NON-SIALATE INHIBITOR OF INFLUENZA A WSN/33 NEURAMINIDASE/

An N-1 strain of influenza A virus neuraminidase (A/WSN/33 NA) was pur ified and used to screen for inhibitors. As a result, a well-known tub erculostatic, 4'-formylacetanilide thiosemicarbazone (or thiacetazone) , was identified. Thiacetazone is a non-sialate compound and inhibits the enzyme in a noncompetitive manner with respect to the substrate si alic acid. Mechanistic studies indicate that the inhibition was due to the competition of thiacetazone with Ca2+, which maintains N-1 neuram inidase in an active conformation. The K-i for the inhibition was esti mated to be about 4 mu M. Equilibrium exchange experiments revealed th at when purified A/WSN/33 NA was incubated with 5 mu M (CaCl2)-Ca-45, 2 mol of Ca-45(2+) ion was exchanged into each mole of NA tetramer and subsequently displaced from the enzyme upon the introduction of the i nhibitor. Inhibition of plaque formation by thiacetazone in an MDCK ce ll culture that had been infected with the influenza A/WSN/33 virus wa s demonstrated. Thiacetazone was highly specific for A/WSN/33 neuramin idase, since little effect was noted when it was tested against NAs fr om the other strains of influenza virus or from bacteria. This compoun d might represent a group of non-sialate inhibitors of influenza NA th at bind to a noncatalytic or an allosteric site on the enzyme.