Identification bioactive compounds from marine microorganism and exploration of structure–activity relationships (SARs)

Marine natural products (MNPs) have become new strong leads for antimicrobial drug discovery and an effective alternative to control drug resistant infections. Herein we report the bioassay guided fractionation of marine extracts from sponges Lendenfeldia, Ircinia and Dysidea that led us to identify novel compounds with antimicrobial properties. Tertiary amines or quaternary amine salts: anilines 1, benzylamines 2, tertiary amines 3 and 4, and quaternary amine salt 5, along with three known compounds (6-8) were isolated from a crude extract and MeOH eluent marine extracts. The absolute configurations of the new compounds were assigned based on tandem mass spectrometry (MS) analysis. Several of the compounds exhibited potent in-vitro antibacterial activity, especially against Methicillin-resistant Staphylococcus aureus (MRSA) (MICs from 15.6 to 62.5 micro g/mL). Herein, we also, report structure activity relationships of a diverse range of commercial structurally similar compounds. The structure activity relationships (SARs) results clearly demonstrate that modification of the amines through linear chain length, and inclusion of aromatic rings, modifies the observed antimicrobial activity towards different biological activity. Several commercially available compounds, which are structurally related to the molecules we discovered showed broad spectrum antimicrobial activity against different test pathogens with an MIC50 range of 50 to 0.01 microM. The results of cross-referencing antimicrobial activity and cytotoxicity establish that these compounds are promising potential lead molecules, with a favourable therapeutic index for antimicrobial drug development. Additionally, the SAR studies show that simplified analogues of the isolated compounds with increased bioactivity


INTRODUCTION
Almost 70 percent of earth's surface is covered by ocean, representing a huge reserve of natural biological and chemical diversity on our planet. 1 Marine ecosystems have long been a rich source of bioactive natural products in the search for interesting molecules and novel therapeutic agents. [2][3][4][5][6] Many interesting and structurally diverse secondary metabolites have been isolated from marine sources over the last 70 years. 7-10 In addition, the preclinical pharmacology of seventy-five compounds isolated from marine organisms have been reported to have biological activities. 11 Yet the first 'drugs from the sea' were only approved in the early 2000s: the cone snail peptide ziconotide (ω-conotoxin MVIIA) in 2004 to alleviate chronic pain, 12 and sea squirt metabolite trabectedin in 2007 for treatment of soft-tissue sarcoma. 13 Marine natural products (MNPs) have displayed exceptional potency and potential as anticancer therapeutics. 14

Interest in
MNPs has continued to grow, 7, 9-10 spurred in part by the spread of antimicrobial resistant pathogens and the need for new drugs to combat them. 4 The most prolific marine organisms are sponges, 15 and the oldest metazoans on earth belong the phylum Porifera. 16 The Demospongiae are the most abundant class of Porifera, representing 83% of described species, [16][17] and has the largest number of bioactive compounds. 14 The genus Lendenfeldia are known as a source of sulfated sterols. 18 The Lendenfeldia species metabolites have an anti-HIV, anti-tumor 19 , anti-inflammatory, antifouling 20 activities but they lack antimicrobial activity. 18 Secondary metabolites of the genus Ircinia and Dysidea are prime candidates for further study to unveil their biological metabolites with antibacterial activity. 14,[21][22][23] Human pathogens are associated with a variety of moderate to severe infections and the recent rise of multi-drug resistant pathogens makes treatment more difficult. The last two decades have seen the emergence of methicillin-resistant Staphylococcus aureus (MRSA) strains resistant even to 'drugs of last resort' such as vancomycin, 24 and Mycobacterium tuberculosis resistant to all first-line agents, 25-27 highlighting the urgent need to find new effective antibiotics with distinct mechanisms of action. Natural products continue to offer a productive source of structural diversity and bioactivity, and are an important source for new drugs. [4][5][7][8] In the search for new antimicrobial agents, we screened a set of marine extracts 28 to determine activity against antibiotic resistant microorganisms using a high-throughput screening (HTS) assay. Fractionation and purification of active components by high-performance liquid chromatography (HPLC), Nuclear magnetic resonance (NMR) and structural elucidation using high resolution and tandem mass spectrometry (MS) led us to a series of potential scaffolds for new, bioactive amine natural products (Figures 1, S20, S21and S24).

Identification of active Marine Extracts.
To identify marine samples with activity against MRSA, 1434 compounds from the AIMS Bioresources Library 28 (provided by the Queensland Compound Library, 29 now called Compounds Australia 30 ) were screened in a resazurin cell viability assay. Of the samples tested, 29 inhibited the growth of MRSA by greater than 50% compared to non-treated controls. Minimum inhibitory concentrations (MICs) were determined for the 23 most promising samples, representing extracts and fractions from the phyla Porifera (90%), Echinodermata (5%) and Chordata (5%) ( Table 1). The five most active samples showed MICs at 31.3 µg mL −1 (all Porifera samples), while another four samples returned MICs of 62.5 µg mL −1 (also all Porifera). Cytotoxicity screens against HepG2, HEK 293, A549 and THP-1 cell lines were performed to define the cytotoxicity profile of the most active samples. Pleasingly, all the samples most active against MRSA were also nontoxic to the cell lines tested (Tables 1).  Aniline/amines 1-5 are all known compounds, 34-36 however they have not previously been identified as natural products, nor has their antibiotic activity been assessed.  Compound 8 has previously been identified and isolated from the marine sponges Theonella swinhoe, Halicondriidea, and Coscinoderma mathewsi and its inhibitory effects on different enzymes reported before [56][57][58] . The bioactivity of 8 was first reported in 1998, when it was identified as a thrombin inhibitor 56 . Antimicrobial activity against S. aureus, C. albicans, and B.
Samples of three extracts (20608, 26051 and 19033) were subjected to HPLC purification and bioassay guided fractionation, enabling bioactive components to be isolated in low yield («1 mg). Then high-resolution MS and tandem MS analysis was used to decipher structures ( Table   2

Purification of Natural Products from Extracts and Structure Elucidation
High Performance Liquid Chromatography (HPLC) Purification. Samples were separated using analytical (Waters 2695 Alliance pump with Waters 2996 PDA, Sunfire reversed-phase column, and WFIII fraction collector) and preparative (Waters 600 HPLC pump, Phenomenex reversed-phase column, Waters 2487 UV detector and WFIII fraction collector) HPLC systems with UV detectors at 254 and 280 nm, employing a gradient of solvents A (dH 2 O) and B (acetonitrile) with trifluoroacetic acid (0.1%). Extract mixtures were kept at 4 °C until injection, then extract sample (100 μ L) was injected onto an analytical Waters X-bridge C18 100 Å (4.6 × 250 mm, 5 µm) reversed-phase column on the same analytical HPLC system described above.
The mobile phase was obtained using binary gradients of solvents A and B at a flow rate of 1 ml min -1 at 30 °C over 80 min. Fractions, separated every 60 s, were collected. Purified fractions were flash-frozen in liquid nitrogen then freeze-dried overnight. The resulting fractionated extracts were re-suspended in DMSO and antibacterial activity versus MRSA was determined as described above.
Fractions identified as active against MRSA were further purified on the preparative HPLC unit described above, using a Phenomenex C18 100 Å (250 × 21.2 mm, 10 µm) reversed-phase column with UV detection at 254 and 280 nm, 7 mL min -1 flow rate with water/acetonitrile gradient containing 0.1% trifluoroacetic acid.
The gradient for AIMS extracts 19033, 20608, 26104, 25641and 26051 was 0% B initially, increased to 40% B over 20 min, then to 100% over 40 min, held at 100% for 10 min. For AIMS extracts 25663 gradient was 0% B initially, increased to 40% B over 60 min, then to 100% over 30 min, held at 100% for 10 min. Compounds thus purified were evaluated for biological activity and analysed by MS to determine potential structures for the bioactive components.

Supporting Information
Bioactivity and toxicity screening data, HPLC fractionation and purification protocols, plus mass spectrometry data (HRMS spectra, tables of daughter ions, and proposed fragmentation pathways) for natural products and synthetic compounds.

Funding Sources NHMRC Project Grant APP1084266
Author Contribution Statement MD conceived and designed the experiments; performed the experiments; analyzed the data; MD wrote the paper.

Notes
The authors declare no competing financial interest