In our project many different tools were used to validate the viability of the inhibition of PLA2 (Phospholipase A2) by the protein γ-PLI and Jararhagin by BJ46a, as well as elucidate the inhibition process and our inhibitors structure, so we can achieve a better end product, the first is responsible for the myonecrosis and the latter for hemorrhagic effects, both present in the venom inoculated by Bothrops jararaca. In order to better understand these inhibition processes we choose tools designed for studying structural modeling, molecular docking and molecular dynamics. The kinetic aspects of this process were also investigated to find the best inhibitory parameters.
Jararhagin has domains in their structure that allow interaction with elements of the cell, being a catalytic domain, a disintegrin-like domain and a cysteine-rich domain (Lima,2013) and four domains in its structure: a pro-peptide region (domain A), a metal ion binding region (domain B), a disintegrin region (domain C), and a carboxyl-terminal region with a cysteine-rich domain (domain D) (Paine et al.,1992). On the topic of structural modeling, the Jararhagin, PLA2, BJ46a and γ-PLI inhibitor were searched in the NCBI database for submission of its sequences, to SWISS-MODEL. The results obtained by SWISS-MODEL, a fully automated protein structure homology modeling server, were favorable, when modeling Jararhagin, we obtained a QMEAN (Qualitative Model Energy Analysis) of 0.8, and in the Ramachandran plot for the inhibitor BJ46a 91.06% of the residuals are located in favorable regions, indicating a good result. Then for BJ46a, which is the inhibitor that we will synthesize, we searched on the SWISS-MODEL webserver the four best templates which were used in MODELLER for the homology modeling. In MODELLER, the best template among the four best templates was found, the template 6ht9. 1. B Fetuin-B. With this template we are able to build a model that we used in further simulations, we validated the model with the help of UCLA-DOE LAB and Zhang Lab server and obtained some further parameters, such as the Ramanchadran graph which indicated that 78% of the amino acids are in favorable regions, thus presenting an excellent result for our model.
Docking is a bioinformatics tool used for analyzing the interaction between two proteins, to predict the behavior of the complex and acquire data such as: binding energies, stability of the complex and protein parameters. We used two docking servers for this purpose, PYDOCK based on disolvation energy and electrostatic interactions and HDOCK which measures the docking score and ligand rmsd, both showed converging results, showing the best docking structure that illustrates how the inhibition process works on structural terms, supporting each other and overall were satisfactory for proving the efficacy of our project.
Molecular dynamics were researched for our inhibitor in order to determine its stability, ability of maintain its therapeutical, physical, chemical properties over time against external factors (temperature, humidity, light, gases, pH, packaging material) and internal factors (oxidation, hydrolysis, drug-drug interactions, presence of impurities). We required this information for evaluating the feasibility of distributing our drug across the country, and for such we utilized two softwares: GROMACS (GROningen MAchine for Chemical Simulation), a versatile and efficient program for Molecular Dynamics analysis, and the CHARMM-GUI (Chemistry at HARvard Macro-molecular Mechanics - Graphical User Interface) server, a website. First, with GROMACS we found some basic parameters about the molecular dynamics simulated in aqueous state with Na+ and Cl- in order to neutralize the inhibitor charge. Initially it was observed that in the start terminator the bond is composed of NH3+ (ASN1: NH3+) and in the end terminator the bond is made with COO- (LEU281: COO-). And also it was calculated to have an atomic mass of 32039.831 a.m.u. (atomic mass unit), lower than the native protein(46kDa), this is due to the removal of the signal peptides. With CHARMM-GUI we were able to determine that our compound is stable at temperatures of 303.15 K or 30°C, ideal for tropical countries, like Brazil.
Finally, the kinetic aspects of our inhibitions of interest were studied, both inhibitors are not allosteric and, therefore, can be modeled according to the Michaelis-Menten mechanism (NEVES-FERREIRA et al., 2015). Furthermore, proteins interact by forming a non-covalent, i.e. reversible, complex in which the inhibitor competes with the substrate for the (competitive) binding site (VALENTE et al., 2001). The parameters present in these equations are not available in the literature for our proteins, therefore we had to use softwares, such as scilab to estimate these values.
In summary, the structural and kinetic modeling performed during our project proves that the inhibitions studied can be achieved in reality, and that it is viable as a complement to traditional serums.
As we were able to synthesize the enzyme responsible for the myonecrotic effect, as well as its inhibitor and predict the interaction between them, we can safely state that our project is viable as a first step towards a completely synthetic serum for snakebite treatments. Traditionally, the snakebite serum is produced via large animals such as horses and sheep, our proposed concept is proven once we expressed the target protein from bacteria and proved that it is present via the SDS PAGE method, to do so, we estimated the size of our target proteins through the EXPASY software and then observed protein bands in the regions which our proteins should be. The fact that we can observe it in the SDS-PAGE gel suggests that our expression was successful, therefore, we were able to express the inhibitor of one of the main components in snake venom through bacteria, once again proving that our motivations to develop this very project were justified.
Figure 1 - SDS PAGE result for the Phospholipase A2
Source: Author (2022)
Figure 2 - SDS PAGE result for 𝛾PLI
Source: Author (2022)
It is possible to observe the efficacy of the inhibition from the graphic of the kinetic model for the PLA2 x 𝛾PLI that we will obtain once the inhibitory assay is done, a classic competitive inhibition (Lehninger)
Due to problems during lab practices such as our iGEM 2022 kit not arriving and our iGEM 2021 kit being completely degraded, it was not possible to perform the inhibitory assay of our protein until Wiki freeze. Despite this, we were able to synthesize the enzyme responsible for the myonecrotic effect, as well as its inhibitor.