Evaluating Distribution Patterns of Cyanobacterial Secondary Metabolism

The phylum Cyanobacteria, comprised of photosynthetic prokaryotes, has been surveyed for bioactive secondary metabolites for roughly the last 30 years resulting in the identification of drug leads to treat a range of diseases identified.1 In particular, synthetic analogues of the freshwater cryptophycin 1 (10a, Figure 6) and the marine dolastatin 10 have reached clinical trials with the dolastatin 10 (15, Figure 8) analogue monomethyl auristatin E (16b, Figure 8) being approved as antibody-drug conjugate anticancer agents.2–5 Our research group identifies bioactive secondary metabolites from freshwater cyanobacterial strains. We have amassed a collected of over 1,200 strains primarily from the Midwestern United States whose metabolomes are subjected to a bioassay-guided fractionation drug discovery strategy. Following this process, two novel laxaphycin type-B metabolites, laxaphycins B5 and B6 (24 and 25, Figure 12) were obtained and demonstrated high nM/low μM IC50 against cancer lines OVCAR3 (ovarian), MDA-MB-231 (breast adenocarcinoma), and MDA-MB-435 (melanoma). The structures were elucidated by two-dimensional NMR spectroscopy using HSQC, COSY, HMBC, band-selective HMBC, TOCSY, and ROESY experiments to determine the planar structures while advanced Marfey’s analysis was performed to determine the absolute configurations of the amino acids. In addition to the novel laxaphycins, the epi-statine-containing phormidepistatin A was obtained from UIC 10484. The statine moiety is an established pharmacophore known to inhibit aspartic proteases, including cathepsin D, which is implicated in multiple types of cancer.6 Due to this, phormidepistatin A (41, Figure 17) was tested against cathepsin D and displayed mild inhibition against the enzyme with an IC50 of 21.4 µM. The structure was elucidated by two-dimensional NMR spectroscopy using HSQC, COSY, HMBC, band-selective HMBC, and HSQC-TOCSY experiments to solve the planar structure. The six amino acid absolute configurations were assessed by advanced Marfey’s analysis. The 3,9-dihydroxy-2-methyl-10-phenyldecanoic acid (DMPD; 42) moiety attached to N-terminal serine is currently being assessed for the absolute configuration of the two secondary alcohols in addition to the methylated chiral carbon. The relative configuration of the methylated C-2 was established as erythro with respect to the hydroxylated C-3 by J-based configurational analysis. NMR experiments psHMBC, HSQMBC, and DQF-COSY were performed for this analysis. The discovery of phormidepistatin A led us to evaluate the phylogenetics of the statine (Sta), or statine-like (Sta-like), γ-amino acid within the phylum Cyanobacteria. Since UIC 10484 is also a laxaphycin type-B producer, we investigated the presence of phormidepistatin A in two other laxaphycin producers: UIC 10045, a closely related cf. Phormidium sp. strain, of the order Oscillatoriales, and UIC 10339 of the order Nostocales. Both strains were found to produce phormidepistatin A. Additionally, a structure search identified 32 known cyanobacterial secondary metabolites containing a Sta/Sta-like moiety. A structure similarity assessment established eight compound classes incorporating a Sta/Sta-like amino acid with all eight produced by at least one strain with a published 16S rRNA sequence. A phylogenetic analysis using the 16S sequence was performed, which found incorporation of the Sta/Sta-like amino acid by a range of cyanobacteria. To broadly assess phylogenetic distribution of cyanobacterial secondary metabolism, a comprehensive literature search was performed to identify strains with published secondary metabolite production and a 16S rRNA sequence. This effort resulted in a dataset comprised of 216 strains collected from freshwater, marine, and other environments known to produce 791 compounds (509 unique structures). A phylogenetic analysis identified a pattern in which exclusively freshwater (along with brackish water strains primarily from the Baltic Sea) and exclusively marine clades interspersed throughout the phylogenetic tree but with limited overlap between the two ecological groups. This coincided with minimal secondary metabolite overlap between marine and freshwater cyanobacteria. Cyanobacteria are a potent source for bioactive secondary metabolites. This was evident with the isolation of laxaphycins B5 and B6 in addition to phormidepistatin A from UIC 10484. To maximize the identification of therapeutically relevant secondary metabolites from the phylum, the 16S rRNA sequence can be used as a genetic barcode to survey secondary metabolite trends. As an example, a phylogenetic assessment of the secondary metabolites containing the biomedically-relevant Sta/Sta-like moiety demonstrated that diverse strains utilize the amino acid in a number of different compound classes. Our findings suggest cyanobacteria are a potential source for aspartic protease inhibitors. As another example, the 16S rRNA sequence preliminarily established that freshwater and marine cyanobacteria thus far evaluated for secondary metabolism are phylogenetically distinct from each other. This dissertation proposes one reason the two ecological groups have been found to produce different chemistry is due to this evolutionary distinction.7 In sum, using the 16S rRNA sequence as a central datapoint for other data, including secondary metabolite production, bioactivity, geographical location and environment, or any other information, could serve as a means to monitor how the phylum Cyanobacteria is being evaluated for drug discovery purposes.