The objective of this grant is the preclinical development of MRS-2541, a novel antibiotic structure targeting Gram+ bacteria by a new mechanism of action. MRS-2541 inhibits bacterial methionyl- tRNA synthetase required for bacterial protein synthesis, which is very different from its mammalian counterpart. MRS-2541 is the result of an arduous testing cascade of over 500 compounds requiring broad-spectrum in vitro and in vivo Gram+ activity, a low resistance frequency and minimal hERG and mitochondrial toxicity. The initial clinical indication will be acute bacterial skin and skin structure infections (ABSSSI) with clear endpoints and relatively easy enrollments. MRS-2541 is highly potent against serious pathogens such as methicillin resistant Staphylococcus aureus (MRSA), vancomycin resistant Enterococcus faecium (VRE), and Streptococcus species, with minimum inhibitory concentrations (MICs) ≤ 0.5 µg/mL, which are below those of widely used drugs such as vancomycin and linezolid. MRS-2541 has minimal cytotoxicity on mammalian cells, is well tolerated in mice at 150 mg/Kg/day, has excellent oral bioavailability, and is as efficacious as linezolid in the S. aureus and S. pyogenes thigh murine models of infection by both the oral and subcutaneous route. Specific Aim 1 will be to optimize the new 5-step synthesis from a commercially available fluorinated precursor and prepare 150 g for Aims 2-4. Aim 2 will characterize the PK/ADME profile of MRS-2541, including metabolic profiling, cytochrome inhibition, and PK in a non-rodent species. Aim 3 will more fully characterize the antibacterial profile with MIC90s vs target and some off-target organisms, as well as the determination of the driver for in vivo efficacy, which is critical for human dose projections. Finally, Aim 4 will support a GLP 28-day toxicology study in rodents along with several safety pharmacology studies. The scientific teams at University of Washington and TSRL have the combined expertise in chemistry, microbiology, pharmacology, and preclinical drug development to execute the proposed research plan. A successful project will bring forward MRS-2541 as a clinical lead candidate, representing a novel antibiotic class to address the critical public health issue of bacterial drug-resistance not only for ABSSSIs, but for a variety of serious Gram+ infections.