Project Summary Cūrza is developing a new class of broad-spectrum antibiotics, focused on multidrug-resistant (MDR) Gram- negative pathogens. The CZ-02 program is directed towards compounds that bind to a unique site on the bacterial ribosome that is not targeted by approved antibiotics and have not encountered cross-resistance to other antibiotics used clinically. Inspired by a natural product identified as a lead for Mycobacterium tuberculosis (Mtb), analogs have been developed that selectively inhibit bacterial protein synthesis, with little effect on mammalian protein synthesis, through a binding interaction with the ribosome at a heretofore un-drugged site. The natural product that inspired CZ-02s has multiple metabolic liabilities and lacks activity against Gram- negative pathogens. However, after re-engineering the natural product’s minimum pharmacophore responsible for activity into new chemical matter the resulting compounds are metabolically stable, exhibit exquisite selectivity and potency for bacterial protein synthesis and are efficacious against MDR pathogens in vitro and in vivo; all while displaying a lack of cytotoxicity toward mammalian cells and sparing mitochondrial function. The proposed SBIR project will ultimately deliver a new antibiotic candidate that is potent with broad spectrum activity and efficacy, focusing on Gram-negative pathogens that will be at the GLP toxicology stage. This Direct Phase II project will advance the CZ-02 program by the following aims. Aim 1 will optimize the lead series to improve activity against Pseudomonas aeruginosa and Acinetobacter baumannii while maintaining coverage of Enterobacterales and Gram-positive pathogens. Rigorous microbiological and biochemical evaluation along with profiling of absorption, distribution, metabolism, excretion and toxicity (ADME-Tox) will guide optimization efforts. Aim 2 will use in vivo pharmacokinetic (PK) evaluation to guide optimization along with maximum tolerated dose (MTD) determination to guide in vivo efficacy initially in mouse septicemia models to select lead compounds for evaluation in pneumonia models of P. aeruginosa and/or A. baumannii for down- selection to a single lead candidate. Aim 3 will provide scale-up chemistry to support in vivo studies. Aim 4 has sophisticated PK/pharmacodynamic (PD) profiling to establish the PD driver of efficacy and definition of the optimal dosing regimen in pneumonia models. At the culmination of the project, a lead compound will be ready for non-GLP toxicology and subsequent IND- enabling studies to ultimately deliver a new antibiotic from a novel class targeting Gram-negative pathogens.