Prospecção, síntese química e caracterização farmacológica de toxinas de peçonhas Elapidae (Micrurus lemniscatus) e Conidae sobre o sistema colinérgico

Abstract

Toxins from animal venoms went through great evolutionary pressure, which undoubtedly contributed for their high affinity and selectivity to their molecular targets. Therefore, they constitute unlimited resources for the discovery of new drug candidates. There is a diversity of possible targets for animal toxins, however here I would like to highlight the cholinergic transmission (CT). Among the protein toxins which have CT as a target, one of the most abundant families correspond to the three fingers toxins (3FTxs) - found mainly in Elapidae snake venoms - and the α-conotoxins, isolated from the venom of marine gastropod molluscs of the genus Conus. This thesis comprehends an introduction about the potential toxins which might modulate CT and 2 different chapters: prospecting of 3FTxs of a Brazilian coral snake specie and prospecting of new α-conotoxin subfamilies. Therefore, for the accomplishment of the first part of the thesis, the venom of Micrurus lemniscatus was fractioned by HPLC and Ml4 toxin (7201.12 Da) was isolated, chemically synthesized and studied on muscarinic and nicotinic receptors by binding techniques and inositol monophosphate (IP1) measurement. Ml4 demonstrated M3 muscarinic and Torpedo nicotinic activity, on the other hand it did not show any cytotoxicity. Surprisingly, the synthetic version of Ml4, MiLTx1, had no effect on CT even at concentrations 100x higher than those tested for the native toxin. Both toxins share the same sequence, mass and fragmentation pattern. However, when the native toxin and its synthetic version were coinjected in reverse phase chromatography, they exhibit 3 min difference in elution time, confirming that they are not identical. One of the hypothesis for this difference is the presence of D-aminoacids in Ml4 or that the major peak of Ml4 is not responsible for the observed cholinergic activity. Some minor contaminant, could explain this contradictory result. MonoS8, the fraction from where Ml4 was firstly purified, showed activity on M3 and Torpedo receptors, as expected. However, the observed muscarinic response was agonism and not antagonism, as previously observed for Ml4. After that, MonoS8 was purified and only 6 toxins obtained could be submitted to activity verification. MonoS8 7 (6782.7 Da) was the only peak / toxin that could compete with the radioligand and then had its concentrationresponse activity confirmed. However, agonist / antagonist character as well as the affinity and concentration of the toxin, were not possible to be determined due to the limited amount of material. The venom of M. lemniscatus has a myriad of 3FTxs toxins which are difficult to purify. However, for the first time, we have detected a M3 muscarinic, probable agonist, toxin through direct binding to the receptor. On the second chapter of this thesis, 18 peptides were selected from the VENOMICS Project, with cysteine pattern CC_C_C (classical α-conotoxins) potentially active on CT. The peptides were chemically synthesized and characterized according to its pharmacological target. None of them showed adrenergic α1, muscarinic M1, or neuronal nicotinic activities (α3β2, α3β4, α4β2 and α7-5HT3 chimera). In addition, the activity of the 18 peptides was evaluated on the neuromuscular junction of rat hemidiaphragm and our data have shown that 2 peptides potentiated and 2 inhibited the contraction. Among those active peptides, the Torpedo nicotinic activity was evaluated in isolated receptors throughbinding and electrophysiology techniques. Only P010378* competed in a concentration dependent manner with the radioligand. Furthermore, both isomers of P020581 reduced the residual current of ACh in Xenopus oocytes incorporated 2α1β1δγ receptors. Five of the initial samples showed a muscle nicotinic activity, although these toxins could not be completely characterized. Our prospecting studies highlights a promising research field for discovering new toxins with different profiles of selectivity and interaction with cholinergic receptors. A major limitation of bioprospecting native compounds is the considerable amount of material required. Therefore, technological advances have contributed significantly to the improvement of venom research, and omics approach play an essential role in the development of toxins as therapeutic tools.