Non-tuberculous mycobacteria (NTM) are emerging pathogens with high intrinsic drug resistance. Mycobacterium abscessus is the most pathogenic refractory NTM member, and infections with this pathogen are associated with especially poor clinical outcomes. The standard of care therapy of amikacin and clarithromycin fails in a high proportion of cases, and thus there is a clear need for new therapeutic options. To approach this challenge, we investigated the synthetic modification of spectinomycin, an aminocyclitol antibiotic that exhibits potent bacterial protein synthesis inhibition but has limited efficacy in mycobacteria due to intrinsic resistance mechanisms. A library of semi-synthetic spectinomycin analogs was profiled for activity against M. abscessus, from which a distinct structural subclass of ethylene linked aminomethyl spectinomycins (eAmSPC) was identified. Initial leads of this subclass display potent anti-M. abscessus activity, while maintaining the desired pharmacological properties of minimal cytotoxicity and hepatic metabolism, low protein binding, and absence of mitochondrial protein synthesis inhibition. These leads have favorable activity against multi-drug resistant M. abscessus clinical isolates, are active against other NTM pathogens, minimally induce the WhiB7 ribosomal stress response pathway, are not substrates for mycobacterial aminoglycoside modifying enzymes and demonstrate robust efficacy in M. abscessus mouse infection models. The results of these preliminary studies suggest that eAmSPCs have the potential to be developed into treatments for M. abscessus and other NTM infections. The key goals of this proposal are to increase the potency and tolerability of the eAmSPCs. This will be achieved through an iterative drug cycle to include: (i) A structure-guided optimization strategy will be applied to the ethyl side chain to reduce its lipophilicity and generate extra binding interactions in the RpsE / 30S helix-34 binding side pocket. (ii) Further rounds of optimization will be guided by mycobacterial ribosomal inhibition, minimum inhibitory concentration (MIC) activity against a panel of non-tuberculous mycobacteria (NTM), and in vitro pharmacokinetic studies. Recently developed Cryo-EM methods in mycobacterial ribosomes will confirm the binding mode and assist in these structure-based drug design efforts. Accumulation studies to investigate permeability and efflux will help define which structural modifications are successful in overcoming intrinsic resistance mechanisms. Whole- genome sequencing and RNAseq studies will ensure compounds remain on target and explore drug resistance and virulence mechanisms. (iii) In vivo pharmacokinetics, safety, and efficacy studies on emerging leads will be determined, in comparison to the standard of care antibiotics, using acute and chronic mouse models of NTM infection that recapitulate the pathology of NTM infected human lung. Late leads will be profiled for in vitro pharmacology ...