Project Summary In 2020 alone, about 200 million oral antibiotic prescriptions were dispensed by outpatient healthcare providers in the United States. Broad-spectrum antibiotics make up most of these prescriptions. However, in addition to targeting the intended pathogen, broad-spectrum antibiotics alter the composition of the human microbiome (dysbiosis). Dysbiosis caused by broad-spectrum antibiotic treatment can increase susceptibility to or worsen the outcome of disease, including inflammatory bowel disease, cancer, and psychiatric and neurodegenerative disorders. Therefore, it is important to develop therapeutic strategies that minimize the collateral damage of broad-spectrum antibiotics. Here we propose to explore molecules that selectively antagonize the activity of broad-spectrum antibiotics as a strategy to minimize dysbiosis. The advantage of selective antidotes over the alternative strategy of developing narrow-spectrum or pathogen-targeted antibiotics is to be able to continue to use already developed broad-spectrum antibiotics. Proof-of-concept for the selective antidote strategy has been recently provided. From the four previously identified antidotes, three are not available in the US due to their toxicity, and the remaining one has poor water solubility and unfavorable pharmacokinetics. We have recently discovered a family of depsipeptide natural products we named pseudovibriamides. Pseudovibriamides are produced by Pseudovibrio bacteria that are part of the healthy microbiome of marine sponges. Interestingly, Pseudovibrio has been proposed to contribute to marine sponge health by producing broad-spectrum antibiotics that can prevent the growth of pathogens. Marine sponges are ancient animals that form important symbiotic relationships with their microbes. Thus, broad-spectrum antibiotics known to be produced by Pseudovibrio and other sponge bacteria would cause dysbiosis in the sponge animal. We hypothesize that pseudovibriamides act as selective antidotes to protect commensal bacteria and the sponge host but allow activity against pathogens. We envisage the ecological role of pseudovibriamides may be translated into pharmaceutical applications. This proposal has two aims. Aim 1 is to improve access to pseudovibriamides using biosynthetic methods and Aim 2 is to explore their antibiotic and strain spectra. We will use sponge bacteria commensals and pathogens as controls to test the hypothesis. We will then explore the spectrum of antidote activity of pseudovibriamides with widely used antibiotics and the human pathogens they are intended to treat, and with prevalent and abundant human commensals. Thus, this proposal will enable facile access to and will explore the antidote spectrum of a family of bacterial natural products shown to have antibiotic antidote activity. What we learn will serve as steppingstones for future studies on their mode of action to enable the discovery of further antidotes.