Structure-Based Inhibitors of Influenza Virus Sialidase. A Benzoic Acid Lead with Novel Interaction

Abstract

Influenza virus sialidase is a surface enzyme that is essential for infection of the virus. The catalytic site is highly conserved among all known influenza variants, suggesting that this protein is a suitable target for drug intervention. The most potent known inhibitors are analogs of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en), particularly the 4-guanidino derivative (4-guanidino-Neu5Ac2en). We utilized the benzene ring of 4-(N-acetylamino)benzoic acids as a cyclic template to substitute for the dihydropyran ring of Neu5Ac2en. In this study several 3-(N-acylamino) derivatives were prepared as potential replacements for the glycerol side chain of Neu5Ac2en, and some were found to interact with the same binding subsite of sialidase. Of greater significance was the observation that the 3-guanidinobenzoic acid derivative (equivalent to the 4-guanidino grouping of 4-guanidino-Neu5Ac2en), the most potent benzoic acid inhibitor of influenza sialidase thus far identified (IC50 = 10 microM), occupied the glycerol-binding subsite on sialidase as opposed to the guanidino-binding subsite. This benzoic acid derivative thus provides a new compound that interacts in a novel manner with the catalytic site of influenza sialidase.

Characterization of Sialidase from an Influenza A (H3N2) Virus Strain: Kinetic Parameters and Substrate Specificity

“…This result is in accordance with data relating a 5-10% correlation between NA 'head' content and complete virion structure. This value does not take in account spike stems nor eventual losses during the purification process [5,42]; if the pronase-treated virus sample is redigested with this enzyme, more sialidase is released. In fact, the amount of NA liberated for this second pronase digestion is equivalent to that obtained with the first treatment (data not shown).…”

“…To obtain NA crystals, the utilization of chromatographic methodologies based on charge properties of the sialidase molecule [5,42] has provided preparations ex-pressing one single band; however, they exhibit loss of that enzyme activity which is considered as essential for our purposes [13,36,47].…”

Protein Flexibility in In Silico Screening

“…In an application to hyaluronate lyase [72], whose size precludes the use of MD to investigate biologically relevant timescales, flexibility (allosteric) information and functional implications were derived by CONCOORD. Furthermore, Mustard and Ritchie [73] showed that docking to multiple structures, which were obtained by an essential dynamics study following a CON-COORD run, generates better docking predictions than docking only to unbound or modeled structures.…”

The Glycobiology of Influenza Viruses

“…The planar intermediate has been exploited in the development of inhibitors of NA and of virus growth; several inhibitors based on planar molecules have been shown to inhibit in the micromolar range [41], for review see [42]. Detection of Neu5Ac2en as a product of cleavage by NA and other sialidases [43, 441 may indicate an additional mechanism, akin to the dehydration mechanism of the hyaluronic acid and proteoglycan lyases, which generates a planar reaction product with a double bond in the sugar ring.…”