Discovery of a New Antibiotic Demethoxytetronasin Using a Dual-Sided Agar Plate Assay (DAPA).

Abstract

For over a century, researchers have cultured microorganisms together on solid support─typically agar─in order to observe growth inhibition via antibiotic production. These simple bioassays have been critical to both academic researchers that study antibiotic production in microorganisms and to the pharmaceutical industry's global effort to discover drugs. Despite the utility of agar assays to researchers around the globe, several limitations have prevented their widespread adoption in advanced high-throughput compound discovery and dereplication campaigns. To address a list of specific shortcomings, we developed the dual-sided agar plate assay (DAPA), which exists in a 96-well plate format, allows microorganisms to compete through opposing sides of a solid support in individual wells, is amenable to high-throughput screening and automation, is reusable, and is low-cost. Herein, we validate the use of DAPA as a tool for drug discovery and show its utility to discover new antibiotic natural products. From the screening of 217 bacterial isolates on multiple nutrient media against 3 pathogens, 55 hits were observed, 9 known antibiotics were dereplicated directly from agar plugs, and a new antibiotic, demethoxytetronasin (1), was isolated from a Streptomyces sp. These results demonstrate that DAPA is an effective, accessible, and low-cost tool to screen, dereplicate, and prioritize bacteria directly from solid support in the front end of antibiotic discovery pipelines.