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.

Keywords: Streptomyces sp; antibiotic bioassay; demethoxytetronasin; dual-sided agar plate assay (DAPA); tetronasin.

Figure 1

Design of DAPA. (i) DAPA plate and the silicone insert. (ii) Sideview of a single DAPA well; T = top and B = bottom. A red silicone insert allows wells to be filled with agar, and custom-engineered interior channels allow the plug to be suspended in the middle of the well upon the removal of silicon. An environmental bacterium (green) is incubated and produces metabolites into the agar plug. (iii) DAPA plate is inverted, and a pathogen (blue) is added. After an appropriate incubation period, growth inhibition may be assessed using orthogonal methods of detection including fluorescence and optical density via a redox dye such as MTT.

Figure 2

(A) One screening plate of environmental bacteria against P. aeruginosa ATCC 10145GFP using fluorescence detection; this represents one of the 37 DAPA plates used in this screen. Extraction/UPLC-MS/MS/GNPS analysis of the duplicate wells highlighted in blue led to dereplication of the known antibiotic bacillomycin LC2 (Figure S2). (B) List of all known antibiotics dereplicated directly from DAPA wells in this screen; three-dimensional structures are putative since our MS analysis is not sufficient to determine the configuration at each stereocenter.

Figure 3

Antibiotic structures isolated from strain K1063 and key 2D NMR correlations of 1.