Publications

The methyl erythritol phosphate (MEP) pathway of isoprenoid biosynthesis is essential for malaria parasites and also for several human pathogenic bacteria, thus representing an interesting target for future antimalarials and antibiotics and for diagnostic strategies. We have developed a DNA aptamer (D10) against Plasmodium falciparum 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), the second enzyme of this metabolic route. D10 binds in vitro to recombinant DXR from P. falciparum and Escherichia coli, showing at 10 mu M a ca. 50% inhibition of the bacterial enzyme. In silico docking analysis indicates that D10 associates with DXR in solvent-exposed regions outside the active center pocket. According to fluorescence confocal microscopy data, this aptamer specifically targets in P. falciparum in vitro cultures the apicoplast organelle where the MEP pathway is localized and is, therefore, a highly specific marker of red blood cells parasitized by Plasmodium vs. naive erythrocytes. D10 is also selective for the detection of MEP+ bacteria (e.g., E. coli and Pseudomonas aeruginosa) vs. those lacking DXR (e.g., Enterococcus faecalis). Based on these results, we discuss the potential of DNA aptamers in the development of ligands that can outcompete the performance of the well-established antibody technology for future therapeutic and diagnostic approaches.

JTD Keywords: 1-deoxy-d-xylulose-5-phosphate reductoisomerase, dna aptamers, plasmodium, 1-deoxy-d-xylulose-5-phosphate reductoisomerase, Apicoplast, Dna aptamers, Drug targets, Evolution, Inhibitors, Isoprenoid biosynthesis, Malaria, Methyl erythritol phosphate pathway, Pathway, Plasmodium, Protein-protein, Web server

The evolution of resistance by the malaria parasite to artemisinin, the key component of the combination therapy strategies that are at the core of current antimalarial treatments, calls for the urgent identification of new fast-acting antimalarials. The apicoplast organelle is a preferred target of antimalarial drugs because it contains biochemical processes absent from the human host. Fosmidomycin is the only drug in clinical trials targeting the apicoplast, where it inhibits the methyl erythritol phosphate (MEP) pathway. Here, we characterized the antiplasmodial activity of domiphen bromide (DB), another MEP pathway inhibitor with a rapid mode of action that arrests the in vitro growth of Plasmodium falciparum at the early trophozoite stage. Metabolomic analysis of the MEP pathway and Krebs cycle intermediates in 20 mu M DB-treated parasites suggested a rapid activation of glycolysis with a concomitant decrease in mitochondrial activity, consistent with a rapid killing of the pathogen. These results present DB as a model compound for the development of new, potentially interesting drugs for future antimalarial combination therapies.

JTD Keywords: antibiotics, antimalarial drugs, domiphen bromide, malaria, plasmodium falciparum, Antibiotics, Antimalarial drugs, Antimalarial-drug, Artemisinin, Combination therapies, Domiphen bromide, Intraerythrocytic stages, Isoprenoid biosynthesis, Malaria, Methyl erythritol phosphate pathway, Nonmevalonate pathway, Plasmodium falciparum, Plasmodium-falciparum apicoplast, Red-blood-cells, Targeted delivery

The use of silver nanoparticles (Ag NPs) in the biomedical field deserves a mindful analysis of the possible inflammatory response which could limit their use in the clinic. Despite the anti-cancer properties of Ag NPs having been widely demonstrated, there are still few studies concerning their involvement in the activation of specific inflammatory pathways. The inflammatory outcome depends on the synthetic route used in the NPs production, in which toxic reagents are employed. In this work, we compared two types of Ag NPs, obtained by two different chemical routes: conventional synthesis using sodium citrate and a green protocol based on leaf extracts as a source of reduction and capping agents. A careful physicochemical characterization was carried out showing spherical and stable Ag NPs with an average size between 20 nm and 35 nm for conventional and green Ag NPs respectively. Then, we evaluated their ability to induce the activation of inflammation in Human Leukemic Monocytes (THP-1) differentiated into M0 macrophages using 1 µM and 2 µM NPs concentrations (corresponded to 0.1 µg/mL and 0.2 µg/mL respectively) and two-time points (24 h and 48 h). Our results showed a clear difference in Nuclear Factor ?B (NF-?b) activation, Interleukins 6–8 (IL-6, IL-8) secretion, Tumor Necrosis Factor-? (TNF-?) and Cyclooxygenase-2 (COX-2) expression exerted by the two kinds of Ag NPs. Green Ag NPs were definitely tolerated by macrophages compared to conventional Ag NPs which induced the activation of all the factors mentioned above. Subsequently, the exposure of breast cancer cell line (MCF-7) to the green Ag NPs showed that they exhibited antitumor activity like the conventional ones, but surprisingly, using the MCF-10A line (not tumoral breast cells) the green Ag NPs did not cause a significant decrease in cell viability. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.

JTD Keywords: activation, biosynthesis, gold nanoparticles, green route, inflammation response, mechanism, metal, nanotechnology, physico-chemical properties, raman-spectroscopy, resonance, silver nanoparticles, surface, Biomedical fields, Cell culture, Cell death, Chemical activation, Chemical routes, Conventional synthesis, Diseases, Green route, Inflammation response, Inflammatory response, Macrophages, Metal nanoparticles, Nf-kappa-b, Pathology, Physico-chemical properties, Physicochemical property, Property, Silver nanoparticles, Sodium compounds, Synthetic routes, Toxic reagents

© 2021 Introduction: Natural products of pharmaceutical interest often do not reach the drug market due to the associated low yields and difficult extraction. Knowledge of biosynthetic pathways is a key element in the development of biotechnological strategies for plant specialized metabolite production. The scarce studies regarding non-model plants impair advances in this field. Erythrina spp. are mainly used as central nervous system depressants in folk medicine and are important sources of bioactive tetracyclic benzylisoquinoline alkaloids, which can act on several pathology-related biological targets. Objective: Herein the purpose is to employ combined transcriptome and metabolome analyses (seeds and leaves) of a non-model medicinal Fabaceae species grown in its unique arid natural habitat. The study tries to propose a putative biosynthetic pathway for the bioactive alkaloids by using an omic integrated approach. Methods: The Next Generation Sequencing-based transcriptome (de novo RNA sequencing) was carried out in a Illumina NextSeq 500 platform. Regarding the targeted metabolite profiling, Nuclear Magnetic Resonance and the High-Performance Liquid Chromatography coupled to a micrOTOF-QII, High Resolution Mass Spectrometer, were used. Results: This detailed macro and micromolecular approach applied to seeds and leaves of E. velutina revealed 42 alkaloids by metabolome tools. Based on the combined evidence, 24 gene candidates were put together in a putative pathway leading to the singular alkaloid diversity of this species. Conclusion: These results contribute by indicating potential biotechnological targets Erythrina alkaloids biosynthesis as well as to improve molecular databases with omic data from a non-model medicinal plant. Furthermore, they reveal an interesting chemical diversity in Erythrina velutina harvested in Caatinga. Last, but not least, this data may also contribute to tap Brazilian biodiversity in a rational and sustainable fashion, promoting adequate public policies for preservation and protection of sensitive areas within the Caatinga.

JTD Keywords: benzylisoquinoline alkaloids, caatinga, codeinone reductase, erythrina velutina, expression, mass-spectrometry, molecular-cloning, morphine biosynthesis, natural-products, opium poppy, papaver-somniferum, plant-metabolism, targeted metabolite profile, transcriptome, Benzylisoquinoline alkaloids, Berberine bridge enzyme, Caatinga, Erythrina velutina, Targeted metabolite profile, Transcriptome

Although coronaviruses (CoVs) have long been predicted to cause zoonotic diseases and pandemics with high probability, the lack of effective anti-pan-CoVs drugs rapidly usable against the emerging SARS-CoV-2 actually prevented a promptly therapeutic intervention for COVID-19. Development of host-targeting antivirals could be an alternative strategy for the control of emerging CoVs infections, as they could be quickly repositioned from one pandemic event to another. To contribute to these pandemic preparedness efforts, here we report on the broad-spectrum CoVs antiviral activity of MEDS433, a new inhibitor of the human dihydroorotate dehydrogenase (hDHODH), a key cellular enzyme of the de novo pyrimidine biosynthesis pathway. MEDS433 in-hibited the in vitro replication of hCoV-OC43 and hCoV-229E, as well as of SARS-CoV-2, at low nanomolar range. Notably, the anti-SARS-CoV-2 activity of MEDS433 against SARS-CoV-2 was also observed in kidney organoids generated from human embryonic stem cells. Then, the antiviral activity of MEDS433 was reversed by the addition of exogenous uridine or the product of hDHODH, the orotate, thus confirming hDHODH as the specific target of MEDS433 in hCoVs-infected cells. Taken together, these findings suggest MEDS433 as a potential candidate to develop novel drugs for COVID-19, as well as broad-spectrum antiviral agents exploitable for future CoVs threats.

JTD Keywords: antiviral activity, biosynthesis, broad-spectrum antiviral, combination treatment, coronavirus, dipyridamole, hdhodh inhibitor, organoids, pyrimidine, pyrimidine biosynthesis, sars-cov-2, target, virus-infection, Antiviral activ-ity, Broad-spectrum antiviral, Combination treatment, Coronavirus, Gene-expression, Hdhodh inhibitor, Organoids, Pyrimidine biosynthesis, Sars-cov-2

The H-NS protein plays a significant role in the modulation of gene expression in Gram-negative bacteria. Whereas isolation and characterization of hns mutants in Escherichia coli, Salmonella and Shigella represented critical steps to gain insight into the modulatory role of H-NS, it has hitherto not been possible to isolate hns mutants in Yersinia. The hns mutation is considered to be deleterious in this genus. To study the modulatory role of H-NS in Yersinia we circumvented hns lethality by expressing in Y. enterocolitica a truncated H-NS protein known to exhibit anti-H-NS activity in E. coli (H-NST(EPEC)). Y. enterocolitica cells expressing H-NST(EPEC) showed an altered growth rate and several differences in the protein expression pattern, including the ProV protein, which is modulated by H-NS in other enteric bacteria. To further confirm that H-NST(EPEC) expression in Yersinia can be used to demonstrate H-NS-dependent regulation in this genus, we used this approach to show that H-NS modulates expression of the YmoA protein.

JTD Keywords: Bacterial Proteins/biosynthesis/genetics/ physiology, DNA-Binding Proteins/biosynthesis/genetics/ physiology, Electrophoresis, Gel, Two-Dimensional, Gene Expression Profiling, Gene Expression Regulation, Bacterial, Genes, Essential, Proteome/analysis, RNA, Bacterial/biosynthesis, RNA, Messenger/biosynthesis, Reverse Transcriptase Polymerase Chain Reaction, Sequence Deletion, Yersinia enterocolitica/chemistry/genetics/growth & development/ physiology