Chemical synthesis and pharmacological characterization of the frog Prokineticin Bv8 and study of the mechanism of chemical defence of cane toads (Rhinella marina)

The development of poisons endowed animals with a crucial survival advantage against a variety of predators. In toxic animals such as frogs and toads, poisons correspond to ensembles of bioactive molecules targeting essential physiological routines (e.g. nerve conduction, nociception, homeostasis, heart frequency and gastrointestinal motility). The understanding of these molecules and the mechanisms evolved for their delivery may provide crucial insights in the development of therapies, molecular tools and novel strategies to control the harmful effects of syndromes, diseases or introduced foreign organisms. In the first chapter, the key features of the chemical defence of frogs are presented. Among many interesting bioactive peptides and proteins found in the skin secretion of frogs is Bv8 or Bombina variegata 8 kDa protein. Bv8 is a 77-residue protein member of the Prokineticin family of cytokines. Prokineticins are involved in key physiological processes, such as neurogenesis, and nociception. We used solid-phase peptide synthesis, native chemical ligation, and in vitro protein folding to establish robust chemical access to this molecule. Synthetic Bv8 was obtained in good yield and exhibited picomolar activity in vitro. The 3D structure of the synthetic Bv8 was determined using NMR spectroscopy. The synthesis and analysis of a N-terminal truncated mutant indicated that the removal of the critical segment for receptor binding did not perturb the core protein structure suggesting that the receptor binding of Bv8 is likely to be highly cooperative. We established a facile and efficient synthesis of this complex molecule, which will enable the future preparation of unique chemical analogues of Prokineticins and the study of many physiological processes linked to them. In chapter two, we present a brief review of the chemical defence of toads, which will be the focus of the last three chapters of the thesis. Unlike frogs, toads have developed a distinct mechanism of defence that does not depend on bioactive polypeptides. Instead, these animals use a variety of small molecular weight molecules to disrupt the cardiovascular system of their predators. The chemical diversity and pharmacological properties of these molecules is discussed. Finally, the benefits and limitations of the classic method to study toad defences is discussed and the methodology used to study the chemical diversity stored inside the parotoid gland is described. In chapter three we revisit the mechanism of chemical defence of cane toads (Rhinella marina). We provide a brief overview of the morphologic and chemical specialization of toad parotoid glands as a delivery apparatus of small molecular weight cardiotoxin and present our results towards the understanding of the complexity of this delivery system. We established a new method to study this delivery apparatus combining microdissection and HPLC-UV/MS to isolate and quantify the bufadienolide content of individual secretory units within the toad parotoid gland. We explored the efficiency of the parotoid gland in releasing toxins and exposed an unknown mechanism that limits the excessive loss of toxins. We demonstrated that the internal modular arrangement of the parotoid gland limits the loss of secretion to secretory units located in the central region of the gland. The process not only allows toads to not become defenceless after surviving the predator attack but also permits them to release secretion in multiple encounters. Chapter 4 discusses the process of recovery of the toxin content of toad parotoid glands after a simulated predator attack. It is long known that toads require a long time to recover their toxin content but no reports exist to explain the reasons for such a delay. Our data indicated that the efficiency of toxin release is operated at the expense of localized damage to the secretory machinery of the organ. We demonstrated that the damage disrupted the vascular endothelium of empty microglands creating a localized bruise. Toads do not lose their entire toxin content during a simulated attack. However, it was clear that the damage associated with the use of this defence make toads significantly less toxic for a month. These findings describe an unexplored limitation of the chemical defence of cane toads and the unknown costs of chemical defences in toxic vertebrates. Finally, in chapter 5 the chemical defence of toads was explored at a molecular level before and after the compression of the parotoid gland. We demonstrated that the swift removal of the bufotoxin content from the toad secretion was mediated by a new esterase enzyme named TSE (or Toad Skin Esterase). TSE was added to the secretion from accessory glands, functionally characterized for the first time in this study. This enzyme was localized within the skin, purified and used to determine the role of bufotoxins in the secretion. The removal of toad bufotoxins showed a crucial role in the re-configuration of secreted toxin granules by reducing their stability, size and fluidity. The process re-models secreted granules generating small multilamelar vesicles of presumably enhanced oral bioavailability. Our findings demonstrated a new biotransformation method, which play an important role facilitating the rapid release of stored toxins and, presumably, the efficacy of toad toxins against predators.