The Arg292-->Lys variant of influenza neuraminidase affects the binding of substrate by modification of the interaction with the substrate carboxylate. This may be one of the structural correlates of the reduced enzyme activity of the variant. Inhibitors that have replacements for the glycerol at position 6 are further affected in the Arg292-->Lys variant because of structural changes in the binding site that apparently raise the energy barrier for the conformational change in the enzyme required to accommodate such inhibitors. These results provide evidence that a general strategy for drug design when the target has a high mutation frequency is to design the inhibitor to be as closely related as possible to the natural ligands of the target.

The first paper in this series (see previous article) described structure-activity studies of carboxamide analogues of zanamivir binding to influenza virus sialidase types A and B and showed that inhibitory activity of these compounds was much greater against influenza A enzyme. To understand the large differences in affinities, a number of protein-ligand complexes have been investigated using crystallography and molecular dynamics. The crystallographic studies show that the binding of ligands containing tertiary amide groups is accompanied by the formation of an intramolecular planar salt bridge between two amino acid residues in the active site of the enzyme. It is 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. In sialidase from type A virus, binding of the carboxamide moeity and salt-bridge formation have only a minor effect on the positions of the surrounding residues, whereas in type B enzyme, significant distortion of the protein is observed. The results suggest that the decreased affinity in enzyme from influenza B is directly correlated with the small changes that occur in the amino acid residue interactions accompanying ligand binding. Molecular dynamics calculations have shown that the tendency for salt-bridge formation is greater in influenza A sialidase than influenza B sialidase and that this tendency is a useful descriptor for the prediction of inhibitor potency.

4-Amino- and 4-guanidino-4H-pyran-6-carboxamides 4 and 5 related to zanamivir (GG167) are a new class of inhibitors of influenza virus sialidases. Structure--activity studies reveal that, in general, secondary amides are weak inhibitors of both influenza A and B viral sialidases. However, tertiary amides, which contain one or more small alkyl groups, show much greater inhibitory activity, particularly against the influenza A virus enzyme. The sialidase inhibitory activities of these compounds correlate well with their in vitro antiviral efficacy, and several of the most potent analogues displayed useful antiviral activity in vivo when evaluated in a mouse model of influenza A virus infection. Carboxamides which were highly active sialidase inhibitors in vitro also showed good antiviral activity in the mouse efficacy model of influenza A infection when administered intranasally but displayed modest activity when delivered by the intraperitoneal route.

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A series of 3-aryl(pyrrolidin-1-yl)butanoic acids were synthesized using a diastereoselective route, via a rhodium catalyzed asymmetric 1,4-addition of arylboronic acids in the presence of ( R)-BINAP to a crotonate ester to provide the ( S) absolute configuration for the major product. A variety of aryl substituents including morpholine, pyrazole, triazole, imidazole, and cyclic ether were screened in cell adhesion assays for affinity against αβ, αβ, αβ, αβ, and αβ integrins. Numerous analogs with high affinity and selectivity for the αβ integrin were identified. The analog ( S)-3-(3-(3,5-dimethyl-1 H-pyrazol-1-yl)phenyl)-4-(( R)-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)butanoic acid hydrochloride salt was found to have very high affinity for αβ integrin in a radioligand binding assay (p K = 11), a long dissociation half-life (7 h), very high solubility in saline at pH 7 (>71 mg/mL), and pharmacokinetic properties commensurate with inhaled dosing by nebulization. It was selected for further clinical investigation as a potential therapeutic agent for the treatment of idiopathic pulmonary fibrosis.