Multiplexing Natural Products Discovery
An organism’s metabolome is the total of all the small molecules that it produces. Efforts to identify bioactive natural products in the metabolomes of microbes, plants, and other organisms are often thwarted by metabolome complexity and the heterogeneous nature of the cell populations to which a potential effector molecule is to be targeted. Thus, natural product discovery would benefit greatly from a rapid and efficient screening system that enables the evaluation of entire metabolomes against a physiologically relevant primary cell population. Now, Vanderbilt Basic Sciences investigator Jonathan Irish and his collaborator Brian Bachmann (Chemistry) describe multiplexed activity metabolomics (MAM), a screening system that achieves those goals. The first step in MAM is to chromatographically separate the metabolites of a “stimulus organism” and distribute them into a microtiter plate while simultaneously analyzing them by UV/visible and mass spectrometry. The result is a metabolite array with associated spectral data that can be used to identify the compounds in each well. The second step is to add target cells from the “response organism” to the wells of the plate and incubate them for a desired period of time. The third step is to “barcode” the cells of each well with distinct concentrations of two fluorescent dyes, combine the cells, and then further label them with fluorescent antibodies that detect cell type or physiological responses (e.g., viability, apoptosis, DNA damage, proliferation). Analysis by flow cytometry characterizes each individual cell on the basis of the combination of fluorescent labels. Barcoding assigns the cell to the correct well, and the fluorescent antibodies identify the cell type, and assess the effects of exposure to the well contents. If cells from a well exhibit an interesting effect, the investigator can use the associated spectral data to identify the compounds in the well. The Bachmann and Irish lab used MAM to identify compounds in microbial extracts that display antitumor activity against acute myeloid leukemia (AML) cells from two patients. From a Streptomyces specus metabolome, they identified compounds that exhibited toxicity in both a cell type- and patient-selective fashion. The most active compounds were the anthracyclines, specumycins A1 and B1. From Nocardiopsis sp. FU40, they identified apoptolidins, which exhibited selective toxicity for normal lymphocytes over tumor cells, and ciromicins A and B, which exhibited the opposite selectivity. Using mass cytometry to differentiate cell types with greater granularity, the researchers further distinguished the activities of ciromicins A and B. Together, the results confirm the power of MAM to identify biologically active natural products in complex metabolomes using heterogeneous mixtures of primary cells and evaluating multiple effects in a single rapid assay. The work is published in the journal Nature Communications [D. C. Earl, et al. Nat. Commun, (2018) published online January 2, DOI: 10.10.1038/s41467-017-02470-8].