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Biolayer interferometry provides a robust method for detecting DNA binding small molecules in microbial extracts

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Abstract

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 product 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 binding, and covalent binding agents including actinomycin D (1), doxorubicin (2), ethidium bromide (3), propidium iodide (4), Hoechst 33342 (5), and netropsin (6). 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.

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Acknowledgements

Thanks to Khaled Attia Mahmoud and Jon Thorson (Center for Pharmaceutical Research and Innovation, Lexington KY) for their kind donation of the bacterial strain RM1-1. Thanks to James A. Strother (UF) for fruitful discussions. We acknowledge the support of the Oregon State University’s NMR Facility funded in part by the National Institutes of Health, HEI grant 1S10OD018518, and by the M. J. Murdock Charitable Trust grant no. 2014162.

Funding

This work was supported by OSU start-up funds and by the National Science Foundation under grant CHE 1808717.

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  1. Sandra Loesgen

    Present address: Whitney Laboratory for Marine Bioscience, Department of Chemistry, University of Florida, St. Augustine, FL, 32080, USA

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  1. Department of Chemistry, Oregon State University, Corvallis, OR, 97331, USA

    Ross D. Overacker, Birte Plitzko & Sandra Loesgen

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Overacker, R.D., Plitzko, B. & Loesgen, S. Biolayer interferometry provides a robust method for detecting DNA binding small molecules in microbial extracts. Anal Bioanal Chem 413, 1159–1171 (2021). https://doi.org/10.1007/s00216-020-03079-5

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