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. 2017 Mar 24;80(3):625-633.
doi: 10.1021/acs.jnatprod.6b00907. Epub 2017 Jan 5.

Integrating Molecular Networking and Biological Assays To Target the Isolation of a Cytotoxic Cyclic Octapeptide, Samoamide A, from an American Samoan Marine Cyanobacterium

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Integrating Molecular Networking and Biological Assays To Target the Isolation of a Cytotoxic Cyclic Octapeptide, Samoamide A, from an American Samoan Marine Cyanobacterium

C Benjamin Naman et al. J Nat Prod. .

Abstract

Integrating LC-MS/MS molecular networking and bioassay-guided fractionation enabled the targeted isolation of a new and bioactive cyclic octapeptide, samoamide A (1), from a sample of cf. Symploca sp. collected in American Samoa. The structure of 1 was established by detailed 1D and 2D NMR experiments, HRESIMS data, and chemical degradation/chromatographic (e.g., Marfey's analysis) studies. Pure compound 1 was shown to have in vitro cytotoxic activity against several human cancer cell lines in both traditional cell culture and zone inhibition bioassays. Although there was no particular selectivity between the cell lines tested for samoamide A, the most potent activity was observed against H460 human non-small-cell lung cancer cells (IC50 = 1.1 μM). Molecular modeling studies suggested that one possible mechanism of action for 1 is the inhibition of the enzyme dipeptidyl peptidase (CD26, DPP4) at a reported allosteric binding site, which could lead to many downstream pharmacological effects. However, this interaction was moderate when tested in vitro at up to 10 μM and only resulted in about 16% peptidase inhibition. Combining bioassay screening with the cheminformatics strategy of LC-MS/MS molecular networking as a discovery tool expedited the targeted isolation of a natural product possessing both a novel chemical structure and a desired biological activity.

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Conflict of interest statement

Notes. The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
LC-MS/MS derived molecular network of extracts and systematically produced VLC fractions from ten Symploca spp. with tentatively identified annotations and examples of clusters correlating to molecules (e.g. chlorophylls) with ubiquitous distribution, (e.g. bastimolides, dolastatin 10, viequeamide B, and unknowns) of intermediate distribution, and (e.g. m/z 911 metabolite = compound 1, and yet-undescribed molecules) that have species-specific occurrence. Single-node clusters were pruned for visualization purposes. Node sizes are scaled to signal intensity, and the cluster containing 1 has been further expanded for legibility. Different node colors represent annotations of different source organisms. See expanded version in Figure S13, Supporting Information.
Figure 2
Figure 2
TOCSY and selected HMBC correlations used to determine the two planar tetrapeptide fragments of samoamide A (1). Bolded bonds represent correlated protons in the TOCSY spectrum. Arrows represent cross peaks from the 1H-13C HMBC spectrum.
Figure 3
Figure 3
Structure of 1 with absolute configurations shown, annotated with observed MS/MS fragmentations. Standard b7-b3 cleavages are shown in purple, and y7-y3 cleavages in orange. Arrows represent direction of sequential fragmentation after initial ring opening adjacent to the prolyl nitrogen atoms during positive mode ESIMS.
Figure 4
Figure 4
Results of in silico docking of 1 with DPP4. Panel A – predicted interaction of a generated samoamide A (1) conformer library with the surface-colored allosteric inhibition site. Panel B – predicted molecular interactions of a low energy conformer at the location shown in panel A; red bond lines denote predicted solvent exposure outside of the interaction cleft of the enzyme. Panel C – legend generated from Molecular Operating Environment (MOE) Software for panel B.

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