Structure-based characterization and optimization of novel hydrophobic binding interactions in a series of pyrrolidine influenza neuraminidase inhibitors.

The structure-activity relationship (SAR) of a novel hydrophobic binding interaction within a subsite of the influenza neuraminidase (NA) active site was characterized and optimized for a series of trisubstituted pyrrolidine inhibitors modified at the 4-position. Previously, potent inhibitors have targeted this subsite with hydrophilic substituents such as amines and guanidines. Inhibitor-bound crystal structures revealed that hydrophobic substituents with sp(2) hybridization could achieve optimal interactions by virtue of a low-energy binding conformation and favorable pi-stacking interactions with the residue Glu119. From a lead methyl ester, investigation of five-membered heteroaromatic substituents at C-4 produced a 3-pyrazolyl analogue that improved activity by making a targeted hydrogen bond with Trp178. The SAR of substituted vinyl substituents at C-4 produced a Z-propenyl analogue with improved activity over the lead methyl ester. The C-1 ethyl ester prodrugs of the substituted C-4 vinyl analogues gave compounds with excellent oral bioavailability (F > 60%) when dosed in rat.

Enteric Bacterial and Viral Pathogens and Indicator Bacteria in Hard Shell Clams.

Enteric bacteria and virus levels were determined in hard shell clams, Mercenaria mercenaria , harvested from areas open or closed for commercial shellfishing on the basis of total coliform levels in water. Four pairs of open and closed stations were sampled seasonally over a 1-year period. Enteric viruses were isolated from 3 of 13 100-g clam samples from open beds and 6 of 15 samples from closed beds. Salmonella was found in 1 of 15 samples from closed areas, but not in any samples from open areas. No Shigella or Yersinia were isolated from clams taken from either open or closed beds. Levels of Vibrio parahaemolyticus , an indigenous estuarine microorganism, were similar in clams from open and closed areas. No statistically significant difference was found in the occurrence of enteric viruses in clams from open and closed areas. Product-moment correlations between concentrations of enteric viruses and bacteria in clams or water demonstrated no statistically significant correlations between virus concentrations in clams and total coliforms or fecal coliforms in water or total coliforms, fecal coliforms, fecal streptococci or aerobic plate counts in clams.

Occurrence of Enteric Bacteria and Viruses in Oysters.

Enteric bacteria and virus levels were determined in oysters from paired stations that were opened or closed for commercial shellfishing on the basis of total coliform levels in the water. Six pairs of stations were sampled quarterly over a 1-year period. Enteric viruses were found in 3 of 24 50-g oyster samples from closed areas and in none of 23 samples from open areas. Salmonella was found in 2 of 47 samples of 40 g each, one from an open and the other from a closed area. Although enteric pathogens of fecal origin were found only in oysters that exceeded the recommended market limit of 230 fecal coliforms per 100 g of meat, the fecal coliform levels in some virus-positive samples were much lower than those in Salmonella -positive samples. Vibrio parahemolyticus levels were similar in oysters from both open and closed beds, indicating no particular association with fecal pollution. However, there was a marked seasonal variation in V. parahemolyticus levels. Total but not fecal coliform levels in oysters from open beds correlated with the occurrence of rainfall 1 or 2 days before sample collection. Neither total nor fecal coliform levels in oysters from closed beds correlated with rainfall. These findings suggest that fecal coliforms levels in oysters are less influenced by rainfall than are total coliforms, and therefore may be a more specific indicator of recent fecal pollution.