Prevention of SARS-CoV viral infection by inhibiting human transmembrane II serine proteases (TMPRSS2) and 3CLpro by a Bowman-Birk protease inhibitor and derived peptides

BTCI is able to cross the plasma membrane and co-localize with the 20S proteasome (as a complex) in the cytoplasm and nucleus of MCF7 cells; it has a natriuretic effect, increasing the glomerular filtration rate and ion flow, and potentiates the action of the guaniline peptide, which has a natriuretic effect on the renal system, in Wistars rat models [4]; it has a hypotensive effect, in models of Wistar and SHR rats (naturally hypertensive), by intravenous administration [5] and by gavage [6], mediated by the inhibitory activity against the angiotensinogen-converting enzyme 1 (ECA1). Therefore, the present proposal is based on the biotechnological and biomedical potential of this inhibitor, as presented, and in recent studies by our group showing that BTCI inhibits the enzymatic activity of a human transmembrane II serinoprotease (TMPRSS2) (article in preparation) on the cell surface of several target cells that allows the activation of S proteins bound to the AEC2 receptor on the surface of the viral host cell, inducing the fusion of the plasma membrane of the viral envelope and the direct entry of SARS-CoV into the cells. At the same time, the 3CLpro protein is chymotrypsin-like, which allows us to suggest a pathway for its inhibition. In the present project we propose to evaluate the inhibitory effect of the Bowman-Birk serinoprotease inhibitor, called BTCI, and its derived peptides, against proteases such as human transmembrane II serinoproteases (TMPRSS2) and the main SARS-CoV protease, called 3CLpro, essential for virus replication. For the development of the project, we will approach two strategies, one theoretical and the other experimental. In the theoretical part, we propose to obtain and analyze the molecular complexes of the serinoproteases cited with BTCI and derived peptides, by computational simulation of molecular dynamics and hybrid methods QM / MM (from English: quantum mechanics / molecular mechanics), molecular docking, as well as determination of the electronic structure of the atoms of the complexes' interface to evaluate the molecular interaction mechanism. Experimentally, serinoproteases will be obtained by gene recombination, from the synthetic gene, and tested enzymatically, in vitro, using spectroscopic methods and isothermal titration calorimetry (ITC), under the action of BTCI and derived peptides. In this context, thermodynamic parameters and binding constants will be determined to assess the affinity and stability of the complexes formed. The effect of these inhibitors will also be evaluated in the presence of protein S, to be obtained by gene recombination, in order, together with the results to be obtained with the other proteases, to constitute data that support the preventive effect of the SARS-CoV infection process. . using spectroscopic methods and isothermal titration calorimetry (ITC), under the action of BTCI and derived peptides. In this context, thermodynamic parameters and binding constants will be determined to assess the affinity and stability of the complexes formed. The effect of these inhibitors will also be evaluated in the presence of protein S, to be obtained by gene recombination, in order, together with the results to be obtained with the other proteases, to constitute data that support the preventive effect of the SARS-CoV infection process. . using spectroscopic methods and isothermal titration calorimetry (ITC), under the action of BTCI and derived peptides. In this context, thermodynamic parameters and binding constants will be determined to assess the affinity and stability of the complexes formed. The effect of these inhibitors will also be evaluated in the presence of protein S, to be obtained by gene recombination, in order, together with the results to be obtained with the other proteases, to constitute data that support the preventive effect of the SARS-CoV infection process. . [1] Joanitti, GA, Azevedo, RB & Freitas, SM Apoptosis and lysosome membrane permeabilization induction on breast cancer cells by an anticarcinogenic Bowman-Birk protease inhibitor from Vigna unguiculata seeds. Cancer letters 293, 73-81, doi: 10.1016 / j.canlet.2009.12.017 (2010). [2] Mehdad, A. et al. A Bowman-Birk inhibitor induces apoptosis in human breast adenocarcinoma through mitochondrial impairment and oxidative damage following proteasome 20S inhibition. Cell death discovery 2, 15067, doi: 10.1038 / cddiscovery.2015.67 (2016). [3] Souza Lda, C. et al. Effects of an anticarcinogenic Bowman-Birk protease inhibitor on purified 20S proteasome and MCF-7 breast cancer cells. PloS one 9, e86600, doi: 10.1371 / journal.pone.0086600 (2014). [4] Carvalho, AF et al. BTCI enhances guanylin-induced natriuresis and promotes renal glomerular and tubular effects. Brazilian journal of biology = Revista brasleira de biologia 68, 149-154 (2008). [5] by Cunha Morales Alvares, A. et al. Bowman-Birk protease inhibitor from Vigna unguiculata seeds enhances the action of bradykinin-related peptides. Molecules (Basel, Switzerland) 19, 17536-17558, doi: 10.3390 / molecules191117536 (2014). [6] Maria Alzira Garcia de Freitas, Nathalia Oda Amaral, Alice da Cunha Morales Álvares, Sandriele Aires de Oliveira, Azadeh Mehdad, Diego Elias Honda, Amanda Sá Martins Bessa, Marcelo Henrique Soller Ramada, Lara Marques Naves, Carolina Nobre Ribeiro Pontes, Carlos Henrique Castro, Gustavo Rodrigues Pedrino and Sonia Maria de Freitas. Blood pressure-lowering effects of a Bowman-Birk inhibitor and its derived peptides in normotensive and hypertensive rats. Scientific Reports (under "minor revision" analysis). 2020.