Quantum Biochemistry Screening and In Vitro Evaluation of Leishmania Metalloproteinase Inhibitors.

Leishmanolysin, also known as major promastigote protease (PSP) or gp63, is the most abundant surface glycoprotein of Leishmania spp., and has been extensively studied and recognized as the main parasite virulence factor. Characterized as a metalloprotease, gp63 can be powerfully inactivated in the presence of a metal chelator. In this study, we first used the structural parameters of a 7-hydroxycoumarin derivative, L1 compound, to evaluate the theoretical-computational experiments against gp63, comparing it with an available metal chelator already described. The methodology followed was (i) analysis of the three-dimensional structure of gp63 as well as its active site, and searching the literature and molecular databases for possible inhibitors; (ii) molecular docking simulations and investigation of the interactions in the generated protein-ligand complexes; and (iii) the individual energy of the gp63 amino acids that interacted most with the ligands of interest was quantified by ab initio calculations using Molecular Fraction with Conjugated Caps (MFCC). MFCC still allowed the final quantum balance calculations of the protein interaction to be obtained with each inhibitor candidate binder. L1 obtained the best energy quantum balance result with -2 eV, followed by DETC (-1.4 eV), doxycycline (-1.3 eV), and 4-terpineol (-0.6 eV), and showed evidence of covalent binding in the enzyme active site. In vitro experiments confirmed L1 as highly effective against L. amazonensis parasites. The compound also exhibited a low cytotoxicity profile against mammalian RAW and 3T3 cells lines, presenting a selective index of 149.19 and 380.64 µM, respectively. L1 induced promastigote forms' death by necrosis and the ultrastructural analysis revealed disruption in membrane integrity. Furthermore, leakage of the contents and destruction of the parasite were confirmed by Spectroscopy Dispersion analysis. These results together suggested L1 has a potential effect against L. amazonensis, the etiologic agent of diffuse leishmaniasis, and the only one that currently does not have a satisfactory treatment.

Trypanosoma brucei Interaction with Host: Mechanism of VSG Release as Target for Drug Discovery for African Trypanosomiasis.

The protozoan Trypanosoma brucei, responsible for animal and human trypanosomiasis, has a family of major surface proteases (MSPs) and phospholipase-C (PLC), both involved in some mechanisms of virulence during mammalian infections. During parasitism in the mammalian host, this protozoan is exclusively extracellular and presents a robust mechanism of antigenic variation that allows the persistence of infection. There has been incredible progress in our understanding of how variable surface glycoproteins (VSGs) are organised and expressed, and how expression is switched, particularly through recombination. The objective of this manuscript is to create a reflection about the mechanisms of antigenic variation in T. brucei, more specifically, in the process of variable surface glycoprotein (VSG) release. We firstly explore the mechanism of VSG release as a potential pathway and target for the development of anti-T. brucei drugs.

Insights of antiparasitic activity of sodium diethyldithiocarbamate against different strains of Trypanosoma cruzi.

Chagas disease is caused by Trypanosoma cruzi and affects thousands of people. Drugs currently used in therapy are toxic and have therapeutic limitations. In addition, the genetic diversity of T. cruzi represents an important variable and challenge in treatment. Sodium diethyldithiocarbamate (DETC) is a compound with pharmacological versatility acting as metal chelators and ROS generation. Thus, the objective was to characterize the antiparasitic action of DETC against different strains and forms of T. cruzi and their mechanism. The different strains of T. cruzi were grown in LIT medium. To evaluate the antiparasitic activity of DETC, epimastigote and trypomastigote forms of T. cruzi were used by resazurin reduction methods and by counting. Different response patterns were obtained between the strains and an IC50 of DETC ranging from 9.44 ± 3,181 to 60.49 ± 7.62 µM. Cell cytotoxicity against 3T3 and RAW cell lines and evaluated by MTT, demonstrated that DETC in high concentration (2222.00 µM) presents low toxicity. Yet, DETC causes mitochondrial damage in T. cruzi, as well as disruption in parasite membrane. DETC has antiparasitic activity against different genotypes and forms of T. cruzi, therefore, representing a promising molecule as a drug for the treatment of Chagas disease.

Variable Surface Glycoprotein from Trypanosoma brucei Undergoes Cleavage by Matrix Metalloproteinases: An in silico Approach.

In order to survive as extracellular parasites in the mammalian host environment, Trypanosoma brucei has developed efficient mechanisms of immune system evasion, which include the abundant expression of a variable surface glycoprotein (VSG) coat. VSGs are anchored in the parasite membrane by covalent C-terminal binding to glycosylphosphatidylinositol and may be periodically removed by a phospholipase C (PLC) and a major surface protein (TbMSP). VSG molecules show extraordinary antigenic diversity and a comparative analysis of protein sequences suggests that conserved elements may be a suitable target against African trypanosomiasis. However, the cleavage mechanisms of these molecules remain unclear. Moreover, in protozoan infections, including those caused by Trypanosoma brucei, it is possible to observe an increased expression of the matrix metalloproteinases (MMPs). To address the cleavage mechanism of VSGs, the PROSPER server was used for the identification of VSG sequence cleavage sites. After data compilation, it was observed that 64 VSG consensus sequences showed a high conservation of hydrophobic residues, such as valine (V), methionine (M), leucine (L) and isoleucine (I) in the fifth position-the exact location of the cleavage site. In addition, the PROSPER server identified conserved cleavage site portions of VSG proteins recognized by three matrix metalloproteases (gelatinases: MMP-2, MMP-3 and MMP-9). However, further biological studies are needed in order to analyze and confirm this prediction.

Biological and Molecular Characterization of Trypanosoma cruzi Strains from Four States of Brazil.

Chagas disease affects between six and seven million people. Its etiological agent, Trypanosoma cruzi, is classified into six discrete typing units (DTUs). The biological study of 11 T. cruzi strains presented here included four parameters: growth kinetics, parasitemia curves, rate of macrophage infection, and serology to evaluate IgM, total IgG, IgG1, IgG2a, and IgG3. Sequencing of small subunit of ribosomal RNA (SSU rRNA)was performed and the T. cruzi strains were classified into three DTUs. When their growth in liver infusion tryptose medium was represented in curves, differences among the strains could be noted. The parasitemia profile varied among the strains from the TcI, TcII, and TcIII groups, and the 11 T. cruzi strains produced distinct parasitemia levels in infected BALB/c. The TcI group presented the highest rate of macrophage infection by amastigotes, followed by TcII and TcIII. Reactivity to immunoglobulins was observed in the TcI, TcII, and TcIII; all the animals infected with the different strains of T. cruzi showed anti-T. cruzi antibodies. The molecular study presented here resulted in the classification of the T. cruzi strains into the TcI (Bolivia, T lenti, Tm, SC90); TcII (Famema, SC96, SI8, Y); and TcIII (QMM3, QMM5, SI5) groups. These biological and molecular results from 11 T. cruzi strains clarified the factors involved in the biology of the parasite and its hosts. The collection of triatomine (vector) species, and the study of geographic distribution, as well as biological and molecular characterization of the parasite, will contribute to the reporting and surveillance measures in Brazilian states.

Use of Zymography in Trypanosomiasis Studies.

Zymography assay is a semiquantitative technique, very sensitive, and commonly used to determine metalloproteinase levels in different types of biological samples, including tissues, cells, and extracts of protein. Samples containing metalloproteinases are loaded onto a polyacrylamide gel containing sodium dodecyl sulphate (SDS) and a specific substrate (gelatin, casein, collagen, etc.). Then proteins are allowed to migrate under an electric current and the distance of migration is inversely correlated with the molecular weight. After migration, the gel is placed in a renaturing buffer to allow proteins to regain their tertiary structure, necessary for enzymatic activity (metalloproteinase activity). In the context of infections caused by trypanosomatids (Leishmania spp., Trypanosoma cruzi, and Trypanosoma brucei), the characterization of metalloproteinase by zymography can contribute to the comprehension of the pathogenesis mechanisms and host-parasite interaction.