title: Microscale Adaptation of In Vitro Transcription/Translation for High-Throughput Screening of Natural Product Extract Libraries.
abstract: Novel antimicrobials that effectively inhibit bacterial growth are essential to fight the growing threat of antibiotic resistance. A promising target is the bacterial ribosome, a 2.5 MDa organelle susceptible to several biorthogonal modes of action used by different classes of antibiotics. To promote the discovery of unique inhibitors, we have miniaturized a coupled transcription/translation assay using E. coli and applied it to screen a natural product library of ~30 000 extracts. We significantly reduced the scale of the assay to 2 μL in a 1536-well plate format and decreased the effective concentration of costly reagents. The improved assay returned 1327 hits (4.6% hit rate) with %CV and Z' values of 8.5% and 0.74, respectively. This assay represents a significant advance in molecular screening, both in miniaturization and its application to a natural product extract library, and we intend to apply it to a broad array of pathogenic microbes in the search for novel anti-infective agents.
authors.affiliation: Life Sciences Institute, University of Michigan, 210 Washtenaw Ave., Ann Arbor, MI, 48109-2216, USA.
authors.forename: Andrew N
authors.initials: AN
authors.lastname: Lowell
authors.affiliation: Life Sciences Institute, University of Michigan, 210 Washtenaw Ave., Ann Arbor, MI, 48109-2216, USA.|Center for Chemical Genomics, University of Michigan, 210 Washtenaw Ave., Ann Arbor, MI, 48109-2216, USA.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Santoro
authors.affiliation: Life Sciences Institute, University of Michigan, 210 Washtenaw Ave., Ann Arbor, MI, 48109-2216, USA.|Center for Chemical Genomics, University of Michigan, 210 Washtenaw Ave., Ann Arbor, MI, 48109-2216, USA.
authors.forename: Steven M
authors.initials: SM
authors.lastname: Swaney
authors.affiliation: Life Sciences Institute, University of Michigan, 210 Washtenaw Ave., Ann Arbor, MI, 48109-2216, USA.|Center for Chemical Genomics, University of Michigan, 210 Washtenaw Ave., Ann Arbor, MI, 48109-2216, USA.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: McQuade
authors.affiliation: Life Sciences Institute, University of Michigan, 210 Washtenaw Ave., Ann Arbor, MI, 48109-2216, USA.
authors.forename: Pamela J
authors.initials: PJ
authors.lastname: Schultz
authors.affiliation: Life Sciences Institute, University of Michigan, 210 Washtenaw Ave., Ann Arbor, MI, 48109-2216, USA.|Center for Chemical Genomics, University of Michigan, 210 Washtenaw Ave., Ann Arbor, MI, 48109-2216, USA.
authors.forename: Martha J
authors.initials: MJ
authors.lastname: Larsen
authors.affiliation: Life Sciences Institute, University of Michigan, 210 Washtenaw Ave., Ann Arbor, MI, 48109-2216, USA.|Department of Chemistry and Medicinal Chemistry, University of Michigan, 210 Washtenaw Ave., Ann Arbor, MI, 48109-2216, USA.|Department of Microbiology and Immunology, University of Michigan, 210 Washtenaw Ave., Ann Arbor, MI, 48109-2216, USA.|Department of Chemistry, University of Michigan, 210 Washtenaw Ave., Ann Arbor, MI, 48109-2216, USA.
authors.forename: David H
authors.initials: DH
authors.lastname: Sherman
mesh_terms: D000890:Anti-Infective Agents / Q000494:pharmacology; D001688:Biological Products / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D057166:High-Throughput Screening Assays / Q000379:methods*; D008156:Luciferases / Q000235:genetics; D008904:Miniaturization / Q000379:methods; D014176:Protein Biosynthesis / Q000187:drug effects; D012270:Ribosomes / Q000187:drug effects / Q000235:genetics; D054852:Small Molecule Libraries; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D000890:Anti-Infective Agents; D001688:Biological Products; D054852:Small Molecule Libraries; D008156:Luciferases
keywords: antimicrobial screening; microscale high-throughput screening; natural products extract library; transcription/translation