Targeted natural products discovery from marine cyanobacteria using combined phylogenetic and mass spectrometric evaluation

Abstract

Combined phylogenetic and HPLC-MS-based natural products dereplication methods aimed at identifying cyanobacterial collections containing the potent cytotoxins largazole, dolastatin 10, and symplostatin 1 were developed. The profiling of the phylogeny, chemical space, and antiproliferative activity of cyanobacterial collections served to streamline the prioritization of samples for the discovery of new secondary metabolites. The dereplication methods highlighted the biosynthetic potential and combinatorial pharmacology employed by marine cyanobacteria. We found that largazole was always coproduced with dolastatin 10 or with symplostatin 1 and consequently tested combinations of these agents against colon cancer cells. Combinatorial regimens of largazole and dolastatin 10 aimed at curbing the growth of HCT116 cancer cells showed cooperative activity.

Figure 1

Potent cytotoxins from marine cyanobacteria identified as Symploca and their mechanisms of action.

Figure 2

Evolutionary tree used to catalog the specimens (indicated in bold font) and place them in phylogenetic perspective with known natural products (NP) producing marine cyanobacteria. All gene sequences for NP-producing marine cyanobacteria available in public databases were included in the phylogenetic inferences. Groups with taxonomic reference strains or type strains were condensed and identified by the genus name. The phylogenetic inferences were based on the SSU (16S) rRNA nucleotide sequences using Bayesian Inference (MrBayes) and Maximum Likelihood (PhyML). The support values are indicated as posterior probability (MrBayes) and bootstrap support (PhyML). The scale bar is indicated at 0.05 expected nucleotide substitutions per site using the GTR+I+G substitution model. The presence of dolastatin 10, symplostatin 1 and largazole is indicated by colored dots.

Figure 3

Summary of bioactivity and natural products screening of cyanobacterial collections. A. The majority of the cyanobacterial collections displayed antiproliferative activity against HT29 human colorectal adenocarcinoma cells as assessed using the MTT reagent. The majority of potent bioactive extracts showed combinations of dolastatin 10, largazole and symplostatin 1 based on mass spectrometry-based dereplication using multiple reaction monitoring. B. Detailed distribution of the three known antiproliferative agents in profiled cyanobacterial collections.

Figure 4

Combinatorial pharmacology of dolastatin 10 and largazole on HCT116 cell growth. HCT116 cells were treated with either single agents or combinations of largazole and dolastatin 10. The predicted additive effects were extrapolated from the dose-response analysis for the single agents and calculated based on Loewe’s additivity. Statistical analysis using two sample t-test indicated significant difference between treatments using single agents versus combination (p < 0.05). However, there was no statistically significant difference between the predicted additive effect and experimental values obtained, indicating an additive effect of largazole and dolastatin 10. Data are presented as mean ± SD (n = 4).