Phenotypic screening puts the drug discovery process to work at the level of the whole cell. Antibiotic drugs work by inhibiting specific targets in a bacterial cell. But in phenotypic screening, libraries of small molecules are added not to individual, isolated targets but to whole cells of Mycobacterium tuberculosis (M.tb). Compounds that kill or stop the growth of M.tb - but do not affect the survival or growth of mammalian cells - become potential lead molecules. This approach has proven effective and led to the discovery of all novel anti-mycobacterial agents which are in clinical development today. Adding compounds to whole cells - as in phenotypic screening - makes every pathway in the cell a potential drug target. This greatly increases the likelihood of finding an effective inhibitor. In addition, compounds that cannot get into cells, or that are rapidly effluxed from cells, will never be identified as potential lead molecules and will not result in wasted effort. Phenotypic screening has traditionally been done with a surrogate bacterial species/strain (because of its safety or rapidity) that does not faithfully reproduce the drug susceptibility profile of M.tb. Screening has also been limited to actively replicating cultures and therefore has not necessarily identified or prioritized compounds with the potential to act against persistent populations and thus shorten therapy. The current project is designed to screen directly against M.tb under both replicating and non-replicating conditions. This overcomes the issue of poor predictability associated with the earlier screening approach and enables prioritization of leads with potential for shortening treatment. The assay against non-replicating cells uses low-oxygen cultures of M.tb that may more closely mimic the persistent infection conditions in human. The phenotypic screening project started recently. As a pilot project, the team has completed the screening of two libraries: a diverse scaffold-based library of 50,000 drug-like compounds and a 1,500 compound library of natural products and semi-synthetic natural product derivatives. Several promising and novel compound series have been identified from both synthetic and natural product collections. Some of these series showed potent anti-mycobacterial activity and low cytotoxicity and have moved to the hit-to-lead phase. The project team has completed screening of the third and forth libraries which all together consisted of 66,000 synthetic and natural-product derived compounds. Approximately 1500 hits have been identified and confirmed, and they are currently being triaged.