demystifying-vivo-bioluminescence-imaging-chagas-disease-mouse-model

Demystifying In Vivo Bioluminescence Imaging of a Chagas Disease Mouse Model for Drug Efficacy Studies

To control and decrease the public health impact of human protozoan diseases such as Chagas disease, leishmaniasis, and human African trypanosomiasis, ...

Universidade de São Paulo

A library of 16 4-substituted 2-(1H-pyrrolo[3,2-c]pyridin-2-yl)propan-2-ols 17-32 has been synthesized for use in biological testing against Trypanosoma cruzi, the protozoan parasite that causes Chagas disease. The 4-substituted 2-(1H-pyrrolo[3,2-c]pyridin-2-yl)propan-2-ols 17-32 were subjected to biological testing to evaluate their efficacy against intracellular Trypanosoma cruzi (Y strain) amastigotes infecting U2OS human cells, with benznidazole as a reference compound. The assay was performed in duplicate (two independent experiments) and submitted to High Content Analysis (HCA) for determination of trypanocidal activity. Three of the tested compounds presented relatively high trypanocidal activity (19, 22 and 29), however severe host cell toxicity was observed concomitantly. Chemical optimization of the highly active compounds and the synthesis of more compounds for biological testing against Trypanosoma cruzi will be required to improve selectivity and so that a structure-activity relationship can be generated to provide a more insightful analysis of both chemical and biological aspects.

Synthesis and trypanocidal activity of a library of 4-substituted 2-(1H-pyrrolo[3,2-c]pyridin-2-yl)propan-2-ols.

The effects on potency of cruzain inhibition of replacing a nitrile group with alternative warheads were explored. The oxime was almost an order of magnitude more potent than the corresponding nitrile and has the potential to provide access to the prime side of the catalytic site. Dipeptide aldehydes and azadipeptide nitriles were found to be two orders of magnitude more potent cruzain inhibitors than the corresponding dipeptide nitriles although potency differences were modulated by substitution at P1 and P3. Replacement of the α methylene of a dipeptide aldehyde with cyclopropane led to a loss of potency of almost three orders of magnitude. The vinyl esters and amides that were characterized as reversible inhibitors were less potent than the corresponding nitrile by between one and two orders of magnitude.

A comparative study of warheads for design of cysteine protease inhibitors.

The current article describes the synthesis, characterization, and application of a designed hybrid fluorescent BTD-coumarin (2,1,3-benzothiadiazole-coumarin) derivative (named ). The use of  for live-cells staining showed excellent results, and lipid droplets (LDs) could be selectively stained. When compared with the commercially available dye (BODIPY) for LD staining, it was noted that the designed hybrid fluorescence was capable of staining a considerable larger number of LDs in both live and fixed cells (ca. 40% more). The new dye was also tested on live  (complex model) and showed an impressive selectivity inside the worm, whereas the commercial dye showed no selectivity in the complex model.

From Live Cells to : Selective Staining and Quantification of Lipid Structures Using a Fluorescent Hybrid Benzothiadiazole Derivative.

Cell-based screening has become the major compound interrogation strategy in Chagas disease drug discovery. Several different cell lines have been deployed as host cells in screening assays. However, host cell characteristics and host-parasite interactions may play an important role when assessing anti- compound activity, ultimately impacting on hit discovery. To verify this hypothesis, four distinct mammalian cell lines (U2OS, THP-1, Vero and L6) were used as  host cells in High Content Screening assays. Rates of infection varied greatly between different host cells. Susceptibility to benznidazole also varied, depending on the host cell and parasite strain. A library of 1,280 compounds was screened against the four different cell lines infected with , resulting in the selection of a total of 82 distinct compounds as hits. From these, only two hits were common to all four cell lines assays (2.4%) and 51 were exclusively selected from a single assay (62.2%). Infected U2OS cells were the most sensitive assay, as 55 compounds in total were identified as hits; infected THP-1 yielded the lowest hit rates, with only 16 hit compounds. Of the selected hits, compound FPL64176 presented selective anti- activity and could serve as a starting point for the discovery of new anti-chagasic drugs.

Drug Discovery for Chagas Disease: Impact of Different Host Cell Lines on Assay Performance and Hit Compound Selection.

The current methodologies used to identify promising new anthelmintic compounds rely on subjective microscopic examination of worm motility or involve genetic modified organisms. We describe a new methodology to detect worm viability that takes advantage of the differential incorporation of the fluorescent molecular marker propidium iodide and the 2,1,3-benzothiadiazole core, which has been widely applied in light technology. The new assay developed could be validated using the "Pathogen Box" library. By use of this bioassay, it was possible to identify three molecules with activity against  that were previously described as effective in in vitro assays against other pathogens, such as , and , accelerating the identification of molecules with anthelmintic potential. The current fluorescence-based bioassay may be used for assessing  viability. The described methodology replaces the subjectivity of previous assays and provides an enabling technology that is useful for rapid in vitro screens of both natural and synthetic compound libraries. It is expected that the results obtained from these robust in vitro screens would select the most effective compounds for follow-up in vivo experimentation with pathogenic helminths.

Expanding the Biological Application of Fluorescent Benzothiadiazole Derivatives: A Phenotypic Screening Strategy for Anthelmintic Drug Discovery Using .

High genetic and phenotypic variability between  species and strains within species make the development of broad-spectrum antileishmanial drugs challenging. Thus, screening panels consisting of several diverse  species can be useful in enabling compound prioritization based on their spectrum of activity. In this study, a robust and reproducible high content assay was developed, and 1280 small molecules were simultaneously screened against clinically relevant cutaneous and visceral species: , , and . The assay is based on THP-1 macrophages infected with stationary phase promastigotes and posterior evaluation of both compound antileishmanial activity and host cell toxicity. The profile of compound activity was species-specific, and out of 51 active compounds, only 14 presented broad-spectrum activity against the three species, with activities ranging from 52% to 100%. Notably, the compounds CB1954, Clomipramine, Maprotiline, Protriptyline, and ML-9 presented pan-leishmanial activity, with efficacy greater than 70%. The results highlight the reduced number of compound classes with pan-leishmanial activity that might be available from diversity libraries, emphasizing the need to screen active compounds against a panel of species and strains. The assay reported here can be adapted to virtually any  species without the need for genetic modification of parasites, providing the basis for the discovery of broad spectrum anti-leishmanial agents.

A Multi-Species Phenotypic Screening Assay for Leishmaniasis Drug Discovery Shows That Active Compounds Display a High Degree of Species-Specificity.

The emergence of the new corona virus (SARS-CoV-2) and the resulting COVID-19 pandemic requires fast development of novel prevention and therapeutic strategies. These rely on understanding the biology of the virus and its interaction with the host, and on agnostic phenotypic screening for compounds that prevent viral infection. In vitro screenings of compounds are usually performed in human or animal-derived tumor or immortalized cell lines due to their ease of culturing. However, these platforms may not represent the tissues affected by the disease in vivo, and therefore better models are needed to validate and expedite drug development, especially in face of the COVID-19 pandemic. In this scenario, human induced pluripotent stem cells (hiPSCs) are a powerful research tool due to their ability to generate normal differentiated cell types relevant for the disease. Here we discuss the different ways hiPSCs can contribute to COVID-19 related research, including modeling the disease in vitro and serving as a platform for drug screening.

Human induced pluripotent stem cells as a tool for disease modeling and drug screening for COVID-19.

Few extracts of plant species from the Brazilian flora have been validated from a pharmacological and clinical point of view, and it is important to determine whether their traditional use is proven by pharmacological effects.  is one of those plants, which belongs to the Fabaceae family that is widely used in traditional medicine and is very rich in tannins. Due to the lack of effective drugs to treat severe cases of Covid-19, the main protease of SARS-CoV-2 (M) becomes an attractive target in the research for new antivirals since this enzyme is crucial for virus replication and does not have homologs in humans. This study aimed to prospect inhibitor candidates among the compounds from  extract, by virtual screening simulations using SARS-CoV-2 M as target. Experimental validation was made by inhibitory proteolytic assays of recombinant M and by antiviral activity with infected Vero cells. Docking simulations identify four compounds with potential inhibitory activity of M present in the extract. The compound pentagalloylglucose showed the best result in proteolytic kinetics experiments, with suppression of recombinant M activity by approximately 60%. However, in experiments with infected cells ethyl acetate fraction and sub-fractions, F2 and F4 of  extract performed better than pentagalloylglucose, reaching close to 100% of antiviral activity. The prominent activity of the extract fractions in infected cells may be a result of a synergistic effect from the different hydrolyzable tannins present, performing simultaneous action on M and other targets from SARS-CoV-2 and host.Communicated by Ramaswamy H. Sarma.

Antiviral activity of  var.  extract and fractions against SARS-CoV-2.

The deaths caused by the covid-19 pandemic have recently decreased due to a worldwide effort in vaccination campaigns. However, even vaccinated people can develop a severe form of the disease that requires ICU admission. As a result, the search for antiviral drugs to treat these severe cases has become a necessity. In this context, natural products are an interesting alternative to synthetic medicines used in drug repositioning, as they have been consumed for a long time through traditional medicine. Many natural compounds found in plant extracts have already been shown to be effective in treating viral and bacterial diseases, making them possible hits to exploit against covid-19. The objective of this work was to evaluate the antiviral activity of different plant extracts available in the library of natural products of the Universidade Estadual de Maringá, by inhibiting the SARS-CoV-2 main protease (M), and by preventing viral infection in a cellular model. As a result, the extract of , obtained by ultrasound, showed a M inhibition capacity greater than 90%. In the infection model assays using Vero cells, an inhibition of 99.6% was observed, with a selectivity index of 42.7. The  molecular docking simulations using the extract compounds against M, suggested Tellimagrandin II as the component of  extract most likely to inhibit the viral enzyme. These results demonstrate the potential of  extract as a promising source of natural compounds with antiviral activity against covid-19.Communicated by Ramaswamy H. Sarma.

Potential of plant extracts in targeting SARS-CoV-2 main protease: an  and  study.

The use of copper as an antimicrobial agent has a long history and has gained renewed interest in the context of the COVID-19 pandemic. In this study, the authors investigated the antimicrobial properties of an alloy composed of copper with a small percentage of silver (Cu-0.03% wt.Ag). The alloy was tested against various pathogens, including , , , , and the H1N1 virus, using contact exposure tests. Results showed that the alloy was capable of inactivating these pathogens in two hours or less, indicating its strong antimicrobial activity. Electrochemical measurements were also performed, revealing that the small addition of silver to copper promoted a higher resistance to corrosion and shifted the formation of copper ions to higher potentials. This shift led to a slow but continuous release of Cu ions, which have high biocidal activity. These findings show that the addition of small amounts of silver to copper can enhance its biocidal properties and improve its effectiveness as an antimicrobial material.

Micro-Addition of Silver to Copper: One Small Step in Composition, a Change for a Giant Leap in Biocidal Activity.

The potential emergence of zoonotic diseases has raised significant concerns, particularly in light of the recent pandemic, emphasizing the urgent need for scientific preparedness. The bioprospection and characterization of new molecules are strategically relevant to the research and development of innovative drugs for viral and bacterial treatment and disease management. Amphibian species possess a diverse array of compounds, including antimicrobial peptides. This study identified the first bioactive peptide from  in a transcriptome analysis. The synthetic peptide sequence, which belongs to the defensin family, was characterized through MALDI TOF/TOF mass spectrometry. Molecular docking assays hypothesized the interaction between the identified peptide and the active binding site of the spike WT RBD/hACE2 complex. Although additional studies are required, the preliminary evaluation of the antiviral potential of synthetic SS-I was conducted through an in vitro cell-based SARS-CoV-2 infection assay. Additionally, the cytotoxic and hemolytic effects of the synthesized peptide were assessed. These preliminary findings highlighted the potential of SS-I as a chemical scaffold for drug development against COVID-19, hindering viral infection. The peptide demonstrated hemolytic activity while not exhibiting cytotoxicity at the antiviral concentration.

Article	Year
Synthesis and trypanocidal activity of a library of 4-substituted 2-(1H-pyrrolo[3,2-c]pyridin-2-yl)propan-2-ols. European journal of medicinal chemistry\| PubMed ID: 28189084	2017
A comparative study of warheads for design of cysteine protease inhibitors. Bioorganic & medicinal chemistry letters\| PubMed ID: 29054358	2017
From Live Cells to : Selective Staining and Quantification of Lipid Structures Using a Fluorescent Hybrid Benzothiadiazole Derivative. ACS omega\| PubMed ID: 30023883	2018
Drug Discovery for Chagas Disease: Impact of Different Host Cell Lines on Assay Performance and Hit Compound Selection. Tropical medicine and infectious disease\| PubMed ID: 31108888	2019
Expanding the Biological Application of Fluorescent Benzothiadiazole Derivatives: A Phenotypic Screening Strategy for Anthelmintic Drug Discovery Using . SLAS discovery : advancing life sciences R & D\| PubMed ID: 31180789	2019
A Multi-Species Phenotypic Screening Assay for Leishmaniasis Drug Discovery Shows That Active Compounds Display a High Degree of Species-Specificity. Molecules (Basel, Switzerland)\| PubMed ID: 32486239	2020
Human induced pluripotent stem cells as a tool for disease modeling and drug screening for COVID-19. Genetics and molecular biology\| PubMed ID: 33275129	2020
Antiviral activity of var. extract and fractions against SARS-CoV-2. Journal of biomolecular structure & dynamics\| PubMed ID: 36069130	2023
Potential of plant extracts in targeting SARS-CoV-2 main protease: an and study. Journal of biomolecular structure & dynamics\| PubMed ID: 36651196	2023
Micro-Addition of Silver to Copper: One Small Step in Composition, a Change for a Giant Leap in Biocidal Activity. Materials (Basel, Switzerland)\| PubMed ID: 38399167	2024
Antiviral Action against SARS-CoV-2 of a Synthetic Peptide Based on a Novel Defensin Present in the Transcriptome of the Fire Salamander (). Pharmaceutics\| PubMed ID: 38399250	2024

Lucio H. Freitas-Junior

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