Trabectedin: Of Sea Squirts and the Deep Blue Sea

A recent press release heralded Trabectedin's approval for treatment of patients with unresectable or metastatic liposarcoma or leiomyosarcoma who received a prior anthracycline – containing regimen.¹The prescribing information describes Trabectedin as an alkylating drug that binds guanine residues in the minor groove of DNA, forming adducts and resulting in a bending of the DNA helix towards the major groove.²Technical descriptions abound in the literature, but there is another aspect to Trabectedin  – its discovery.³

At a time when drugs are being designed for specific targets in oncology, Trabectedin is a naturally occurring compound found in a sea squirt called Ecteinascidiaturbinate(E.turbinate).³What’s more, relating its discovery and development invokes the unique evolutionary position of the sea squirts, from its phenomenon of symbiosis and adventures on the high seas to skirmishes with aqua-culture, a period of research stasis for lack of sufficiently powerful analytic equipment, and finally a complex process of synthesis.^3-8

Ecteinascidiaturbinateis a Sea Squirt: What are Sea Squirts?

Sea squirts are assigned to the class ascideacea, found in the phylum Chordata under the sub-phylum Tunicata. Members of the ascideacea are sessile and can be single or live in colonies. Individuals in ascideacea, as well as pelagic appendicularia and thaliacea, are referred to as tunicates because of their outer protective covering (tunic) composed of cellulose, which can be clear or opaque.⁴

E.turbinateis an ascidian and lives in colonies of individuals known as zooids. Each zooid is about 2.0 to 2.5 cm in height and the colonies can be up to 14 cms in diameter. Colonies are interconnected via root-like stolons, which provide them with blood and ensure attachment to the substrate and other zooids. They are found in warm shallow waters in the Caribbean and elsewhere and the adult forms are filter feeders. They have inhalant and exhalant siphons, and food particles drawn in by ciliary beating are trapped on a mucous net secreted by the endostyle. The rims of the siphons are bright orange in color due to the presence of carotenoids, but this can vary between populations, with some being pink or yellow.⁹

So far, with this brief description, one might (wrongly) conclude these seem akin to exotic sea anemones or sponges. What makes these animals so interesting is that the ascidians, appendicularians and some thaliaceans have tadpole-like larvae with a notocord. By 1866, the presence of the notocord and other structural relationships discovered by some of the 18^thand 19^thcentury’s most outstanding biologists (among them Thomas H. Huxley, who became known as Darwin’s bulldog), led to the grouping of the tunicates with the vertebrates, in a new phylum, the Chordata. The relationship has recently been further underpinned by molecular studies which concluded the tunicates are the sister group of the vertebrates, displacing the cephalochordate, which were previously favored in this role on the basis of cladistic analyses. The genomes of several ascidians have now been sequenced, in the hope that an in depth study of ascidian embryos will inform on vertebrate origins.⁴

Sea Squirts and Trabectedin

Studies of sea squirts took a new direction when, in the 1950s and 1960s attention turned to marine sources of new possible pharmaceutical agents. The US National Cancer Institute conducted a 15-year analysis of the pharmacological activity of compounds extracted from marine species About 4% of marine species (majority animal) contained antitumor compounds. They were screened by an in vivo bioassay employing P388 murine leukemia.¹⁰

In a paper published in 1990, Rinehart et al described the structures of the ecteinascidins isolated from E.turbinate, including the most abundant and potent, ecteinascidin 743, (later to become ET-743 or Trabectedin).⁷They explained that earlier experiments performed in 1969 with the extracts of E.turbinate, which demonstrated remarkable anticancer activity and powerful immunomodulatory effects, could not proceed to isolation of the active compounds for want of more powerful techniques. In 1978 during the Alpha Helix Caribbean Expedition scientists were able to show cytotoxicity of E.turbinateon board the research vessel. Reinhart and his team set about analyzing the extracts in 1981, taking advantage of new techniques, and by 1986 had succeeded in isolating six ecteinascidins. They found that ecteinascidins were structurally related to safracins and saframycins which are found in microbes.⁷

But where were the ecteinascidins coming from? The sea squirts themselves apparently do not synthesize it, but it is instead produced by a bacterial symbiont (one of many) living in the sea squirts called Candidatus Endoecteinascidiafrumentensis.³Early attempts to farm the sea squirts were unsuccessful, and the yield was very small. Ten pounds of sea squirts would provide only millionths of an ounce of ET-743.⁸Unfortunately it has also proven extremely difficult to culture the bacteria, and currently ET-743 is manufactured by a semi-synthetic process from cyanosafracin.^3,8

Perhaps one reason for the extreme difficulty in culturing the bacteria is the genomic streamlining that occurs in a symbiont once it has adapted to a host. For example, functions required for independent existence outside the host are lost, and those conferring an advantage to the host, such as the production of defensive toxins, are retained.⁶It has been proposed that chemicals like the ecteinascidins, produced by these symbionts possibly serve a protective function, and natural selection leads to symbiosis with bacteria producing these defensive compounds and hence a higher fitness for survival for the animal.⁵Experiments with fish have shown that another compound called didemnin B isolated from a different species of tunicate, will alter predator-prey relationships. Fish would immediately regurgitate mosquito larvae previously treated with didemnin B and learned to associate species of larvae with “distaste.”¹¹For sessile tunicates vulnerable to predation, the survival value of endogenous toxins becomes very apparent.

Conclusion

Trabectedin is set on its clinical trajectory, and its value and niche(s) will be studied and developed by the medical community to the benefit of patients.¹²But the spotlight will not be leaving the tunicates, any time soon either. It has been proposed that aside from medically relevant avenues, tunicates and the study of their embryos may provide original and pivotal insights on animal evolution.⁴

References

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5. Tianero MD, Kwan JC, Wyche TP, et al. Species specificity of symbiosis and secondary metabolism in ascidians.ISME J. 2015;9:615-28
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7. Rinehart KL, Holt TG, Fregeau NL et al. Ecteinascidins 729, 743, 745, 759A, 759B and 770: Potent Antitumor Agents from the Caribbean TunicateEcteinascidia turbinate. J Org Chem. 1990;55:4512-4515
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10. Rinehart KL. Antitumor compounds from tunicates.Med Res Rev. 2000;20:1-27
11. Joullié MM, Leonard MS, Portonovo P, et al. Chemical defense in ascidians of the didemnidae  family.Bioconjug Chem. 2003;14:30-7
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