The Pharmacological Potential of Non-ribosomal Peptides from Marine Sponge and Tunicates

Marine biodiversity is recognized by a wide and unique array of fascinating structures. The complex associations of marine microorganisms, especially with sponges, bryozoans, and tunicates, make it extremely difficult to define the biosynthetic source of marine natural products or to deduce their ecological significance. Marine sponges and tunicates are important source of novel compounds for drug discovery and development. Majority of these compounds are nitrogen containing and belong to non-ribosomal peptide (NRPs) or mixed polyketide–NRP natural products. Several of these peptides are currently under trial for developing new drugs against various disease areas, including inflammatory, cancer, neurodegenerative disorders, and infectious disease. This review features pharmacologically active NRPs from marine sponge and tunicates based on their biological activities.


INTRODUCTION
Nature provides a wide and structurally diverse array of active biomolecules that have proved vital for the development of novel pharmaceuticals. The marine world, covering more than 70% of the Earth's surface, is the home of tremendous biodiversity. Due to very diverse oceanic environments, marine organisms have developed the capacity to produce unique compounds (Steele, 1985;Mehbub et al., 2014). This rich and unprecedented chemo diversity of marine natural products provides an unlimited resource of novel biomolecules in the field of drug development. The importance of marine metabolites in current drug research is driven by the fact, that during 1981-2002, around half of US FDA-approved drugs consisted of either marine metabolites or their synthetic analogs (Vinothkumar and Parameswaran, 2013). Interestingly, the majority of these natural products involved in clinical or preclinical trials are produced by invertebrates, that is, sponges, tunicates, bryozoans, or molluscs. Sixty per cent of these natural products belong to non-ribosomal peptide (NRP) families, which are biosynthesized by poly-functional megasynthases called NRP synthetases (NRPSs) (Finking and Marahiel, 2004;Mehbub et al., 2014). The excellent binding properties, low off-target toxicity, and high stability of NRPs make them a promising molecule for development of new therapeutics. Currently, only a handful of NRPs are used as drug (Table 1).
Marine sponges (Phylum porifera) represent the most primitive multicellular animals, with origins dating back to the Precambrian era (Hentschel et al., 2002). There are about 9000 reported species of sponges and (perhaps twice as many unreported species) available in the ocean (Brusca et al., 1990;Wörheide et al., 2005). These have been broadly categorized in 3 classes : Calcarea (5 orders and 24 families), Demospongiae (15 orders and 92 families), and Hexactinellida (6 orders and 20 families). Till date, more than 5300 different natural products have been isolated from marine sponges, and each year more than 200 additional new metabolites are being discovered (Laport et al., 2009;Mehbub et al., 2014). There are several sponge derived metabolites currently available in market and many in clinical studies ( Table 2).
It is proposed that some of the bioactive compounds isolated from sponges are produced by functional enzyme clusters originated from the sponges and their associated microorganisms (Laport et al., 2009;Thomas et al., 2010). It has been observed that bacterial phyla such as Proteobacteria, Nitrospira, Cyanobacteria, Bacteriodetes, Actinobacteria, Chloroflexi, Planctomycetes, Acidobacteria, Poribacteria, and Verrucomicrobia besides members of the domain Archaea are most sponge-associated bacterial community (Hentschel Umezawa et al., 1966 Daptomycin Lipopeptide Streptomyces roseosporus Antibiotic/disrupts the cell membrane Miao et al., 2005 Cyclosporine A Cyclic peptide Tolypocladium inflatum Immunosuppressant /lower the activity of T cells Murthy et al., 1999 Actinomycin D Polypeptide Streptomyces sp. Antitumor/inhibit transcription Waksman and Woodruff, 1940 Romidepsin Depsipeptide Chromobacterium violaceum Antitumor/Histone deacetylase inhibitor Ueda et al., 1994 TABLE 2 | Sponge secondary metabolites that are FDA-approved agents in clinical trial (Mayer et al., 2010;Newman and Cragg, 2016 Olson and McCarthy, 2005). However, fungi and microalgae also symbiotically inhabit sponges. It has been recognized that one host sponge can possess diverse symbionts. For example, unicellular heterotrophic bacteria, unicellular cyanobacteria, and filamentous heterotrophic bacteria all grow together in sponge Theonella swinhoei (Bewley et al., 1996). Likewise, a sponge belonging to Aplysina includes heterogeneous bacteria Bacillus sp., Micrococcus sp., Arthrobacter sp., Vibrio sp., Pseudoalteromonas sp., and so on (Hentschel et al., 2001). Sponge Rhopaloeides odorabile has β-Proteobacteria, γ -Proteobacteria, Cytophaga, Actinobacteria, and green sulfur bacteria (Webster et al., 2001). Besides this, species-specific symbiotic relationship has also been observed. For example, sponge T. swinhoei and δ-proteobacteria have shown a specific association with each other (Schmidt et al., 2000). A species of α-proteobacteria dominates in sponge R. odorabile over various habitats but is not detected from seawater, which strongly suggests that the symbiont is species specific (Lee Y. K. et al., 2001). On the other hand, one symbiont occurs commonly in various sponges from different regions indicating its wide host range (Wilkinson et al., 1981). For example, cyanobacteria Aphanocapsa sp., Phormidium sp., or Oscillatoria spongeliae are found in numerous sponges (Wilkinson, 1978). Symbiotic associations between sponges and marine microorganisms might be involved in nutrient acquisition, stabilization of sponge skeleton, processing of metabolic waste, and secondary metabolite synthesis. It is assumed that symbiotic marine microorganisms harbored by sponges are the original producers of some of these bioactive compounds (Newman and Hill, 2006). For example, antibiotic polybrominated biphenyl ether isolated from the sponge Dysidea herbacea (Demospongiae) are actually produced by endosymbiotic cyanobacterium O. spongeliae (Unson et al., 1994). A symbiotic bacterium Micrococcus sp. produces diketopiperazines previously ascribed to the host sponge Tedania ignis (Stierle et al., 1988). Another symbiotic bacterium Vibrio sp. produces brominated biphenyl ethers formerly attributed to the host sponge Dysidea sp. (Elyakov et al., 1991). Symbiotic bacterium Vibrio sp. produces an anti-Bacillus peptide andrimid that was found in the sponge Hyatella sp. extract (Oclarit et al., 1994). Antimicrobial activity is detected in Micrococcus luteus isolated from the sponge Xestospongia sp. (Bultel-Poncé et al., 1998). Antimicrobial compounds such as quinolones and phosphatidyl glyceride are isolated from a Pseudomonas sp. collected at the surface of the sponge Homophymia sp. (Bultel-Poncé et al., 1999). However, the mutual mechanism between sponge and its microbial associate, in metabolite production, is not well-understood. Thus, it is extremely relevant to highlight the therapeutic potential of various secondary metabolites synthesized by the microbial flora inhabiting sponges. This is because they open up the possibility of providing a continuous supply of the biologically active compounds by laboratory cultivation of the producer (Thomas et al., 2010). Tunicates include a wide variety of invertebrates that are classified within the Phylum chordata based on presence of a larval notochord during early development. Tunicates contains about 2150 described species that are divided into 4 classes: Ascidiacea (Aplousoobranchia, Phlebobranchia, Stolidobranchia) Thaliacea (Pyrosomida, Doliolida, Salpida), Appendicularia (Larvacea), and Sorberacea (Ruppert and Fox, 2004). Amongst these, Ascidacea (commonly known as the ascidians) are highly studied due to their biologically active metabolites that serve as antineoplastic agents. Geranyl hydroquinone was first ascidian metabolite isolated from Aplidium sp. which displayed chemo protective activity against some forms of leukemia, rous sarcoma, and mammary carcinoma in test animals (Fenical, 1976) (Menna, 2009). Since then, ascidians are known as the source of numerous marine natural products. The biologically active metabolites originated from tunicates which are approved by FDA or in clinical trials along with their biological properties are given in Table 3.
To date, significant biological activities, such as antimicrobial, anticancer, neurotoxic, antiprotozoal and their associated cellular targets have been reported for several NRPs from the marine sponges and tunicates. These NRPs have unique structures as compared with those from other sources. It is this attribute that makes marine sponge-and tunicate-derived NRPs highly attractive as potential drug and molecular probes. In this review, we survey the discoveries of NRPs derived from marine sponges and tunicates, which have shown in vivo efficacy or potent in vitro activity against various human diseases. Our objective is to highlight NRPs that have the greatest potential to be clinically useful. The details of sponge-and tunicate-derived NRPs along with biological properties is given Table 4.

BIOLOGICAL ASPECTS, CHALLENGES, AND FUTURE PERSPECTIVES
Like their structural diversity, metabolites produced from marine sponges and tunicates bind to a variety of cellular targets to elicit their effects. Numerous articles published in recent years highlighting the significance of these metabolites in disease control, the details of their biological significance from molecular recognition perspective have been rather scarce. Although some promising leads have been obtained, the discovery of their cellular targets, molecular interactions, and adverse effects are lacking. In cases where the therapeutic potential has been reported, details of a proper screening approach to identify nucleic acid or protein targets are missing. However, some established metabolites from these sources (see Tables 1, 2) and their derivatives have been examined extensively and their molecular targets are varied. One of the earliest examples in this class is FDA-approved drug Ara-C (cytarabine), which is known to elicit anticancer properties by inhibiting the functions of DNA polymerase (Furth and Cohen, 1968), which ultimately results in stalling DNA synthesis. Another FDA approved related compound Ara-A(vidarabine), which is known to have antiviral properties (active against herpes simplex and varicella zoster viruses), targets viral DNA polymerase (Chadwick et al., 1978) by functioning as mimic of natural nucleotides. Both Ara-C and Ara-A resemble natural cytidine and adenine nucleosides where the structural differences are in the sugar components of the two (arabinose vs. deoxyribose). The natural nucleoside mimics Ara-A and Ara-C are easily phosphorylated as their triphosphate derivatives by kinases and act as terminators of DNA synthesis. Ara-A is also known to impede 3 ′ -end processing of pre-mRNAs by inhibiting cleavage and polyadenylation (Ghoshal and Jacob, 1991;Rose and Jacob, 1978).
Several other molecules that are either FDA approved or in early stages of clinical trials have been identified as anticancer agents with microtubules as their primary molecular targets. The predominance of natural metabolites being microtubule binding agents has been hypothesized as evolutionary response to predation by plants and animals (Dumontet and Jordan, 2010). Some of these molecules, such as discodermolide, are among the first non-taxane stabilizers of microtubules . The microtubule stabilizers act by enhancing microtubule polymerization at high concentrations. Discodermolide has been known to bind to tubulin dimers in a stoichiometric ratio. Competitive binding experiments have shown that it blocks taxol binding and is a much stronger binder of microtubules than taxol (Kowalski et al., 1997). The microtubule binding of Tau proteins is interfered by discodermolide (Kar et al., 2003). Similarly, laulimalide showed properties very similar to paclitaxel where it helped in enhancing tubulin assembly (Gapud et al., 2004). However, laulimalide modulation of microtubule assembly in C. elegans is dose dependent where it stabilization effects were observed only at concentrations higher than 100 nM (Bajaj and Srayko, 2013).
The antiviral effect of homophymine A has been established by measuring the reverse transcriptase activity in HIV-infected primary peripheral blood mononuclear cells (Zampella et al., 2008). The reverse transcriptase activity is exhibited by 2 classes of molecules: one that directly competes with natural nucleotide triphosphates and the other that either directly blocks the catalytic reactions or by allosteric binding that leads to structural changes in the viral enzyme. Since homophymine A lacks structural features to act as mimics of natural nucleotide triphosphates, it is likely to impede the catalytic activity of the enzyme by direct binding.
A tunicate-derived metabolite trabectedin (ET-743) uses DNA binding to exert its anticancer properties. Trabectin binds to the GC rich regions in the B-DNA where it uses its carbolinamine moiety to form adduct with the exocylic amine (N-2) of guanine (Pommier et al., 1996) and covers 3 base pairs during this process (Marco et al., 2006). Unlike B-DNA minor groove binders, such as Hoechst 33258, which binds snugly along the minor groove curvature with high-affinity (Haq et al., 1997), trabectedin only uses part of its structure to make necessary contacts for the antitumor action (D'Incalci and Galmarini, 2010).
Despite these advances in determining the mode of their binding, a large number of recently discovered metabolites are still not explored to assess it functional capabilities. In the past, well-known anti-retroviral drug zidovudine, which was initially thought to be functionally inert, turned out as excellent therapeutic agent. Such discoveries are possible only when a rational screening design is aimed to asses it full potential as a drug. For example, compounds that have structural regions favorable for protein binding should be screened against all potential protein targets. Similarly, compounds that show preference toward nucleic acid binding should be screened using assays such as competition dialysis that establish a preferential nucleic acid target. Such approaches not only determine the best target for a particular compound but also shed light to its secondary targets, which may be helpful in dealing with toxicity issues. Current target design of marine and tunicate metabolites clearly need to take these approaches.
Some of the metabolites that have weaker binding to a target or have poor bioavailability can be improved by nano-encapsulation techniques. Additionally, DNA binding metabolites can be chemically modified to enhance their affinity using multi-recognition of the target (Willis and Arya, 2010), which has led to remarkable enhancement in the affinity of double, , triple (Arya and Willis, 2003), and four-stranded DNA helical structures (Ranjan et al., 2013).

CONCLUSION
Extreme environment of the ocean plays a vital role in exploring and studying marine bio-resources and their bio-actives. The large biodiversity of the sea serves as a huge resource for developing potential drugs with promising pharmacological activities. The significance of marine-derived secondary metabolites has recently been highlighted by introduction of Prialt and Yondelis to the market. In the past three decades, numerous NRPs with unique chemical structures and varied biological activities have been discovered from marine sponges and tunicates as described in this. Some of these exhibit strong potential to be developed as a new drug. However, none of the NRPs highlighted in this review have been successfully marketed as therapeutics. To translate bioactivity of these important metabolites into therapeutically significant outcomes, it is crucial to further unravel their modes of action and measure their toxicity. Since the majority of these studies have been focused on in vitro bioassays and elucidation of the chemical structures only, a complete examination of their biological target selectivity is required. Nevertheless, large-scale production of these NRPs for clinical use is a real challenge. Therefore, environmentally sound and economically feasible alternatives are required. To counter these challenges, many strategies have been established.
Chemical synthesis of NRPs is among the first strategies to be used. However, the structural complexity limits its chemical synthesis and has resulted in only a few successful achievements (e.g., analgesic drug ziconotide; Olivera, 2000). A second strategy uses screening the pharmacological significance of NRPs and subsequently attempting to define the critical pharmacophore that can result in practical drugs based on a marine prototype via chemical synthesis, degradation, modification, or a combination of these. Aquaculture of the source organisms has also been used to secure a sustainable supply of active compounds. However, in most cases, the biomass currently generated is still far from the requirement from an industrial perspective (Mendola, 2000). Identification and large-scale culturing of true producers that are known to thrive within the tissues of marine invertebrates (sponge or tunicate) is an intriguing strategy. However, to date only 5% or less of the symbiotic microbes present in marine specimens can be cultivated under standard conditions. Consequently, molecular approaches such as transfer of biosynthetic gene clusters to a vector suitable for large-scale fermentation could be used to avoid obstacles in culturing symbiotic bacteria. Enzyme technology and solid-phase peptide synthesis offer particularly promising alternatives to generate variety of unique peptides using native peptide as a template. Besides, combinations of chemical synthesis and biosynthetic technologies have potential to accelerate the discovery of novel drugs derived from sponge and their microbial association in future.