Discovery of new antibiotics is limited both by targets available for screening and by the effectiveness of current screening paradigms. The goal of the proposed Phase II work is to complete development of a novel discovery technology, referred to as the TargetArray, which allows all bacterial essential gene targets to be screened simultaneously, in mixed-culture format, with an array of highly sensitive whole-cell assays. The foundation for this technology is a proprietary collection of Staphylococcus aureus strains constructed at Elitra Pharmaceuticals, each engineered to under-express or over-express individual essential gene products, in its current proof-of-concept form, this collection consists of 169 strains that down-regulate target gene products as the result of inducible expression of target-specific antisense (AS) RNAs. These AS expressing clones address approximately 80% of the broadly conserved essential gene targets known by Elitra to exist in S. aureus. To comprehensively address all essential genes of S. aureus, and to enhance the resolution of the TargetArray as a tool for identifying target-specific mechanisms of growth inhibition, we propose to supplement the AS-based collection with promoter-replacement (PR) constructions that achieve either under-expression or over-expression of specified target genes. These constructions will be engineered into S. aureus using a novel gene-manipulation method developed under Phase I funding for this grant. This method allows efficient integration of regulatable promoter cassettes by recET-mediated recombination into S. aureus target genes propagated in E. coil on bacterial artificial chromosomes (BACs). Return to S. aureus by electroporation, followed by chromosomal integration and resolution, provides a very rapid means for recovering the desired constructions. Under Phase II support, we will construct and validate approximately 200 PR strains and carry out full-scale screens with the completed TargetArray using the Elitra synthetic compound library. Chemically attractive hit compounds demonstrated by the TargetArray to have whole-cell, target-specific inhibitory activity will be advanced into lead-optimization chemistry and evaluated for pre-clinical development.
