This mode-of-action (MOA) core will support all 3 projects in our CETR. Our core?s purpose is to divine the MOA of novel lead compounds identified by the projects and filtered through the in vitro and in vivo cores. An- timicrobial development requires knowledge of a drug target and MOA to enable downstream refinements and improvements of leads. We propose to emphasize chemical-genetic strategies together with cutting edge tools and approaches to discern MOA of drug leads. We will exploit the principle of fitness testing where loss of one or both copies of a gene renders a mutant fungus or bacterium hyper- or hypo-sensitive to a specific drug. The hits from this analysis include genes encoding the specific drug target, or effectors of the drug's metabolism. We will capitalize on available mutant libraries that span the genomes of pathogenic fungi and bacteria, in- cluding Candida albicans and Escherichia coli. Our strategy will allow us to screen leads across a comprehen- sive set of drug targets rather than just a single target. Thus, for antifungal candidates, drug-induced growth alteration of strains in a library of heterozygous deletion mutants will reveal a potential drug target and MOA; a so-called haploinsufficiency profile where strain-specific barcodes uniquely mark mutants, and those with al- tered-fitness are detected by Illumina sequencing. For antibacterial leads, high throughput screens will be used to assay mutant libraries of Gram-negative species for drug sensitivity. For E. coli, this includes a two-allele barcoded deletion library and a CRISPRi library that enables conditional expression control of essential genes. In a parallel approach, drug-resistant mutants will be selected, genome sequenced, SNPs identified and tar- gets implicated. Drug targets and MOAs will be confirmed with downstream genetic and biochemical studies. We have several leads in hand to begin our studies. From ongoing work by our group, we expect to study two leads annually for MOA (up to 10 total). Our core is significant because it will generate vital new knowledge about promising antimicrobial drug leads. The insight on drug MOA will be needed to fuel development of new drugs against resistant infectious disease. Our research findings will exert a sustained and powerful influence on the field because it will improve the care of patients infected with resistant fungal and bacterial pathogens. These achievements will help the NIH CETR program realize its goals of improving the public?s health.
SUMMARY Fungal and bacterial pathogens have emerged for which there are no longer effective antimicrobial therapies. The Mode of Action Core will support all three projects in this CETR by analyzing the mode of action and drug target(s) of natural product leads that show promise based on their structural novelty and potent activity in the in vitro and in vivo cores.