Inhibitor Development

Relenza

Relenza was developed through the use of the program GRID [32], which revealed a potentially strong binding site for an NH+ group with a calculated binding energy of -16 kcal/mol in the vicinity of the position normally occupied by the O4 hydroxyl of sialic acid [33]. Using Neu5Ac2en as the scaffold, substitution of O4 with an amino group gained two orders of magnitude of binding over Neu5Ac2en, whereas substitution by a guanidino group (4-guanidino-Neu5Ac2en, Relenza) (Fig. 1, structure 3) gained five orders of magnitude of binding over Neu5Ac2en [33]. In complexes of Relenza with both influenza A [34] (Fig. 4b) and influenza B [35] virus NA (PDB codes 1NNC and 1A4G, respectively), the guanidino group interacts almost ideally with Asp151 and Glu227. Glu119 is also close enough to make a charge-charge interaction, although one study suggests that Glu119 may be neutral in the case of 4-amino-Neu5Ac2en binding [36]. Relenza-resistant mutants have been isolated in vitro, with mutations mainly in Glu119 [37-39], and, in one case, one of the catalytic arginines, Arg292 [40]. A resistant mutant has also been isolated in vivo, with the mutation of Arg152 ^ Lys [41]. Relenza is a successful inhibitor of influenza A and B virus NA, but its highly polar nature (calculated log P of -7) has necessitated administration as a powder, requiring an inhaler with all the inherent problems of such use. Replacement of the glycerol group of Relenza by a series of hydrophobic dihydropyrancaboxamides have provided inhibitors with a binding affinity similar to Relenza for influenza A NA, but with only micromolar inhibition of influenza B NA [35].

Tamiflu

The starting point for the development of Tamiflu was replacement of the dihydropyran ring with a cycohexene, which is chemically more stable and retains the ability to

Figure 4 Interactions of the ligands with the active site of the tern N9 influenza virus NA: (a) Neu5Ac2en (PDB code 1F8B); (b) Relenza (PDB code 1NNC); (c) Tamiflu (PDB code 2QWK); and (d) BCX-1812. (e) Stereo view of a superposition of all four ligands reveals a rigid active site with only Glu276 altering position. Neu5Ac2en complex is yellow, Relenza complex is magenta, Tamiflu complex is cyan, and the BCX-1812 complex is green.

Figure 4 Interactions of the ligands with the active site of the tern N9 influenza virus NA: (a) Neu5Ac2en (PDB code 1F8B); (b) Relenza (PDB code 1NNC); (c) Tamiflu (PDB code 2QWK); and (d) BCX-1812. (e) Stereo view of a superposition of all four ligands reveals a rigid active site with only Glu276 altering position. Neu5Ac2en complex is yellow, Relenza complex is magenta, Tamiflu complex is cyan, and the BCX-1812 complex is green.

Figure 4 (Continued)

alter the stereochemistry of ring substituents [42]. The best inhibition was obtained with the double bond in the position equivalent to that in Neu5Ac2en, mimicking the carbonium cation intermediate. The carboxylate and acetamido groups were kept at C: and C4, respectively, and an amino group at C5 in light of the success of the Relenza development. In order to improve the lipophilicity, the glycerol group was substituted by a series of alkyl ethers. There is a remarkable correlation between the length, geometry, and rigidity of the alkyl chains and NA inhibitory activity, suggesting an incremental entropy gain. The crystal structure of the best inhibitor, with a 3-pentyl group (GS4071, later named Tamiflu carboxy-late) (Fig. 1, structure 4a), showed that Glu276 is rotated away from the active site to extend the hydrophobic pocket (Fig. 4c). The prodrug of GS4071, an ethyl ester derivative (GS4104, Oseltamivir, Tamiflu) (Fig. 1, structure 2a; Fig. 4b) exhibits good oral efficacy [43].

Biocryst Compound (BCX-1812)

The starting point for the development of BCX-1812 was a furanose-based compound (Fig. 1, structure 5) that had the same ring substituents as sialic acid and Neu5Ac2en and inhibited influenza virus NA with a potency similar to Neu5Ac2en [44]. The structure of a complex of (Fig. 1, structure 5) with N9 influenza NA (Fig. 4d) showed that, although the fura-nose ring is significantly displaced compared to the pyranose ring of DANA, all of the ring substituents have very similar interactions with the enzyme. This reflects a feature of the active site, namely that there is little interaction with the ring itself. Consequently, a cyclopentane ring was chosen as the scaffold for chemical stability, with a carboxylic acid group placed at C1. An interesting route in the development was the synthesis of racemic mixtures with a guanadino group at C4 and an n-butyl at Cr, followed by inspection of the highresolution difference electron density maps to ascertain the stereochemistry of the active isomer [26]. The «-butyl chain bound in two different modes in influenza A and B virus NA, reflecting the slightly different environments around the sialic acid glycerol binding pocket in the two enzymes. Again, Glu 276 moves, and in influenza A NA forms a salt bridge with Arg224 as had been observed in the binding of GS4071 [35]. In order to take advantage of both hydrophobic pockets, BCX-1812 (Fig. 1, structure 6) was developed, again as a racemic mixture, and the active isomer identified crystallographically. An interesting feature of BCX-1812 (Fig. 4d) is that the orientation of the guanidino group in the active site is different from that seen for Relenza (Fig. 4b). This may be why BCX-1812 remains effective against a Relenza-resistant mutant (Glu119 ^ Gly) [45], as is also true for Tamiflu, which has only an amino group at this position. BCX-1812 shows great promise as an oral treatment for influenza [46-48].

Abbott Compounds

Abbott published a series of inhibitors based on a pyrro-lidine core, the best of which (A-192558) (Fig. 1, structure 7) had an IC50 of 0.28 \xM against influenza A NA [28,49]. One feature of the development of these inhibitors was the creation of focused combinatorial libraries by automated solid-phase synthesis, in one case containing 550 analogs [28]. Recently, a new compound with Ki of between 0.024 and 0.31 nM against a range of influenza virus NAs has been reported [27]. This compound, A-315675 (Fig. 1, structure 8), retains the carboxyl and acetamido groups, but does not have an amino or guanidino group. No details are available as to how this compound binds in the active site, but it is reported that only Glu276 moves in the active site upon binding, as in other complexes of other inhibitors with a hydrophobic moiety in place of the glycerol group [27].

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