Towards a high throughput screening of inhibitors for Streptococcus pneumoniae

The H-N-H motif characterizes the active site of a large number of different nucleases such as homing endonucleases, restriction endonucleases, structure specific and non-specific nucleases. Several biochemical studies revealed an essential catalytic function for the first amino acid of this motif. This Histidine residue was identified as the general base which activates a water molecule for a nucleophilic attack on the sugar phosphate backbone of nucleic acids. Replacing His by an inert amino acid missing the catalytically active side chain leads to a dramatic decrease in the nucleolytic activity for members of this family of nucleases. We could restore the activity of inactive H-N-H nucleases, viz. EndA (Streptococcus pneumoniae), SmaNuc (Serratia marcescens) and NucA (Anabaena sp.) by adding an excess of imidazole to the enzymes. Imidazole obviously replaces the missing side chain and can restore nucleolytic activity. Significantly, the chemical rescue effect could also be detected in vivo (E. coli). The in vivo assay might be a promising starting point to develop a simple HTS system for functional EndA inhibitors since toxic EndA wt cannot be expressed in E. coli. Isolated inhibitors should block the nucleolytic activity of the surface exposed nuclease of S. pneumoniae which is involved in destroying the innate immune response NETs (Neutrophil extracellular traps). They are composed of neutrophilic DNA which captures pathogens and stimulates their destruction.