Microscale Adaptation of In Vitro Transcription/Translation for High‐Throughput Screening of Natural Product Extract Libraries

Antibiotic resistance is a growing international threat, with 23 000 deaths, 2 million patients, and $20 billion expended in the clinical setting annually in the United States alone (1). The pharmaceutical industry's approach to this problem, combinatorial libraries, has not proven as promising as once hoped (2). Combinatorial synthesis has produced millions of compounds, but the consistent production of potent leads and eventual therapies has remained elusive (3). In an effort to identify new, promising antimicrobials, we sought to develop a high-throughput screening assay for inhibitors of prokaryotic ribosomes that we could apply to natural product extracts. Over the past decade, our laboratory has isolated phylogenetically unique pure-culture microbial strains that we have grown and extracted to create a prefractionation natural products extract library (NPEL) of more than 30 000 samples, a collection housed in the Center for Chemical Genomics (CCG) at the University of Michigan (4). The NPEL has already proven to be a potent resource in high-throughput screening, furnishing promising leads toward antivirals (4), effective inhibitors of siderophore virulence-factor biosynthesis for MRSA and Bacillus anthracis (5), and potent aggregation inhibitors for neurodegenerative disorders (6). Our intention was to develop a high-throughput screen for the identification of new antibacterial agents and apply it to the NPEL. We envisioned adapting a coupled transcription/translation assay used previously for screening industrial chemistry libraries to the NPEL (7,8). A transcription/translation screen is desirable because it enables rapid screening of libraries against the bacterial ribosome, an established target for several classes of antibiotics including macrolides, aminoglycosides, tetracyclines, and oxazolidinones (9). Because different classes of ribosome inhibitors have orthogonal molecular modes of action, resistance by a pathogen to one class does enable it to counter other classes (10,11). Hence, new chemical scaffolds uncovered by a high-throughput screen are likely to be effective against drug resistant bacterial pathogens. This study was motivated by the potential cost and throughput advantages of reducing the scale of a direct ribosome inhibitor screen using natural product extracts as the source of chemical diversity. Previous studies successfully optimized a coupled transcription/translation screen in 384-well plates at a total scale of 16 μL using a luminescence-based reporter on a small-molecule library, which resulted in the discovery of a potential lead for a new, orthogonal class of ribosome inhibitor (7,8). This type of assay is amenable for use in 1536-well plates (12), and a number of secondary and counter screens are available to enable the decoupling of transcription from translation (7,8,13), remove intercalative inhibitors (14), discriminate compounds that interfere with luminescence (7,8), and identify general (eukaryotic) ribosome inhibitors (7,8,14). Our objectives were to create an improved method by significantly increasing efficiency through a reduction in reaction scale and to apply this screening technology directly to natural product extracts.