The alarming increase in incidence of infections caused by drug-resistant bacteria has created an urgent need for new antibacterial agents. We are developing a novel class of small molecule antibiotics (TZUs) targeting PolC, the replicative DNA polymerase in Gram-positive bacteria. These agents exhibit broad-spectrum activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), penicillin-resistant Streptococcus pneumoniae (PRSP), Streptococcus pyogenes (Group A strep), vancomycin resistant enterococcus (VRE), Bacillus anthracis, Listeria monocytogenes and Clostridium difficile. TZUs demonstrate bactericidal activity, oral bioavailability, in vivo efficacy, a low propensity for toxicity and a low rate of spontaneous resistance. TZUs inhibit a novel bacterial target, thereby circumventing existing mechanisms of antibiotic resistance. We have made compelling progress during Phase I, having already met several key goals. In particular, we have identified several TZUs with in vitro potency exceeding that of linezolid, the only orally active drug approved for MRSA. We seek Phase II funding to enable completion of pivotal studies required for filing of an Investigational New Drug (IND) application. The research plan is divided into three stages. Aim 1 focuses on in-depth characterization of three top TZUs; each exhibits key attributes including in vivo efficacy following oral administration and outstanding microbiological potency. Aim 1 studies will include in vivo pharmacodynamics (PD) dose response studies, determination of pharmacokinetic (PK) parameters in two species, determination of the PK-PD index, evaluation of in vitro and in vivo toxicity, and determination of rates of resistance development. Based on these results, the TZU that best exemplifies the target product profile will be selected as the IND candidate. In Aim 2, process chemistry work will commence, focusing on improving synthetic yields, lowering cost, and making the synthetic process more scalable (e.g., by elimination of chromatographic steps). At this stage, the goal will be to develop a synthetic route appropriate for 1-10 kilogram scale synthesis at a cost of less than $25,000 per kg. Exploratory toxicology and toxicokinetic studies will be performed under non-GLP conditions in two species to support dose selection, toxicology endpoints and toxicokinetic time points for GLP-compliant studies. Analytical and bioanalytical methods will be validated under Good Laboratory Practice (GLP) conditions. Comprehensive microbiological characterization will be performed, including MIC90 testing in several Gram-positive species, evaluation of cross-resistance, post antibiotic effect (PAE) and synergy with other antibiotics. In Aim 3, a 5-10 kg GLP-toxicology batch will be synthesized, and GLP-compliant toxicology and safety pharmacology studies will be performed. Successful completion of these IND-enabling studies would support advancement of this program into human clinical trials. This project represents a compelling opportunity to combat the rising tide of antibiotic-resistant infections, providing a much-needed public health benefit.