SBIR-STTR Award

A novel method is proposed for the identification of essential genes whose products will be developed as new antibiotic targets. A combination of transposon mutagenesis and Bacterial Artificial Chromosomes (BACs) in Staphylococcus aureus and Enterococcus faecalis will yield cells that require BAC complementation of transposon mutations in essential genes. Phase I of this project includes the steps involved in constructing BAC libraries of S. aureus and E. faecalis. Phase II of the project will entail using the BAC libraries with transposon mutagenesis to identify essential genes and to develop screens for new antibiotics based on these essential genes. The method proposed for identifying essential genes is rapid, applicable to haploid cells whether prokaryotic or eukaryotic, and is amenable to automation. PROPOSED COMMERCIAL APPLICATIONS: Elitra is creating a unique relational database of both targets & drug screens for major gene/protein targets across multiple pathogens. This database will markedly enhance the ability of Elitra and its corporate partners to make informed decisions about which novel targets to pursue. Elitra is building a unique, ultra rapid Gene-to-Screen technology platform that will allow miniaturized drug screens to be developed for any validated target in its proprietary database within 2-3 weeks. Screening for new antibiotics will begin by the 4Q.99 when the in-house chemical screening library will be in excess of 250,000 compounds

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.

Thesaurus Terms:
antibiotic, chromosome, gene expression, genetic library, method development Staphylococcus aureus, chromosome complement, transfection /expression vector, transposon /insertion element biotechnology, cell line, polymerase chain reaction