SARS-CoV-2 Proteins Bind to Hemoglobin and Its Metabolites.

(1) Background: coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been linked to hematological dysfunctions, but there are little experimental data that explain this. Spike (S) and Nucleoprotein (N) proteins have been putatively associated with these dysfunctions. In this work, we analyzed the recruitment of hemoglobin (Hb) and other metabolites (hemin and protoporphyrin IX-PpIX) by SARS-Cov2 proteins using different approaches. (2) Methods: shotgun proteomics (LC-MS/MS) after affinity column adsorption identified hemin-binding SARS-CoV-2 proteins. The parallel synthesis of the peptides technique was used to study the interaction of the receptor bind domain (RBD) and N-terminal domain (NTD) of the S protein with Hb and in silico analysis to identify the binding motifs of the N protein. The plaque assay was used to investigate the inhibitory effect of Hb and the metabolites hemin and PpIX on virus adsorption and replication in Vero cells. (3) Results: the proteomic analysis by LC-MS/MS identified the S, N, M, Nsp3, and Nsp7 as putative hemin-binding proteins. Six short sequences in the RBD and 11 in the NTD of the spike were identified by microarray of peptides to interact with Hb and tree motifs in the N protein by in silico analysis to bind with heme. An inhibitory effect in vitro of Hb, hemin, and PpIX at different levels was observed. Strikingly, free Hb at 1mM suppressed viral replication (99%), and its interaction with SARS-CoV-2 was localized into the RBD region of the spike protein. (4) Conclusions: in this study, we identified that (at least) five proteins (S, N, M, Nsp3, and Nsp7) of SARS-CoV-2 recruit Hb/metabolites. The motifs of the RDB of SARS-CoV-2 spike, which binds Hb, and the sites of the heme bind-N protein were disclosed. In addition, these compounds and PpIX block the virus's adsorption and replication. Furthermore, we also identified heme-binding motifs and interaction with hemin in N protein and other structural (S and M) and non-structural (Nsp3 and Nsp7) proteins.

Optimization of 1,4-Naphthoquinone Hit Compound: A Computational, Phenotypic, and In Vivo Screening against Trypanosoma cruzi.

Chagas disease (CD) still represents a serious public health problem in Latin America, even after more than 100 years of its discovery. Clinical treatments (nifurtimox and benznidazole) are considered inadequate, especially because of undesirable side effects and low efficacy in the chronic stages of the disease, highlighting the urgency for discovering new effective and safe drugs. A small library of compounds (1a-i and 2a-j) was designed based on the structural optimization of a Hit compound derived from 1,4-naphthoquinones (C2) previously identified. The biological activity, structure-activity relationship (SAR), and the in silico physicochemical profiles of the naphthoquinone derivatives were analyzed. Most modifications resulted in increased trypanocidal activity but some substitutions also increased toxicity. The data reinforce the importance of the chlorine atom in the thiophenol benzene ring for trypanocidal activity, highlighting 1g, which exhibit a drug-likeness profile, as a promising compound against Trypanosoma cruzi. SAR analysis also revealed 1g as cliff generator in the structure-activity similarity map (SAS maps). However, compounds C2 and 1g were unable to reduce parasite load, and did not prevent mouse mortality in T. cruzi acute infection. Phenotypic screening and computational analysis have provided relevant information to advance the optimization and design of new 1,4-naphthoquinone derivatives with a better pharmacological profile.

Heme metabolism as a therapeutic target against protozoan parasites.

Neglected tropical diseases caused by protozoan parasites affect the life of millions of people worldwide, causing mortality, morbidity and high economic and social burden. The search for new drug targets and therapeutic strategies to fight these pathogens are necessary, since many current drugs have limited effects, cause severe side effects and their use has resulted in pathogen resistance. Heme (iron protoporphyrin IX) is a ubiquitous molecule important in many biological processes, including the homeostasis, growth and development of human pathogens such as trypanosomatids (Trypanosoma cruzi, Trypanosoma brucei and Leishmania spp.) and Plasmodium spp. In this review, several chemotherapy approaches and strategies are discussed that target heme transport, catabolism, crystallization and hemeproteins.

The involvement of FAK and Src in the invasion of cardiomyocytes by Trypanosoma cruzi.

The activation of signaling pathways involving protein tyrosine kinases (PTKs) has been demonstrated during Trypanosoma cruzi invasion. Herein, we describe the participation of FAK/Src in the invasion of cardiomyocytes by T. cruzi. The treatment of cardiomyocytes with genistein, a PTK inhibitor, significantly reduced T. cruzi invasion. Also, PP1, a potent Src-family protein inhibitor, and PF573228, a specific FAK inhibitor, also inhibited T. cruzi entry; maximal inhibition was achieved at concentrations of 25µM PP1 (53% inhibition) and 40µM PF573228 (50% inhibition). The suppression of FAK expression in siRNA-treated cells and tetracycline-uninduced Tet-FAK(WT)-46 cells significantly reduced T. cruzi invasion. The entry of T. cruzi is accompanied by changes in FAK and c-Src expression and phosphorylation. An enhancement of FAK activation occurs during the initial stages of T. cruzi-cardiomyocyte interaction (30 and 60min), with a concomitant increase in the level of c-Src expression and phosphorylation, suggesting that FAK/Src act as an integrated signaling pathway that coordinates parasite entry. These data provide novel insights into the signaling pathways that are involved in cardiomyocyte invasion by T. cruzi. A better understanding of the signal transduction networks involved in T. cruzi invasion may contribute to the development of more effective therapies for the treatment of Chagas' disease.

Current understanding of the Trypanosoma cruzi-cardiomyocyte interaction.

Trypanosoma cruzi, the etiological agent of Chagas disease, exhibits multiple strategies to ensure its establishment and persistence in the host. Although this parasite has the ability to infect different organs, heart impairment is the most frequent clinical manifestation of the disease. Advances in knowledge of T. cruzi-cardiomyocyte interactions have contributed to a better understanding of the biological events involved in the pathogenesis of Chagas disease. This brief review focuses on the current understanding of molecules involved in T. cruzi-cardiomyocyte recognition, the mechanism of invasion, and on the effect of intracellular development of T. cruzi on the structural organization and molecular response of the target cell.

Biological and immunological characterization of recombinant Yellow Fever 17D viruses expressing a Trypanosoma cruzi Amastigote Surface Protein-2 CD8+ T cell epitope at two distinct regions of the genome.

BACKGROUND: The attenuated Yellow fever (YF) 17D vaccine virus is one of the safest and most effective viral vaccines administered to humans, in which it elicits a polyvalent immune response. Herein, we used the YF 17D backbone to express a Trypanosoma cruzi CD8+ T cell epitope from the Amastigote Surface Protein 2 (ASP-2) to provide further evidence for the potential of this virus to express foreign epitopes. The TEWETGQI CD8+ T cell epitope was cloned and expressed based on two different genomic insertion sites: in the fg loop of the viral Envelope protein and the protease cleavage site between the NS2B and NS3. We investigated whether the site of expression had any influence on immunogenicity of this model epitope. RESULTS: Recombinant viruses replicated similarly to vaccine virus YF 17D in cell culture and remained genetically stable after several serial passages in Vero cells. Immunogenicity studies revealed that both recombinant viruses elicited neutralizing antibodies to the YF virus as well as generated an antigen-specific gamma interferon mediated T-cell response in immunized mice. The recombinant viruses displayed a more attenuated phenotype than the YF 17DD vaccine counterpart in mice. Vaccination of a mouse lineage highly susceptible to infection by T. cruzi with a homologous prime-boost regimen of recombinant YF viruses elicited TEWETGQI specific CD8+ T cells which might be correlated with a delay in mouse mortality after a challenge with a lethal dose of T. cruzi. CONCLUSIONS: We conclude that the YF 17D platform is useful to express T. cruzi (Protozoan) antigens at different functional regions of its genome with minimal reduction of vector fitness. In addition, the model T. cruzi epitope expressed at different regions of the YF 17D genome elicited a similar T cell-based immune response, suggesting that both expression sites are useful. However, the epitope as such is not protective and it remains to be seen whether expression of larger domains of ASP-2, which include the TEWETGQI epitope, will elicit better T-CD8+ responses to the latter. It is likely that additional antigens and recombinant virus formulations will be necessary to generate a protective response.

Trypanosoma cruzi infection induces a global host cell response in cardiomyocytes.

Chagas' disease, caused by the hemoflagellate protozoan Trypanosoma cruzi, affects millions of people in South and Central America. Chronic chagasic cardiomyopathy, the most devastating manifestation of this disease, occurs in approximately one-third of infected individuals. Events associated with the parasite's tropism for and invasion of cardiomyocytes have been the focus of intense investigation in recent years. In the present study, we use murine microarrays to investigate the cellular response caused by invasion of primary murine cardiomyocytes by T. cruzi trypomastigotes. These studies identified 353 murine genes that were differentially expressed during the early stages of invasion and infection of these cells. Genes associated with the immune response, inflammation, cytoskeleton organization, cell-cell and cell-matrix interactions, apoptosis, cell cycle, and oxidative stress are among those affected during the infection. Our data indicate that T. cruzi induces broad modulations of the host cell machinery in ways that provide insight into how the parasite survives, replicates, and persists in the infected host and ultimately defines the clinical outcome of the infection.

Regulation of extracellular matrix expression and distribution in Trypanosoma cruzi-infected cardiomyocytes.

Alterations in the extracellular matrix have been observed in the cardiomyopathy of Chagas disease caused by Trypanosoma cruzi infection. However, the mechanism of extracellular matrix regulation in T. cruzi-infected cultured cardiomyocytes (CMs) is unclear. Using confocal laser microscopy, we demonstrated that treatment of these cultures with transforming growth factor beta (TGF-beta) and tumor necrosis factor alpha (TNF-alpha) leads to an enhancement of the fibronectin matrix only in uninfected CMs, while infected myocytes displayed low fibronectin expression. Digital image analysis also revealed low superposition of the fibronectin signal with parasite nests in cytokine treated and untreated cultures. Cytochalasin D treatment resulted in microfilament disarray that induced a disturbance in the fibronectin network of CMs, suggesting that cytoskeleton disruption caused by T. cruzi infection disorganizes the fibronectin matrix. Western blot analysis revealed a 2-fold increase in the fibronectin expression in CM cultures after cytokine treatment, whereas T. cruzi infection significantly reduced fibronectin levels in all conditions. In contrast, no change in the laminin expression was detected after cytokine treatment. Laminin distribution was altered in T. cruzi-infected CMs, with intense laminin labeling only at the cell periphery even after cytokine treatment. Our observations indicate that TGF-beta and TNF-alpha stimulates fibronectin expression only in uninfected cells of the T. cruzi-infected cultures, whereas the cells harboring the parasites display low or no fibronectin fibrils.