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| Author(s): |
Roberto Tadashi Kodama
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI) |
| Defense date: | 2019-10-22 |
| Examining board members: |
Fernanda Calheta Vieira Portaro;
Anita Mitico Tanaka Azevedo;
Adriana Karaoglanovic Carmona;
Kerly Fernanda Mesquita Pasqualoto;
Carlos Alberto da Silva;
Patrick Jack Spencer
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| Advisor: | Fernanda Calheta Vieira Portaro |
| Abstract | |
Peptidases play an important role for the proper physiological functions of the most diverse organisms. When unregulated, they are associated to pathologies, for example, the exacerbated activity of elastase-1 is related to pancreatitis, and, for the cathepsin L, it is involved in the tumors metastasis. Therefore these peptidases have become potential therapeutic targets regarding the search for inhibitors. The venoms of snakes are composed by complex mixtures of molecules that can feature diverse functions, including the inhibition of enzymes. Considering this, the present works objective was to purify, characterize and to study in vitro and in silico new inhibitors that are present in the low molecular weight fraction of the venoms from three African snakes genera, Naja, Dendroaspis and Bitis. For that, the low molecular weight fraction from each venom (<10 kDa) were submitted to RP-HPLC chromatographic fractioning, using a C-18 column. The fractions were them collected, and utilizing fluorescent substrates, the screening test was performed to verify the inhibition by the fractions over the peptidases studied in this work. The identification of the fractions of interest was carried out by mass spectrometry, among them, molecules from the three-finger-toxins class was identified, and, for the first time, described as peptidase inhibitors. Other molecules from Kunitz class, that were previously described as Na+ and K+ channel blockers, but not as peptidase inhibitors, were also identified. The peptide DPSA8P (from Dendroaspis polylepis venom) stood out due to the fact that it had similar cathepsin L and elastase-1 inhibition on the screening step. This peptide, classified as a Kunitz one, was characterized as an incompetitive inhibitor for elastase-1, presenting a Ki of 8 µM. The docking (ZDOCK) analysis indicated interaction models of this peptide with elastase-1, in the catalytic site and in other regions of this enzyme. Using these interaction models, two peptides were designed to be studied as possible inhibitors of both elastase-1 and cathepsin L. The first one did not show inhibitory activity, however, the second, a cyclic peptide named as PEP2, which contains 30 amino acid residues, showed to be a competitive inhibitor, with Ki of 1.96 µM over cathepsin L. Furthermore, we performed re-docking analyses of the BPPs, from the Bitis spp venom, using the ACE (angiotensin converting enzyme) as receptor. These results showed high relation of the scores obtained in the in silico studies with the Kis previously determined. This work describes some low molecular weight toxins with a new function as peptidase inhibitors, and this could contribute to further understand the envenomation process. In addition, the efficiency of the docking technique to design new molecules was shown, aiming at the search molecular docking was used to the design new of peptidase inhibitors, which led to the PEP2, a peptide that is able to inhibit the cathepsin L activity with a low inhibition constant. Showing once again that this methodology may be used for the search of new biotechnological entities. (AU) | |
| FAPESP's process: | 15/13124-5 - Purification and characterization of peptides present in the venom of African snakes: searching for peptidase inhibitor of medical importance |
| Grantee: | Roberto Tadashi Kodama |
| Support Opportunities: | Scholarships in Brazil - Doctorate |