Bioactivities of Halometabolites from Marine Actinobacteria

Natural halogenated compounds (halometabolites) are produced mainly by marine organisms, including marine Actinobacteria. Many commercially important compounds for pharmaceuticals contain halogen, and the halogen is responsible for the physical and chemical properties as well as bioactivities and toxicities. In the exploration of marine environment that is supported by advanced structure elucidation, varied panel bioassays and high-throughput screening have accelerated number of halometabolites isolated from marine Actinobacteria to date. The metabolites exhibited unique structures and promising bioactivities. This review focuses on the chemodiversity and bioactivities of marine halometabolites from marine Actinobacteria reported in the last 15 years (2003–2018).


Introduction
Halometabolites are a group of compounds contain halogen substituents (F, Cl, Br, I). To date, there are more than 5000 halogenated compounds with high degree of structural variability containing a single or several halogen atoms [1]. Halogen elements are found in several forms in nature. Chloride, iodine, and bromide salts are present in the oceans, while the Earth's crust is rich in fluorine. Natural organohalogens (or halometabolites) are produced from two sources: abiogenic and biogenic. Natural abiogenic organohalogen is formed during geothermal processes such as volcano, hot springs, or earthquake. Biomass burning and soil chemistry have also contributed to the enormous number of abiogenic halometabolites [2]. Biogenic halometabolites are produced by bacteria, fungi, plants, marine invertebrates, and macroalgae [1][2][3][4][5][6][7][8].
Halometabolites in nature have several functions in physiological, biochemical, or defensive role for their host including communication (quorum sensing) and production of growth hormones, sex pheromone, toxin, or antibiotics. The role of substituent halogen in organic compounds is related to the bioactivity, bioavailability, and stability of the compounds.
Chlorinated antibiotics were discovered from the exploration of soil Actinobacteria since the discovery of streptomycin from Streptomyces griseus. A number of drugs derived from Actinobacteria such as antibiotic and anticancer are on the market today. Chlorinated antibiotics such as chloramphenicol and vancomycin played important roles for the eradication of infectious diseases in human. Chloramphenicol is a broad-spectrum antibiotic used to treat bacterial infections. Chloramphenicol antibiotic is on the WHO (World Health Organization) list of essential medicine. Chlortetracycline is a member of the tetracycline family and produced by Streptomyces aureofaciens. Chlortetracycline was used clinically in 1948 and is used to prevent, control, and treat animal health problems and increase growth rate in chickens, turkeys, ducks, swine, calves, beef cattle, and anticancer produced by Micromonospora echinospora. Linking calicheamicin to monoclonal antibody is used for therapy of acute myeloid leukemia. Rebeccamycin is produced by Streptomyces sp. and is a derivate of staurosporine with chlor attached to it. Rebeccamycin showed in vitro antitumor activity at Inhibitory Concentration (IC50) 480 nM against P388 leukemia cells. Complestatin is a cyclic halogenated peptide produced by Streptomyces lavendulae anti and exhibited activity as HIV-1 integrase inhibitor. Vancomycin is a halogenated glycopeptide active against Staphylococcus aureus (including methicillin-resistant strains), S. epidermidis (including multiple-resistant strains), Streptococcus pneumoniae (including multiple-resistant strains), S. pyogenes, S. agalactiae, S. bovis, S. mutans, Clostridium spp., Listeria monocytogenes, Actinomyces spp., and Lactobacillus spp. [9][10][11].

Research Methodology
In the studies on bioactive marine natural products, we look for the potential of marine Actinobacteria as halometabolites producers. This comprehensive review illustrates the chemistry and biological activities of halometabolites produced by marine Actinobacteria reported in the last 15 years (2003-2018). Mining and searching for data of compounds and bioactivities were obtained from reports in the database MarinLit, Google Scholar, ScienceDirect, Dictionary of Marine Natural Products, and Marine Natural Product Review. Herein, we grouped the halometabolites based on class of compounds.

Halometabolites Isolated from Marine Actinobacteria
The marine environment is home for wide diversity of organisms and sources of structurally diverse secondary metabolites and drug leads. Halometabolites were produced mainly by marine organisms because seawater contained ion chloride and bromine. Marine organisms have the capability to oxidize bromide more easily than chlorine in the biosynthesis of organic compounds, thus bromometabolites are higher than chlorometabolites as observed in sponge and red algae [4,5,7].
The phylum Actinobacteria is Gram-positive bacteria with high G-C content in DNA. Terrestrial Actinobacteria has been explored for decades as sources of pharmacologically active compounds, and more than 70% of antibiotics used today are derived from Actinobacteria. Other bioactive compounds such as anticancer, antifungal, anthelminthic, antidiabetic, etc., were discovered from terrestrial Actinobacteria as well. Figure 1 shows diverse commercial halometabolites isolated from terrestrial Actinobacteria to prove Actinobacteria play important roles for biomedicine and biotechnology applications [9,12]. Marine environment is different from terrestrial so that marine Actinobacteria have special characteristic and adapted to stress in marine environment. As a result, marine Actinobacteria produce new type of secondary metabolites that differs from terrestrial one. Marine Actinobacteria can be found in any part of the ocean such as water column, sediment, deep sea, and in association with seaweed, sponges, and marine organisms [13][14][15]. Marine Actinobacteria have been explored and yielded structurally unique secondary metabolites with varied biological activities [15][16][17]. Searching for bioactive halometabolites was focused on many members of this genera such as Streptomycetes, Actinoplanes, Nocardia, and other rare Actinobacteria [18][19][20]. One study showed that Marine environment is different from terrestrial so that marine Actinobacteria have special characteristic and adapted to stress in marine environment. As a result, marine Actinobacteria produce new type of secondary metabolites that differs from terrestrial one. Marine Actinobacteria can be found in any part of the ocean such as water column, sediment, deep sea, and in association with seaweed, sponges, and marine organisms [13][14][15]. Marine Actinobacteria have been explored and yielded structurally unique secondary metabolites with varied biological activities [15][16][17]. Searching for bioactive halometabolites was focused on many members of this genera such as Streptomycetes, Actinoplanes, Nocardia, and other rare Actinobacteria [18][19][20]. One study showed that marine Actinomycetes was a major producer of antibacterial compounds compared to Bacilli and Gammaproteobacteria [21].

Lynamicins
Marine Actinomycete NPS 12745 (Marinispora sp.) was isolated from marine sediment of Mission Bay, San Diego, coast yielded bisindole pyrrole compounds named lynamicins A-E ( Figure 2). The series of compounds were tested against series of panel test bacteria that were resistant and sensitive to antibiotics. However, lynamicin E exhibited broad-spectrum activity and potency for treatment of nosocomial infection at Minimum Inhibition Concentration (MIC) 1.8-36 µg/mL [22].

Marinopyrroles
An obligate marine Actinomycetes CNQ-418 related to Streptomyces was isolated from 51 m sediment of La Jolla, California, produced two unique halogenated metabolites with uncommon 1,3 -bipyrrole pharmacophore called marinopyrrole A and B ( Figure 2). The compounds were active against methicillin-resistant Staphylococcus aureus at MIC 90 0.61 and 1.1 µM for marinopyrrole A and B, respectively. The IC 50 against HCT-16 (human colon cancer cell line) for marinopyrrole A was 8.8 µM and marinopyrrole B was 9.0 µM [23]. Further examination of broth fermentation of Streptomyces strain CNQ-418 yielded marinopyrroles A-F. The compounds showed variation of substitution chlorine and bromine. Marinopyrrole A-C had significant activity against MRSA (methicillin-resistant Staphylococcus aureus) with MIC 90 at less than 1 µg/mL [24]. Marinopyrrole A showed activity against S. aureus strains with MIC 0.188-1.5 µg/mL. This activity was better than available antibiotics vancomycin and linezolid. In addition, marinopyrrole A was active against H. influenzae at MIC 2 µg/mL. The toxicity against mammalian cell line was more than 20 times of the MIC value [25]. Marinopyrrole A is reported to be an antagonist of Myleoid Leukemia (Mcl-1), a member of the anti-apoptotic B-cell Lymphoma-2 (Bcl-2) family, which is a well-validated drug target for cancer treatment. The cell-based assay shows a high selectivity of marinopyrrole A. Treatment with marinopyrrole A inhibits the viability of K562 cells transfected with Mcl-1 gene with Effective concentration (EC 50 ) value of 1.6 µM. The selectivity is more than 40-fold greater over the cells transfected with Bcl-XL gene. Moreover, marinopyrrole A can decrease Mcl-1 expression by increasing the cleavage of caspase-3 and Poly (ADP-ribose) polymerase (PARP). Marinopyrrole A is also reported to completely restore the sensitivity of multidrug-resistant leukemia cells to ABT-737 [26].

Lodopyridone
Lodopyridone ( Figure 2) is a unique alkaloid isolated from an obligate marine Saccharomonospora CNQ-490 collected from sediment in La Jolla Submarine Canyon, California. The compound has interesting carbon skeleton properties with ethanolamine, thiomethyl with substitution of pyridine, thiazole, and chloroquinoline. The bioactivity was modest against cancer cell line HCT-116 at IC 50 3.6 µM, but there was no activity against MRSA [27].

Indimicin
Deep sea Streptomyces sp. SCS10 03032 from South China produced chlorinated bisindole alkaloid indimicin A-E ( Figure 2) along with lynamicin F and G. The antibacterial activity was tested against E. coli ATCC 25922, S. aureus ATCC 29213, B. thuringiensis SCSIO BT01, B. subtilis SCSIO BS01, and C. albicans ATCC10231 with MIC > 128 µg/mL and was considered as inactive. The cytotoxicity was tested against cancer cell line, and only indimicin B was active against MCF-7 cell line at IC 50 10 µM [31].

Nitropyrrolins
Marine Actinomycetes strain CNQ-509 isolated from marine sediment of La Jolla, California, produced a new set of nitrophyrrolins ( Figure 3). The compounds were hybrid isoprenoid composed of linear sesquiterpenoid and α farnesyl nitropyrrole. Two compounds had chlorin moieties Nitropyrolins C and E. The compounds showed no activity against MRSA and cancer cell line HCT-116 [34].

Merochlorins
Bioassay-guided fractionation has led to discover novel meroterpen merochlorins (Figure 3) from marine Streptomyces CNH-189 from coastal sediment in California. The compounds displayed unrelated skeleton to available antibacterial agents [35]. Merochlorin A was active against Gram-positive bacteria but inactive against Gram-negative bacteria and showed no cross-resistance to Gram-positive bacteria. Merochlorins A was active against MRSA, MSSA (methicillin-sensitive Staphylococcus aureus), VSSA (vancomycin-sensitive Staphylococcus aureus), and VRSA (vancomycin-resistance Staphylococcus aureus) at concentration 2-4 µg/mL, and merochlorin A was active against Clostridium difficile [36].

Piperazimycins
Cyclic chlorinated hexadepsipeptide piperazimycin A-C ( Figure 4) were isolated and purified from an ethyl acetate extract of culture fermentation of Streptomyces sp. isolated from Guam. The compounds were assayed for bioactivity against human colon carcinoma and 60 cancer cell lines. Each compound exhibited significant cytotoxicity with an average GI (Growth Inhibition)50 76 ng/mL against HCT-16 (human colon carcinoma). Piperazimycin A was the most potent and 3 times more active against solid tumor compared to other piperazimycins [43].

Totopotensamides
Streptomyces sp. 1053 U.I. Ia.Ib cultivated from gastropod Lienardia totopotens collected near Mactan Island, Cebu, Philippines, produced hybrid peptide-polyketide glycoside totopotensamide A and B (Figure 4). The compounds had interesting features but showed no activity in wide-range bioassay including DRG panel assay for neurological activity [45].

Piperazimycins
Cyclic chlorinated hexadepsipeptide piperazimycin A-C ( Figure 4) were isolated and purified from an ethyl acetate extract of culture fermentation of Streptomyces sp. isolated from Guam. The compounds were assayed for bioactivity against human colon carcinoma and 60 cancer cell lines. Each compound exhibited significant cytotoxicity with an average GI (Growth Inhibition) 50 76 ng/mL against HCT-16 (human colon carcinoma). Piperazimycin A was the most potent and 3 times more active against solid tumor compared to other piperazimycins [43].

Totopotensamides
Streptomyces sp. 1053 U.I. Ia.Ib cultivated from gastropod Lienardia totopotens collected near Mactan Island, Cebu, Philippines, produced hybrid peptide-polyketide glycoside totopotensamide A and B (Figure 4). The compounds had interesting features but showed no activity in wide-range bioassay including DRG panel assay for neurological activity [45].

Salinosporamides
Salinosporamide A ( Figure 5) was discovered from culture broth of Salinispora tropica CNB. The compound has unique and unusual structure consists of fused γ-lactam β-lactone ring structure.

Salinosporamides
Salinosporamide A ( Figure 5) was discovered from culture broth of Salinispora tropica CNB. The compound has unique and unusual structure consists of fused γ-lactam β-lactone ring structure. Salinosporamide A and B inhibited selectively the proteolytic activity of the 20S subunit of the proteasome. Both compounds also inhibited human colon carcinoma HCT-116 but had no activity against antibiotic-resistant strain Staphylococcus aureus, Enterococcus faecium, Candida albicans, and herpes simplex virus. Salinosporamide A inhibited proteasomal chymotrypsin-like proteolytic at IC 50 1.3 nM. Cytotoxicity of salinosporamide A was observed against HCT-116 at IC 50 11 ng/mL. The strong potency was examined against NCI-H226 (non-small cell lung cancer), SF-539 (CNS cancer), SK-MEL-28 (melanoma), and MDA-MB-435 (breast cancer) LC 50 less than 10 nM. Salinosporamide A shares structure similarity to omuralide A but is more potent than omuralide A. This is due to methylation at C-3, chloroethyl group at C-2, and cyclohexene at C-5. B-lactone moiety is the key for bioactivity. Mechanism of action of salinosporamide A induces apoptosis, suppresses osteoclastogenesis, and inhibits invasion through down-modulation of NF-kB regulated gene products [46,47].

Marmycins
Marmycins, an angucycline class of compounds, were isolated from marine Actinomycetes belonging to Streptomyces CNH-990. The compounds have no significant activity as antibiotic against MRSA and VREF and antifungal against ARCA. Marmycin A ( Figure 5) showed activity against HCT-16 at IC 50 60.5 nM, but the chlorinated analog (marmycin B) was less potent at IC 50 1.09 µM [52]. This fact is in opposite that halogenation usually responsible for bioactivity and enhance the bioactivity.

Fijiolides
Marine-derived Actinobacteria genus Nocardiopsis isolated from sediment near Beqa Island in Beqa Lagoon, Fiji, produce Fijiolide A and B ( Figure 5). Fijiolide A enhanced the activity of quinone reductase 1 (QR1), an enzyme that converts quinone to hydroquinone at concentration 28.4 µM. In addition, Fijiolide A reduced TNF-α-induced NF-kB activation to 70.3% and IC 50 0.57 µM. In contrast, Fijiolide B did not exhibit activities suggesting that substitution on the nitrogen atom affects activity [53]. Arctic marine Actinomycetes identified as Streptomyces strain ART 5 was isolated from the arctic region, eastern Siberia during the RV Araon Arctic Expedition (ARA 03B). Profiling chemistry of fermentation broth yielded identification of fijiolide A and B along with articoside, C-1027-chromophore V, and C-1027-chromophore-III. The compounds were tested for bioactivity against Candida albicans and antiproliferative activities against human carcinoma cell lines. C-1027-chromophore V and C-1027-chromophore-III showed bioactivity against C. albicans at IC 50 37.9 µM and 25.6 µM, respectively, but fijiolides showed no activity. The difference in the benzoxazine that counts for the activity.  [53]. Arctic marine Actinomycetes identified as Streptomyces strain ART 5 was isolated from the arctic region, eastern Siberia during the RV Araon Arctic Expedition (ARA 03B). Profiling chemistry of fermentation broth yielded identification of fijiolide A and B along with articoside, C-1027chromophore V, and C-1027-chromophore-III. The compounds were tested for bioactivity against Candida albicans and antiproliferative activities against human carcinoma cell lines. C-1027chromophore V and C-1027-chromophore-III showed bioactivity against C. albicans at IC50 37.9 μM and 25.6 μM, respectively, but fijiolides showed no activity. The difference in the benzoxazine that counts for the activity. Antiproliferative activities of compounds were ranged from moderate to strong against cancer cell lines HCT-116, A549, SNU638, SK-HEP1, K562, and MDA-MB231 at IC50 0.6-44 μM [54].

Streptochloride
Chlorinated polyketide compound streptochlorides ( Figure 5) were isolated from ethyl acetate extract of fermentation broth of Streptomyces sp. OUCMDZ-1703 associated with unidentified soft coral. Both compounds have modest antimicrobial activity against P. aeruginosa, E. coli, and S. aureus but no activity against MRSA. Streptochloride A and B demonstrated cytotoxicity against MCF-7 cell line at IC50 9.9 and 20.2 μmol/L, respectively [55].

Future Direction and Conclusion
Marine Actinobacteria have shown as producer of varied diversity of halometabolites compounds. The compounds range from simple to complicated structures with group under polyketides, peptides, alkaloid, and terpenoid. Some compounds demonstrate intriguing structure special for marine compounds. The compounds exhibited enormous potential for the discovery of new therapeutic leads in the development of drugs to fight the current antibiotic resistance threats,

Future Direction and Conclusions
Marine Actinobacteria have shown as producer of varied diversity of halometabolites compounds. The compounds range from simple to complicated structures with group under polyketides, peptides, alkaloid, and terpenoid. Some compounds demonstrate intriguing structure special for marine compounds. The compounds exhibited enormous potential for the discovery of new therapeutic leads in the development of drugs to fight the current antibiotic resistance threats, anticancer, and other bioactivities. Marine Actinobacteria produce more chlorometabolites than bromometabolites in contrast with sponges and red algae which are rich in bromometabolites.
To date, there are several bioprospecting programs with target marine biodiversity for novel bioactive metabolites including halometabolites. FADH 2 -dependent halogenase is the biggest group of halogenating enzymes, thus can be used as target in the bioprospecting of halometabolites from marine Actinobacteria. Genome mining by employing gene that encodes FADH 2 -dependent halogenase as an indicator has enabled to screen 555 genetic potentials of actinomycetes for halogenated natural products [56]. Gao and Huang employed the same approach to screen 228 Actinomycetes to find distribution of the gene and secondary metabolites [57]. Screening mangrove-derived Actinomycetes using FADH 2 -dependent halogenase resulted in 26 halogenase-positive strain among 163 isolates [58]. PCR-based marker gene screening was employed to detect FADH 2 -dependent halogenase gene of Arctic marine Actinobacteria. The study concluded that Arctic marine Actinobacteria are potential in halometabolites production [59]. Three novel halogenase gene clusters were identified in microbial metagenome of marine sponge indicated that the microbial consortia of sponges including marine Actinobacteria are a valuable resource for novel halogenation [60]. There is a correlation between the distribution of FADH 2 -dependent halogenase gene in filamentous actinomycetes and the potential for producing halometabolites.
Comparative genome studies showed that Actinobacteria are rich in secondary metabolites genes that never been explored, so the chance to discover new bioactive halometabolites is still wide open. Further research into mechanisms of biological halogenation will provide insight and a greater understanding of biosynthesis of halometabolites. Furthermore, understanding the genes encoding halogenase enzymes may be used to generate recombinant organisms to produce derivative new natural product. Advance technology in exploration and collection, compound isolation, purification, structure elucidation, bioassay, and high-throughput screening will ensure and enable to identify potential halometabolites from marine Actinobacteria for benefit to humankind.