New Phocoenamicin and Maklamicin Analogues from Cultures of Three Marine-Derived Micromonospora Strains.

Antimicrobial resistance can be considered a hidden global pandemic and research must be reinforced for the discovery of new antibiotics. The spirotetronate class of polyketides, with more than 100 bioactive compounds described to date, has recently grown with the discovery of phocoenamicins, compounds displaying different antibiotic activities. Three marine Micromonospora strains (CA-214671, CA-214658 and CA-218877), identified as phocoenamicins producers, were chosen to scale up their production and LC/HRMS analyses proved that EtOAc extracts from their culture broths produce several structurally related compounds not disclosed before. Herein, we report the production, isolation and structural elucidation of two new phocoenamicins, phocoenamicins D and E (1-2), along with the known phocoenamicin, phocoenamicins B and C (3-5), as well as maklamicin (7) and maklamicin B (6), the latter being reported for the first time as a natural product. All the isolated compounds were tested against various human pathogens and revealed diverse strong to negligible activity against methicillin-resistant Staphylococcus aureus, Mycobacterium tuberculosis H37Ra, Enterococcus faecium and Enterococcus faecalis. Their cell viability was also evaluated against the human liver adenocarcinoma cell line (Hep G2), demonstrating weak or no cytotoxicity. Lastly, the safety of the major compounds obtained, phocoenamicin (3), phocoenamicin B (4) and maklamicin (7), was tested against zebrafish eleuthero embryos and all of them displayed no toxicity up to a concentration of 25 µM.

Exploring Micromonospora as Phocoenamicins Producers.

Over the past few years, new technological and scientific advances have reinforced the field of natural product discovery. The spirotetronate class of natural products has recently grown with the discovery of phocoenamicins, natural actinomycete derived compounds that possess different antibiotic activities. Exploring the MEDINA's strain collection, 27 actinomycete strains, including three marine-derived and 24 terrestrial strains, were identified as possible phocoenamicins producers and their taxonomic identification by 16S rDNA sequencing showed that they all belong to the Micromonospora genus. Using an OSMAC approach, all the strains were cultivated in 10 different media each, resulting in 270 fermentations, whose extracts were analyzed by LC-HRMS and subjected to High-throughput screening (HTS) against methicillin-resistant Staphylococcus aureus (MRSA), Mycobacterium tuberculosis H37Ra and Mycobacterium bovis. The combination of LC-UV-HRMS analyses, metabolomics analysis and molecular networking (GNPS) revealed that they produce several related spirotetronates not disclosed before. Variations in the culture media were identified as the most determining factor for phocoenamicin production and the best producer strains and media were established. Herein, we reported the chemically diverse production and metabolic profiling of Micromonospora sp. strains, including the known phocoenamicins and maklamicin, reported for the first time as being related to this family of compounds, as well as the bioactivity of their crude extracts. Although our findings do not confirm previous statements about phocoenamicins production only in unique marine environments, they have identified marine-derived Micromonospora species as the best producers of phocoenamicins in terms of both the abundance in their extracts of some major members of the structural class and the variety of molecular structures produced.

Phocoenamicins B and C, New Antibacterial Spirotetronates Isolated from a Marine Micromonospora sp.

Phocoenamicins B and C (1 and 2), together with the known spirotetronate phocoenamicin (3), were isolated from cultures of Micromonospora sp. The acetone extract from a culture of this strain, isolated from marine sediments collected in the Canary Islands, displayed activity against methicillin-resistant Staphylococcus aureus (MRSA), Mycobacterium tuberculosis H37Ra and Mycobacterium bovis. Bioassay-guided fractionation of this extract using SP207ss column chromatography and preparative reversed-phased HPLC led to the isolation of the new compounds 1 and 2 belonging to the spirotetronate class of polyketides. Their structures were determined using a combination of HRMS, 1D and 2D NMR experiments and comparison with the spectra reported for phocoenamicin. Antibacterial activity tests of the pure compounds against these pathogens revealed minimal inhibitory concentration (MIC) values ranging from 4 to 64 µg/mL for MRSA, and 16 to 32 µg/mL for M. tuberculosis H37Ra, with no significant activity found against M. bovis and vancomycin-resistant Enterococcus faecium (VRE) at concentrations below 128 µg/mL, and weak activity detected against Bacillus subtilis grown on agar plates.