**4. Marine possibilities for natural protein folding modulators**

Natural products from the land include promising as sources of novel protein folding modulators for the reasons outlined earlier. By extension, marine sources may hold propor‐ tionally greater opportunity for discovery than their terrestrial counterparts because, until this decade, much of the ocean biota was unexplored. The Census of Marine Life uncovered remarkable and previously unknown plant and animal biodiversity in the oceans (reviewed in [102]). In the Gulf of Maine alone, the current species count identifies at least 652 fish species, 184 species of birds, 733 different species of microscopic plants and algae and 32 mammalian species [103]. Microbial diversity is also extensive worldwide, as inferred from the large number of new protein families uncovered from marine microbes [104].

Marine resources offer largely untapped diversity that may offer new options for natural product development [105]. Only six marine natural products and 14 synthetic compounds based upon the structures of natural marine products are FDA-approved agents or in clinical trial (reviewed in [106]). However, this list is likely to grow. In 2010, 895 new citations were reported on marine-derived compounds [107]. Marine species are already being examined for molecules that inhibit β-secretase 1, an enzyme that processes the amyloid precursor protein to give Aβ and other fragments [108]. Nonetheless, the development of inhibitors has been very difficult elsewhere [108] and there may be other biological consequences to the modula‐ tion of this enzyme. Therefore, it is ideal to focus more specifically on protein misfolding, which appears to be the linchpin in most of the neurodegenerative diseases. There is a wide variety of challenging and variable marine environments that would naturally select for traits that include protein folding modulation due to temperature or pressure, chemical defense mole‐ cules and other products that may be undersampled or undiscovered because of accessibility. Marine plants and sessile marine animals may produce chemical defenses that are remarkable in comparison to those of species that can move away from danger. In addition, species that inhabit highly variable or extreme environments, such as freezing waters, have adaptations to maintain proteostasis during exposure to extreme temperatures and solution properties [109-111]. For these reasons, they may represent ideal sources of natural molecules to prevent or ameliorate protein misfolding.

The discovery, characterization and production of marine-derived molecules appears prom‐ ising, but with complexity and risk, nonetheless. A general overview of the process is shown in Fig. 2.

#### **4.1. Natural protein folding modulators from marine sources**

#### *4.1.1. Marine polyphenols*

Polyphenols are abundant and varied in marine algal species (reviewed in [112]). Like their terrestrial counterparts, algal polyphenols have shown folding modulation of Aβ. The brown seaweed (macroalgae) *Ecklonia cava* is found in the waters around Japan and Korea, where it is used as a herbal remedy. A butanol extract from *E. cava* has been shown to prevent produc‐ tion and aggregation of Aβ and to reduce amyloid plaques [113]. Electron microscopy showed the Aβ oligomers to be reduced and dye binding as well, indicating an inhibition of fibril formation. The polyphenolic phlorotannins are considered to be the active compounds responsible for the biological activities of *E. cava* [114, 115]. Nonetheless, other molecules cannot be strictly ruled out [113].

**Figure 2.** Schematic summary of key steps in the identification and development of marine-sourced protein folding modulators. Blue boxes are steps involved in discovery and characterization of protein folding modulation and the molecule responsible. Green boxes are steps involved in production of the molecule (or of an extract containing it). For more detail, see chapter text. Structural elucidation is not included here as it is not covered in this chapter; it is covered in another chapter of this book.

#### *4.1.2. Marine carotenoids*

**4. Marine possibilities for natural protein folding modulators**

106 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

number of new protein families uncovered from marine microbes [104].

or ameliorate protein misfolding.

*4.1.1. Marine polyphenols*

in Fig. 2.

Natural products from the land include promising as sources of novel protein folding modulators for the reasons outlined earlier. By extension, marine sources may hold propor‐ tionally greater opportunity for discovery than their terrestrial counterparts because, until this decade, much of the ocean biota was unexplored. The Census of Marine Life uncovered remarkable and previously unknown plant and animal biodiversity in the oceans (reviewed in [102]). In the Gulf of Maine alone, the current species count identifies at least 652 fish species, 184 species of birds, 733 different species of microscopic plants and algae and 32 mammalian species [103]. Microbial diversity is also extensive worldwide, as inferred from the large

Marine resources offer largely untapped diversity that may offer new options for natural product development [105]. Only six marine natural products and 14 synthetic compounds based upon the structures of natural marine products are FDA-approved agents or in clinical trial (reviewed in [106]). However, this list is likely to grow. In 2010, 895 new citations were reported on marine-derived compounds [107]. Marine species are already being examined for molecules that inhibit β-secretase 1, an enzyme that processes the amyloid precursor protein to give Aβ and other fragments [108]. Nonetheless, the development of inhibitors has been very difficult elsewhere [108] and there may be other biological consequences to the modula‐ tion of this enzyme. Therefore, it is ideal to focus more specifically on protein misfolding, which appears to be the linchpin in most of the neurodegenerative diseases. There is a wide variety of challenging and variable marine environments that would naturally select for traits that include protein folding modulation due to temperature or pressure, chemical defense mole‐ cules and other products that may be undersampled or undiscovered because of accessibility. Marine plants and sessile marine animals may produce chemical defenses that are remarkable in comparison to those of species that can move away from danger. In addition, species that inhabit highly variable or extreme environments, such as freezing waters, have adaptations to maintain proteostasis during exposure to extreme temperatures and solution properties [109-111]. For these reasons, they may represent ideal sources of natural molecules to prevent

The discovery, characterization and production of marine-derived molecules appears prom‐ ising, but with complexity and risk, nonetheless. A general overview of the process is shown

Polyphenols are abundant and varied in marine algal species (reviewed in [112]). Like their terrestrial counterparts, algal polyphenols have shown folding modulation of Aβ. The brown seaweed (macroalgae) *Ecklonia cava* is found in the waters around Japan and Korea, where it is used as a herbal remedy. A butanol extract from *E. cava* has been shown to prevent produc‐ tion and aggregation of Aβ and to reduce amyloid plaques [113]. Electron microscopy showed

**4.1. Natural protein folding modulators from marine sources**

Marine algae and invertebrates harbour large quantities of carotenoids. This is of interest be‐ cause, as noted above, carotenoids are among the molecules showing promise in modula‐ tion of protein misfolding. Algal carotenoids are widely varied (reviewed in [112]). These appear to be particularly accessible through enzyme-assisted extraction (reviewed in [116]) and they may have interesting activities. Shrimp and crab processing wastes are also excel‐ lent sources of carotenoids. Crustacean shells left over during processing contain the carote‐ noid astaxanthin (reviewed in [112]), which gives them their striking colour. This is in addition to the chitin in the shells, and its component glucosamine, which is well known in other contexts. There are no reports of evaluation of marine-derived carotenoids in protein folding modulation, as there are for terrestrial counterparts. With the numerous and varied sources of marine carotenoids, these could be compared with terrestrial versions previously reported to modulate protein folding.

#### *4.1.3. Marine toxins*

There is a plethora of marine toxins with distinctive neurological effects. These toxins are mainly known for the poisoning risk they carry. Nonetheless, a few of them have valuable biological activities as research chemicals and one has intriguing effects relative to AD. Using cell-based assays, the toxin 13-desmethyl spirolide C from the dinoflagellate *Alexandrium ostenfeldii* was shown to reduce Aβ accumulation in cells by just over 40% [117]. The antibody used (6E10, Covance) detects Aβ without distinguishing between monomeric or oligomeric or fibril forms. It would therefore be valuable to repeat these analyses using fold-specific anti-Aβ antibodies or another fold-sensitive method to see if any effect there could be detected. Otherwise, it may act on Aβ in other beneficial ways that would also be of interest.

#### *4.1.4. Marine-sourced chemical chaperones and conflicting results*

A variety of chemical chaperones, which are loosely defined as small molecules that promote folding of many proteins, have gained interest in terms of wide-spectrum protection of proteins from misfolding (reviewed in [118]). In marine species faced with protein misfolding risk, the accumulation of one or more chemical chaperones is a common adaptation because these small chaperoning molecules promote general proteostasis [111, 119]. Therefore, at first glance, these molecules would appear to be ideal in terms of proteins such as Aβ and αS. They could be produced naturally or synthetically, depending upon their molecular features, and they may stabilize many different proteins. However, some chemical chaperones may have problematic effects with respect to amyloidotic protein misfolding. Glycerol at supraphysiological concen‐ trations (molar range) and trimethylamine oxide (TMAO) at moderate concentrations were both shown to favour the transition of Aβ from its unfolded conformation to the β-sheet form requisite for fiber formation [120]. Protofibril to fibril conversion was also enhanced [120]. The situation appears more complex for αS, with elevated concentrations (molar range) of TMAO favouring a partially folded form with high propensity for fibril formation and even higher concentrations of TMAO favouring an oligomeric α-helical conformation [121], which may be consistent with a native α-helical form of the protein that resists misfolding [29]. Although these chaperone concentrations used were far in excess of those that would be reached *in vivo*, the effects suggest a possibility to be aware of if a novel folding modulator appears to be non-specific. Stabilization by chemical chaperones may bring an increased risk of aggregation for some proteins [119]. Furthermore, stabilization of a wider range of cell proteins or protein complexes may cause unanticipated problems. Therefore, examination of unusual and offtarget effects would be prudent in the evaluation of chemical chaperones.

#### **4.2. Sources for marine products**

A challenge presented by many rare or remote marine species that may produce pharmaceut‐ ical or nutraceutical molecules is the difficulty in obtaining sufficient product in a sustainable manner, from both the environmental and economic standpoints [122]. Options for these products include the development of culture methods for the species or synthesis of the molecule, although in many cases the molecules are too complex to be efficiently synthesized [122, 123]. In contrast, an advantage for nutraceutical or drug development from many accessible marine species, both plant and animal, is that they are already supplied as human food sources. Additionally, a large number of marine species are currently farmed, and availability of a standard supply for natural product development would be more straight‐ forward and predictable than it would be for their wild counterparts. Sustainable production or harvesting of species for natural marine products would be a key consideration in produc‐ tion of folding modulators, just as it is for the production of foods.

In the case of some seaweeds, marine harvesting can be carried out. However, on-land cultivation offers the additional benefits of ideal traceability and process control, albeit with substantial costs [124]. For microalgae, this would be the only option. Algal processing wastes are significant and they are of interest for the development of by-products [125].

For commercially fished animal species, processing for food production is a frequent practice. These wild finfish and shellfish species, as well as those grown in aquaculture, can generate substantial waste during processing for food production and this waste often go to landfills or composting. The identification of valuable properties or activities in waste materials allows them to be used to produce co-products and, with green chemistry initiatives, these could be produced in a sustainable fashion from a healthy wild harvesting or aquaculture operation. There is growing interest in co-product development, as highlighted by two contrasting examples. Snow crab (*Chionoecetes opilio*) are fished across the Maritime provinces of Canada. Crab processing waste currently contributes to landfills, but the potential for additional product development with the identification of sufficiently valuable uses is being considered [126] as is the case elsewhere, likely based upon chitin but possibly upon other products as well. Innovative solutions to waste challenges are also being pursued in salmon aquaculture. Integrated multi-trophic aquaculture of Atlantic salmon (*Salmo salar*) with macroalgal species and mussels (*Mytilus edulis*) in the Bay of Fundy, Canada, minimizes waste accumulation by allowing nutrient and resource recovery with diminished impact on the local environment and this process is advancing toward commercial production [127]. The economic viability of this integration initiative will rely upon the development of foods along with other high-value products from the cultured species [128]. A neuroprotective protein folding modulator from such a resource could enhance markets for the food, while offering the possibility of producing sustainable nutraceuticals and eventual pharmaceuticals as co-products if appropriate.
