Crystallographic studies of sialidase and structure-based design of inhibitors

Presented is the X-ray crystallographic structural analysis of influenza virus sialidase (neuraminidase, EC 3.2.1.18) when complexed to several inhibitors and the use of structure-based methods to develop specific bacterial sialidase inhibitors. The crystal structure of influenza virus type B sialidase (B/Mem/89 and B/Lee/40) was solved using molecular replacement phasing methods. From the complexes, the eleven strictly conserved active site residues that are critical for binding and stabilization of the substrate were identified. Crystallographic studies were also performed on the complex of influenza virus type A (N2 and N9 subtypes) and type B sialidases complexed to a sialic acid derived phosphonate analog, (4-acetamido-2,4-dideoxy-D-glycero-D-galacto-1-octopyranosyl) phosphonic acid or PANA, by difference Fourier techniques. Compared to sialic acid, PANA has a phosphonyl group substituted for the carboxylate group and does not possess a C2 hydroxyl. The crystal structures of the complexes between the equatorial phosphonate enantiomer, ePANA, and the type A and type B influenza virus sialidases indicated that the ePANA compound was bound to all three viral sialidases in a chair-iike conformation. By adopting a chair-like conformation, ePANA can mimic the inhibitor-protein interactions observed in the sialic acid and DANA complexes. The structural studies also indicated that the lower inhibition activity of ePANA toward the type A N9 sialidase is not due to differences in the relative binding mode of ePANA. The crystal structure of the complex between the type A N2 influenza sialidase and the axial phosphonate enantiomer, aPANA, was also solved. In this complex, the axial enantiomer must bind in a boat-like conformation to retain the inhibitor-protein interactions observed in the sialic acid and DANA complexes. Finally, the knowledge derived from analyzing the influenza virus sialidase-inhibitor complexes was applied to the structure-based design of novel bacterial sialidase inhibitors. The coordinates of the Salmonella typhimurium-DANA inhibitor complex and two benzoic acid based compounds and the program GRID were used to construct three classes of bacterial specific sialidase inhibitors. The program DELPHI was used to analyze the effect of the inhibitor modifications upon the calculated free energy change of complex formation.