Analysis and characterization of a promising therapeutic target identified in Leishmania spp

Leishmaniasis is a neglected disease caused by protozoa of the genus Leishmania spp., which affects about 1.6 million individuals each year and 500,000 present themselves in the visceral form. In Brazil there are about 30,000 new cases each year. In addition, the country is responsible for 90% of reported cases of Visceral Leishmaniasis, and this is more severe form of the disease. Allied to these facts, the current treatment is ineffective, contributing to the establishment of resistant strains. Currently, the treatment has several side effects and permanent damage to the health of patients, this fact has contributed to the search for new drugs against leishmaniasis. The enzyme oligopeptidase B (OPB) has been studied as a possible therapeutic target in the development of antiparasitic agents. Thus, the objective of this work is to construct the three-dimensional model of the enzyme Oligopeptidase B of different species of Leishmania spp. and compare them to each other. For this purpose, the comparative modeling method was used. In this method, the models of the species L. brasiliensis, L. donovani, L. infantum, L. mexicana and L. panamensis were constructed using the MODELLER program. Once the models were ready, the validation process was carried out and subsequently characterized, which was possible to verify a promising degree of similarity between the models. Finally, these models were submitted to the method of analysis by normal modes, which obtained a similar movement pattern, thus it was possible to verify a movement in a specific region of an alpha-helix, consequently leading to the triad of the enzyme being exposed, which may be indicative of a mechanism of action. Finally, it is expected to use the models built to assist in the development of a promising new therapy for the treatment of leishmaniasis.


INTRODUCTION
According to this validation for a predicted model to be considered of excellent quality, it must have more than 90% of amino acid residues in the most favorable region (SANTOS-FILHO and ALENCASTRO, 2003).
In addition, it needs to have the majority of its residues in the most favorable regions, as well as not having residues in the non-permissive regions, except the amino acids glycine (Gly) and proline (Pro) that are exceptions for this area. These two residues present variations in the lateral chain that confer greater rigidity, in the case of Pro, and greater flexibility, in the case of Gly, thus being can assume unexpected angulations.
For this reason they are accepted in the non-permissive regions of the chart. The existence of the regions not allowed is due to the fact that there are steric effects among the residues (side chains) of amino acids. (MORRIS et al., 1992).
Verify 3D analyzes the compatibility of an atomic (3D) model with its own amino acid (1D) sequence. Each residue receives a structural class based on its location and environment (alpha, beta, loop, polar, apolar, etc.). Soon after, a database generated from good structures is used to get a score for each of the 20 amino acids in this structural class. The vertical axis on the graph represents the mean score of the 3D-1D profile for each residual in a sliding window of 21 residuals and the results in the form of scores range from -1 (poor score) to +1 (good score) (EISENBERG et al., 1997).
ProSa-web calculates an overall quality score for a specific input structure. If the score of this is outside a range characteristic for native proteins, the structure probably contains errors. A lot of local quality scores point to problematic parts of the model that

PHYLOGENETIC TREE
Finally, the degree of evolutionary kinship between Leishmanias species was analyzed using the MEGA (Molecular evulutionary genetics analysis) program, using the Neighbor-joining methods, which allows the construction of the phylogenetic tree in order to define the evolutionary proximities between populations of sequences previously defined by the user (KUMAR et al., 2004).

NORMAL MODES
To perform the normal modes, the files generated in the stages of energy minimizações made by GROMACs version 5.1.2 were used. The first 4 steps related to the molecular dynamics process were performed. The first was the generation of topology files, with the addition of hydrogens to the protein. In the second, the water box was created, and this is a very important step for the calculation of the interaction between the protein and the solvent. In the third, ions were added to establish a neutral system. Finally, in the fourth stage, energy minimizations were performed, in which the AMBER99SB force field was inserted. From this point on, the normal modes of the models were performed using the ANM (Anisotropic Network Model) (http://anm.csb.pitt.edu/) server in order to analyze the movement of enzymes of Leishmania species and also to observe some important characteristics for enzyme, such as possible movements related to the mechanism of action (EYAL et al., 2015).

SEQUENCES
We obtained 100 primary structures of the OPBs of leishmania spp. using the UniProt server, with 5 species of Leishmanias selected. The revised amino acid sequences  and A0A088RJA7, respectively These species were selected, due to their high incidence in South America and their resistance against current treatment for leishmaniasis (GHORBANI and FARHOUDI, 2018).
For the construction of 3D models, the BLAST program was used to compare the amino acid sequences of the target sequences with protein sequences of experimentally elucidated three-dimensional structures. Based on the identity between the sequences and the number of gaps, the ENZYME OPB of L. major (code PDB 2XE4) was selected as a mold protein (MCLUSKEY et al., 2010). The identity between the target enzymes and their respective mold presented values between 86% and 96% (Table 1). The percentage of identity between two sequences refers to the presence of the same amino acid in the same position between the aligned sequences. For the construction of a model of a protein with more than 80 amino acid residues, the percentage of identity between the primary structures of the mold and the model should be above 25%. In addition, the percentage of gaps must be low of 20% to be considered a good alignment (SANTOS-FILHO and ALENCASTRO, 2003). Thus, the probability of similarity of the three-dimensional structures of proteins is high. In the phylogenetic tree analysis, it was possible to explain the degree of identity among leishmania species. It was observed that the species with higher degree of identity, such as L. infantum and L. donovani, both with 96%, presented a proximity to each other, besides being closer to L.major. The other species presented lower values such as L.mexicana (90%), which was in the median in relation to the other models.
Finally, the models with the lowest percentage of identity, L. brasiliensis and L.
panamensis, both with 86%, were more distant evolutionarily from their mold, but were very close to each other ( Figure 2). This analysis allowed the understanding of the difference between the degree of identity between species and the understanding of some important characteristics of the enzyme among the species. where group 1 is highlighted in red and group 2 in green.
Source: Prepared by the author based on the results of MEGA.