2, 10,52,55 and NEU3; 43 as a result, caution should be used when testing inhibitors that could contain this contaminant.Detection of neuraminidase activity oen exploits the uorogenic substrate 2 0 -(4methylumbelliferyl)-a-D-N-acetylneuraminic acid (4MU-NANA; 4). 56,57Alternative methods have been developed that detect the release of free sialic acid, 58,59 or that use a coupled assay to detect the newly exposed terminal sugar aer neuraminidase action. 60here are few detailed studies of hNEU substrate specicity, and additional work is needed to develop the full picture of differences in substrate activity among the isoenzymes.Studies of enzyme specicity with synthetic analogs of sialosides are extremely valuable for the design of inhibitors or enzyme probes, 61 and known substrate activities of each hNEU isoenzyme are briey summarized below.
NEU1 targets glycoprotein substrates and has been shown to prefer a(2,3) over a(2,6) linkages (i.e. 1 vs. 2). 62The enzyme has reduced activity for ganglioside substrates. 4NEU1 has been shown to target cell surface components such as the toll-like receptor, 62 insulin receptor, 26 and others. 30ynthetic substrates with variation in the Neu5Ac residue and its glycosidic linkage have been tested with NEU2. 60Similar Fig. 1 Binding site interactions of the hNEU enzymes NEU2 and NEU3.Schematics of the key interactions for DANA in the active site of NEU2 (left). 38,47The interactions proposed for the NEU3 active site are based on mutagenesis and homology modeling studies (right). 43Residues that form the arginine triad in both enzymes are colored in blue.The proposed Tyr nucleophile is shown in magenta for each binding site.The active site nucleophiles act as a catalytic pair with E218 (NEU2) and E225 (NEU3), not shown in this figure .to NEU1, NEU2 shows a strong preference for a(2,3) linkages.Testing with substrates containing N-glycolyl-neuraminic acid (Neu5Gc) residues (6) found that this substrate was preferred over the native Neu5Ac analog (5).Further modication of the Neu5Gc residue at the C9 position to an azido group also resulted in a substrate with improved activity (7). 60Modication at the C9 of Neu5Gc-or Neu5Ac-a(2,3) to O-methyl, -uoro, or -deoxy analogs greatly reduced activity, suggesting that this position is critical for recognition.The NEU2 active site has been proposed to recognize portions of the glycan substrate distal to the Neu5Ac glycosidic linkage.Site directed mutagenesis and molecular modeling have implicated D358 and Y359 in the recognition of 6 0 -sialyllactose analogs, 63 and Q112 and Q270 may interact with the Gal or Glc residues of sialyllactose substrates. 47nactivating substrates of hNEU have been tested against the NEU2 isoenzyme.Kai et al. tested a 2-diuoromethylphenyl sialoside (12) against NEU2. 64They conrmed that the compound could inactivate the enzyme in vitro and used an azide tag to generate a library of potential inhibitors.The most active compound identied had a reported inhibition constant of 200 mM.Quinone methide based inactivators may have long lifetimes, and the rate of formation and consumption of the reactive intermediate may inuence the specicity of these reagents. 65Another class of inactivators, 2-deoxy-2,3-diuoroderviatives of Neu5Ac (13), have been tested against NEU2 but had weaker activity against the enzyme when compared to viral and bacterial enzymes. 66Crystallographic data on the inactivated complex between 2,3-diuoro sialic acid analogs and NEU2 has recently been reported. 67luorogenic or inactivating substrates for hNEU are potentially useful for sensitive detection and imaging of enzyme activity. 61Zamora et al. have tested a series of 4MU-NANA (4) derivatives against hNEU from cell lysates. 68Among the N5 0 -modied derivatives of 4, uoromethyl and glycolyl derivatives showed improved activity.Notably, the activity of hNEU against the modied substrates was distinct from bacterial enzymes, raising the possibility of species-or isoenzyme-specic forms of 4MU-NANA.A uorogenic benzothiazolylphenol-sialoside has been used to detect sialidase activity in mammalian tissues. 69nactivating 2-deoxy-2,3-diuoro derivatives of Neu5Ac have been used as imaging probes for hNEU.Tsai et al. synthesized compound 14 (used as its per-acetylated form in cells) for labeling of NEU with an alkyne tag that could be easily visualized using Cu-catalyzed azide-alkyne cycloaddition (CuAAC). 70irect labeling of all four hNEU isoenzymes and viral enzymes was observed by blotting.Fluorescent labeling of cells with these probes was also performed to visualize enzyme activity.
The NEU3 isoenzyme has been found to generally prefer glycolipid over glycoprotein substrates. 3,4,71The substrate spec-icity of NEU3 has been analyzed for gangliosides, demonstrating that the enzyme possesses both a(2,3) and a(2,8) sialidase activity. 3,72,73Additionally, the enzyme does not cleave a(2,3) linkages at internal sites, for example both GM1 and GM2 (which contain a single branched a(2,3)-Neu5Ac-[GlcNAcb(1,4)]-Gal residue) are poor substrates. 3,42,71This observation has been rationalized using the homology model of NEU3. 43urthermore, neoglycoproteins containing GD1a oligosaccharides are substrates of NEU3. 74he substrate activity of synthetic analogs of GM3 with NEU3 have been analyzed using a mass spectrometry-based kinetic assay. 75These studies revealed that the isoenzyme requires a hydrophobic aglycone; however, there is no requirement for diacyl or sphingosine-based lipids.Thus, the native GM3 substrate (8) can be simplied to an octyl-glycoside (9)  providing identical substrate activity.The N5-azidoacetate (10) showed slightly improved activity over the native trisaccharide (9).NEU3 did not tolerate larger modications at the N-acyl sidechain of Neu5Ac and showed minimal activity with triazole derivatives of 10.Interestingly, the C9-azido analog (11) retained signicant substrate activity even aer modication to a triazole.These ndings suggest that modication of substrates at Neu5Ac may be better tolerated at C9 over N5.
NEU4 is able to hydrolyze both glycoprotein and glycolipid substrates. 4,76The short isoform of NEU4 has been shown to cleave the a(2,3) linkage of sialyl-Lewis a and sialyl-Lewis x antigens, which are critical for leukocyte adhesion mediated by Eselectin. 77,78NEU4 has also been shown to selectively cleave poly-sialic acids, containing a(2,8) linkages, in neuronal cells. 79

Small molecule inhibitors of hNEU
Potent and selective small molecule inhibitors of hNEU will be valuable tools for research into the function of specic isoenzymes.Signicant progress has been made in the design of inhibitors for glycosyl hydrolases. 80Neuraminidase inhibitors which target the vNEU enzymes have been extremely successful, 33 but known vNEU inhibitors appear to have very weak activity against hNEU.Additionally, the hNEU system is complicated by the number of isoenzymes in the family which may impede the design of selective inhibitors.Studies of hNEU inhibitors are summarized below, paying particular attention to examples that provide isoenzyme selectivity.

Non-carbohydrate inhibitors
Relatively few non-carbohydrate derived inhibitors have been tested against hNEU, and most of these have shown limited inhibitory activity.The cyclohexene-based oseltamivir and cyclopentane-derived peramivir are both potent vNEU inhibitors but show limited activity against hNEU. 34,39Derivatives of 2-uorobenzoic acid were designed to inhibit hNEU; the most active of these had a K i of $300 mM. 81A furan-2,5-dicarboxylic acid has been tested but was found to have weak inhibitory activity against NEU3 and NEU4. 82

Neu5Ac-and DANA-based inhibitors of individual isoenzymes
The most commonly used pan-selective neuraminidase inhibitor is the 2-deoxy-2,3-didehydro-N-acetyl-neuraminic acid (DANA; 15) (Fig. 3).It is interesting to note that DANA can be synthesized by the NEU enzymes when Neu5Ac is present in high enough concentrations.The formation of DANA is likely the result of dehydration by Neu5Ac when bound in the active site. 83The hNEU enzymes are also capable of catalyzing this dehydration reaction. 75,84arly work with puried hNEU isoenzymes conrmed that DANA (15) and C9-modied derivatives were active inhibitors. 85his was the rst study to identify the low mM activity of these compounds; however, neither 15 nor 17 was found to have selectivity for isoenzymes of hNEU.Thioglycosides have been investigated as potential inhibitors of NEU2. 52,63The NEU2 enzyme shows a strong preference for a(2,3)-linked substrates, 55,60 and a thioglycoside(methyl-2-deoxy-2-thio-Neu5Ac-a(2,3)-b-galactoside) exhibited mild inhibitory activity. 52everal reports have tested DANA-analogs for inhibition of the NEU2 isoenzyme.Li et al. found an IC 50 of 18 AE 1 mM for 15 with a recombinant form of the enzyme. 55DANA itself was more potent as an inhibitor of a(2,3)-Neu5Ac substrates over a (2,6), and we will focus on results against this substrate.Modication of DANA at the N5 acyl group resulted in improved potency for compound 18 (IC 50 of 8.3 AE 1 mM).The viral inhibitor, zanamivir ( 16), was also tested against NEU2 and was one of the most potent inhibitors in the study (IC 50 of 5.3 AE 0.7 mM.) Khedri et al. explored modications of DANA at C9 in combination with a modied N-acyl group. 86The N5-glycolyl-C9-azido analog ( 19) had relatively low potency.However, the N5-azidoacetate-C9-azido analog (20) showed improved activity as an inhibitor of NEU2 (IC 50 of 13 AE 4 mM) over DANA (IC 50 of 32 AE 6 mM).Other modications at C9, including O-methyl and alkylamine derivatives showed reduced potency.DANA derivatives with the glycerol side chain replaced by a C6-ether showed moderate potency against NEU2, though these were all lower activity than the parent, 15. 39 Testing of a small library of inhibitors against NEU3 was reported by Zou et al. 87 Compounds included azide derivatives of DANA, which could be easily converted to triazole analogs. 88odications at both the N5 and C9 positions were used to explore the structure activity relationships of these compounds.In common with reports for NEU2, this study found that compound 18 had improved potency (IC 50 of 21 AE 8 mM) relative to DANA (15; IC 50 of 48 AE 5 mM). 87Modication at C9 to an azide, as in compound 17, resulted in decreased activity.However, conversion to a C9-triazole maintained activity when an alkyl group was included (21; IC 50 of 20 AE 4 mM).Similar triazole modications of 18 universally reduced potency, leading the authors to conclude that the active site of the enzyme could tolerate large modications at C9 better than at N5. Similar observations were found in substrate studies of NEU3. 75

Selectivity of inhibitors among the hNEU family
Recent studies have begun to realize isoenzyme-selective inhibitors of hNEU.It should be noted that even the well-known inhibitor, DANA (15), is typically found to have a range of activity among the hNEU isoenzymes that varies between 2 to 10-fold (comparing the most active to least active inhibitors). 34,54,70,89However, studies that have tested DANA against all hNEU typically nd it to be most active against the NEU3 or NEU4 isoenzymes. 54,70esting of C9-amido derivatives of DANA has identied NEU1-selective inhibitors.Magesh et al. synthesized a library of compounds and tested these against lysates from hNEU transiently transfected cell lines. 89The design of the compounds tested was based on homology models of the four isoenzymes. 41everal compounds were selective inhibitors of NEU1, with the most potent compounds containing small alkyl or aryl groups (22; NEU1 IC 50 13 mM).Notably, these compounds were signicantly more active than the parent DANA (15) in the same assays.The NEU1-active inhibitors showed very little potency towards the other isoenzymes tested.Compound 22 showed minor activity for NEU3, with approximately 25-fold selectivity for NEU1 based on the reported IC 50 data.These ndings suggested the possibility that the binding pocket for the glycerol sidechain of Neu5Ac (C7-C9) may show variability among the isoenzymes that could be exploited for the design of selective inhibitors.
A recent study examined the activity of a library of DANA derivatives containing C4 and C7 modications which allowed the identication of NEU2 and NEU3 selective inhibitors. 54This strategy was able to identify a 12-fold selective inhibitor for NEU2, 23 (IC 50 86 AE 17 mM).Combining C4-azido and C7-benzyloxyamine modications to the DANA scaffold generated a nearly 40-fold selective inhibitor for NEU3 (24; IC 50 24 AE 2 mM, K i 8 AE 1 mM). 54Albohy et al. identied oseltamivir analogs that were moderately active against NEU3 with partial selectivity for NEU3 over NEU4. 90Tsai et al. tested the activity of compound 14 against puried recombinant hNEU isoenzymes. 70They reported the compound was most active against NEU3 (IC 50 5 AE 3 mM), although the selectivity was approximately 2-fold (based on the second most potent IC 50 among hNEU).
The most potent and selective inhibitors for NEU4 were recently identied through only minor modications of DANA. 44Albohy et al. tested a set of compounds against all four hNEU which had previously shown only moderate activity in assays of NEU3 alone. 44However, in testing against a panel of puried and recombinant hNEU, several of these compounds were found to have nanomolar activity against the NEU4 isoenzyme.The most potent compounds were C9-triazole derivatives of DANA containing an alkoxy group (25; K i 30 AE 19 nM).Importantly, these compounds are the most potent inhibitors of any hNEU isoform reported to date and also exhibit unprecedented selectivity, with approximately 500-fold selectivity for NEU4 over all other isoenzymes.The activity of the compound was tested in assays using GM3 as a substrate to conrm that the activity was not an artifact of the 4MU-NANAbased assays used in screening.

Activity of NEU inhibitors across species
The hNEU enzymes share a great deal of homology with NEU of other species.Additionally, NEU enzymes of bacteria and viruses are important virulence factors and targets for the development of therapeutics. 33,91The active site topology and interactions between the enzyme and 15 are generally similar to the bacterial and viral enzyme.However, some notable differences are found in the interactions at the glycerol sidechain, and a dynamic loop containing D46 (NEU2). 38,39esting of clinically active vNEU inhibitors has shown that these compounds have weak to moderate potency for the hNEU.Hata et al. tested a panel including DANA (15), zanamivir (16), and oseltamivir with puried isoenzymes. 34Although zanamivir and oseltamivir are reported to have exceptional potency against vNEU, 92 against hNEU only zanamivir shows low micromolar activity.This nding may be benecial for the use of antivirals, as these compounds should have limited effects on native hNEU.Mutations of NEU2 have been found to alter its susceptibility to oseltamivir, which may be a concern for side effects in some individuals. 93,94In the case of oseltamivir, the glycerol sidechain is replaced by a 3-pentyl group.This feature of the inhibitor benets activity against some viral enzymes due to a rearrangement in the active site. 51Other features of the viral active sites, such as the 150-loop, 95 have also been used to target vNEU. 96,97However, compounds designed to exploit features such as the 150-loop of vNEU are not potent inhibitors of hNEU. 90,96he key structural differences between the active sites of vNEU and hNEU are the binding pockets that complex the glycerol side chain (C7-C9 pocket) and the C4 substituent (Fig. 1).The C7-C9 binding pocket of NEU2 features two Tyr residues (Y179, Y181) that complex O7 and O9. 38This is distinct from the Glu residue that forms H-bond contacts with this portion of DANA in vNEU. 98Additionally, computational studies have suggested that the O4-binding pocket residues are a key factor in the decreased activity for C4-amino/guanidino inhibitors for hNEU. 99everal examples conrm that hNEU have distinct requirements for inhibition relative to bacterial NEU enzymes.In a library of compounds with modied C7-substituents Zhang et al. found very little activity for the V. cholerae NEU, but many of these compounds were active against hNEU. 54Compound 20, while active against NEU2, was found to have much lower activity against the V. cholerae and C. perfringens neuraminidase enzymes.Interestingly, the C9-azido-N5-glycolyl analog of DANA (19) had the reverse selectivity, showing approximately 10-fold selectivity for V. cholerae neuraminidase over C. perfringens and NEU2. 86Diuoro inhibitors which target vNEU were found to have signicantly lower potency against NEU2. 66

Conclusions and future prospects
The hNEU enzyme family has begun to gain the attention of medicinal chemists as a potential target for the development of tools for glycobiology and as a source for therapeutics.A large body of work has focused on the study of hNEU enzyme function using genetic tools; however, the availability of small molecule inhibitors with known specicity for hNEU family members will provide for new approaches in this complex system.Consider that a family of enzymes with similar mechanisms may compensate for the deletion of a single isoenzyme.However, isoenzyme-selective inhibitors of sufficient potency could be used to dissect the contributions of individual enzymes.Signicant progress has been made on the identication of inhibitors, yet there remains a distinct need for compounds that can target each of the hNEU isoenzymes with greater specicity and potency.The most useful compounds will have activity both in vitro and in vivo, but currently few in vivo studies for the inhibitors discussed here have been reported.Additionally, the most potent compounds currently available are based on the DANA scaffold, and organic scaffolds that are more synthetically accessible or which are conformationally restricted could be an important asset to the eld. 100