Mining the kds Pathways - New Source of Novel Antibacterial Targets

University Of Michigan At Ann Arbor, Ann Arbor MI

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Abstract

DESCRIPTION (provided by applicant): In order to overcome the major human health problems posed by the continual emergence of drug-resistant bacteria, it is necessary to discover novel targets for the development of new and innovative antibiotics so as to prevent cross-resistance with present agents. Gram-negative bacteria are intrinsically more resistant to antibiotics and antibacterial compounds due to their rather unique outer cell envelope structure when compared the Gram-positive microorganism. The presence of the macromolecule, lipopolysaccharide (LPS) which surrounds and protects the bacteria from its environment has long been recognized to be responsible for the diverse immunological and biological properties attributed to "endotoxin". Thus, both the biosynthesis of this complex molecule and the inhibition of key enzymes in its construction have been the subject of numerous efforts by both academic and pharmaceutical investigators. We propose to identify targets for the development of a new class of Gram-negative antibiotic, namely inhibitors of the kds gene family that are responsible for the synthesis of the novel bridging carbohydrate 3-deoxy-D-manno-oct-2-ulosonic acid, KDO, a key component in LPS biosynthesis, by ultimately developing a sensitive, robust HTS assay to interrogate the gene products of kdsD, kdsA, kdsC and kdsB simultaneously;however, the present proposal seeks to develop an individual HTS assay for each of the four enzymes as well as the coupled pairs kdsD with kdsA and kdsC with kdsB. In collaboration, the University of Michigan Center for Chemical Genomics (CCG) staff will assist in validation of the high throughput screening assays developed in our laboratory to screen a trainer set of diverse small molecules from their library of pharmacologically active compounds before we send our "final" assay procedures to an appropriate Molecular Libraries Probe Production Centers most likely the Broad Institute Comprehensive Screening Center. Any "hits" identified via these screens will be validated in our secondary screening procedures and the confirmed "hits" will be subjected to more detailed evaluation, both in vitro kinetically and in vivo against a panel of microorganisms. Ultimately, the compounds identified could serve as leads for further cycles of optimization and evaluation in subsequent work. PUBLIC HEALTH RELEVANCE: In order to overcome the major human health problems posed by the continual emergence of drug-resistant bacteria, it is necessary to discover novel targets for the development of new and innovative antibiotics so as to prevent cross-resistance with present agents. We propose to identify targets for the development of a new class of Gram-negative antibiotic, namely inhibitors of the kds gene family by developing a sensitive, robust High Throughput Screening assay.