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Biolayer Interferometry Provides a Robust Method for Detecting DNA-Binding Small Molecules in Microbial Extracts
preprintsubmitted on 19.08.2020, 22:15 and posted on 20.08.2020, 12:20 by Ross D. Overacker, Birte Plitzko, Sandra Loesgen
DNA replication is an exceptional point of therapeutic intervention for many cancer types and several small molecules targeting DNA have been developed into clinically used antitumor agents. Many of these molecules are naturally occurring metabolites from plants and microorganisms, such as the widely used chemotherapeutic doxorubicin. While natural product sources contain a vast number of DNA binding small molecules, isolating and identifying these molecules is challenging. Typical screening campaigns utilize time-consuming bioactivity-guided fractionation approaches, which use sequential rounds of cell-based assays to guide the isolation of active compounds. In this study, we explore the use of Biolayer Interferometry (BLI) as a tool for rapidly screening natural products sources for DNA targeting small molecules. We first verified that BLI robustly detected DNA binding using designed GC and AT rich DNA oligonucleotides with known DNA intercalating, groove-, and covalent-binding agents including actinomycin D (1), doxorubicin (2), ethidium bromide (3), propidium iodide (4), Hoechst 33342 (5), netropsin (6), and cisplatin (7). Although binding varied with the properties of the oligonucleotides, measured binding affinities agreed with previously reported values. We next utilized BLI to screen over 100 bacterial extracts from our microbial library for DNA binding activity and found three highly active extracts. Binding-guided isolation was used to isolate the active principle component from each extract, which were identified as echinomycin (8), actinomycin V (9), and chartreusin (10). This biosensor-based DNA binding screen is a novel, low cost, easy to use, and sensitive approach for medium-throughput screening of complex chemical libraries.