scholarly journals Structural characterization of cocktail-like targeting polysaccharides from Ecklonia kurome Okam and their anti-SARS-CoV-2 activities invitro

2021 ◽  
Author(s):  
Shihai Zhang ◽  
Rongjuan Pei ◽  
Meixia Li ◽  
Hao Sun ◽  
Minbo Su ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Monosaccharide Composition ◽  
Structure Study ◽  
Exchange Chromatography ◽  
Spike Protein ◽  
Drug Candidate ◽  
Mannuronic Acid ◽  
Guluronic Acid ◽  
The Impact

AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent responsible for the worldwide coronavirus disease 2019 (COVID-19) outbreak. Investigation has confirmed that polysaccharide heparan sulfate can bind to the spike protein and block SARS-CoV-2 infection. Theoretically, similar structure of nature polysaccharides may also have the impact on the virus. Indeed, some marine polysaccharide has been reported to inhibit SARS-Cov-2 infection in vitro, however the convinced targets and mechanism are still vague. By high throughput screening to target 3CLpro enzyme, a key enzyme that plays a pivotal role in the viral replication and transcription using nature polysaccharides library, we discover the mixture polysaccharide 375 from seaweed Ecklonia kurome Okam completely block 3Clpro enzymatic activity (IC50, 0.48 µM). Further, the homogeneous polysaccharide 37502 from the 375 may bind to 3CLpro molecule well (kD value : 4.23 × 10−6). Very interestingly, 37502 also can potently disturb spike protein binding to ACE2 receptor (EC50, 2.01 µM). Importantly, polysaccharide 375 shows good anti-SARS-CoV-2 infection activity in cell culture with EC50 values of 27 nM (99.9% inhibiting rate at the concentration of 20 µg/mL), low toxicity (LD50: 136 mg/Kg on mice). By DEAE ion-exchange chromatography, 37501, 37502 and 37503 polysaccharides are purified from native 375. Bioactivity test show that 37501 and 37503 may impede SARS-Cov-2 infection and virus replication, however their individual impact on the virus is significantly less that of 375. Surprisingly, polysaccharide 37502 has no inhibition effect on SARS-Cov-2. The structure study based on monosaccharide composition, methylation, NMR spectrum analysis suggest that 375 contains guluronic acid, mannuronic acid, mannose, rhamnose, glucouronic acid, galacturonic acid, glucose, galactose, xylose and fucose with ratio of 1.86 : 9.56 : 6.81 : 1.69 : 1.00 : 1.75 : 1.19 : 11.06 : 4.31 : 23.06. However, polysaccharide 37502 is an aginate which composed of mannuronic acid (89.3 %) and guluronic acid (10.7 %), with the molecular weight (Mw) of 27.9 kDa. These results imply that mixture polysaccharides 375 works better than the individual polysaccharide on SARS-Cov-2 may be the cocktail-like polysaccharide synergistic function through targeting multiple key molecules implicated in the virus infection and replication. The results also suggest that 375 may be a potential drug candidate against SARS-CoV-2.

scholarly journals Dalbavancin binds ACE2 to block its interaction with SARS-CoV-2 spike protein and is effective in inhibiting SARS-CoV-2 infection in animal models

Cell Research ◽  
2020 ◽  
Vol 31 (1) ◽  
pp. 17-24 ◽  
Author(s):  
Gan Wang ◽  
Meng-Li Yang ◽  
Zi-Lei Duan ◽  
Feng-Liang Liu ◽  
Lin Jin ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Spike Protein ◽  
Drug Candidate ◽  
Peptide Drug ◽  
Converting Enzyme ◽  
Angiotensin Converting Enzyme 2 ◽  
High Affinity ◽  
Plasma Half Life

AbstractInfection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic worldwide. Currently, however, no effective drug or vaccine is available to treat or prevent the resulting coronavirus disease 2019 (COVID-19). Here, we report our discovery of a promising anti-COVID-19 drug candidate, the lipoglycopeptide antibiotic dalbavancin, based on virtual screening of the FDA-approved peptide drug library combined with in vitro and in vivo functional antiviral assays. Our results showed that dalbavancin directly binds to human angiotensin-converting enzyme 2 (ACE2) with high affinity, thereby blocking its interaction with the SARS-CoV-2 spike protein. Furthermore, dalbavancin effectively prevents SARS-CoV-2 replication in Vero E6 cells with an EC50 of ~12 nM. In both mouse and rhesus macaque models, viral replication and histopathological injuries caused by SARS-CoV-2 infection are significantly inhibited by dalbavancin administration. Given its high safety and long plasma half-life (8–10 days) shown in previous clinical trials, our data indicate that dalbavancin is a promising anti-COVID-19 drug candidate.

scholarly journals From Human Pluripotent Stem Cells to 3D Cardiac Microtissues: Progress, Applications and Challenges

2020 ◽  
Vol 7 (3) ◽  
pp. 92
Author(s):  
Mariana A. Branco ◽  
Joaquim M.S. Cabral ◽  
Maria Margarida Diogo
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
High Throughput Screening ◽  
Cardiac Development ◽  
Human Pluripotent Stem Cells ◽  
Cardiac Cells ◽  
Cardiac Disorders ◽  
The Impact

The knowledge acquired throughout the years concerning the in vivo regulation of cardiac development has promoted the establishment of directed differentiation protocols to obtain cardiomyocytes (CMs) and other cardiac cells from human pluripotent stem cells (hPSCs), which play a crucial role in the function and homeostasis of the heart. Among other developments in the field, the transition from homogeneous cultures of CMs to more complex multicellular cardiac microtissues (MTs) has increased the potential of these models for studying cardiac disorders in vitro and for clinically relevant applications such as drug screening and cardiotoxicity tests. This review addresses the state of the art of the generation of different cardiac cells from hPSCs and the impact of transitioning CM differentiation from 2D culture to a 3D environment. Additionally, current methods that may be employed to generate 3D cardiac MTs are reviewed and, finally, the adoption of these models for in vitro applications and their adaptation to medium- to high-throughput screening settings are also highlighted.

Abstracts

2008 ◽  
Vol 27 (6) ◽  
pp. 405-405
Author(s):  
David J. Dix
Keyword(s):  
Risk Assessment ◽  
High Throughput ◽  
Environmental Protection Agency ◽  
High Throughput Screening ◽  
Human Health Risk ◽  
Environmental Chemicals ◽  
In Vitro Assays ◽  
The Impact ◽  
Toxicity Pathways

The U.S. Environmental Protection Agency (EPA), National Toxicology Program (NTP), and National Institutes of Health (NIH) Chemical Genomics Center (NCGC) have complementary research programs designed to improve chemical toxicity evaluations by developing high throughput screening (HTS) methods that evaluate the impact of environmental chemicals on key toxicity pathways. These federal partners are coordinating an extension of the EPA’s ToxCast program, the NTP’s HTS initiative, and the NCGC’s Molecular Libraries Initiative into a collaborative research program focused on identifying toxicity pathways and developing in vitro assays to characterize the ability of chemicals to perturb those pathways. The goal is to develop new paradigm for high throughput toxicity testing that collects mechanistic and quantitative data from in vitro assays measuring chemical modulation of biological processes involved in the progression to toxicity. As toxicity pathways are identified, the in vitro assays can be optimized for comparison to in vivo animal studies, and for predicting effects in humans. Subsequent computational modeling of toxicity pathway responses and appropriate chemical dosimetry will need to be developed to make these predictions relevant for human health risk assessment. This work was reviewed by EPA and approved for publication but does not necessarily reflect official Agency policy. Index Terms: Toxicogenomics, High Throughput Screening/Testing, EPA ToxCast, Chemical Risk Assessment

scholarly journals Structural Characterization and Immune Activity Screening of Polysaccharides With Different Molecular Weights From Astragali Radix

2020 ◽  
Vol 11 ◽  
Author(s):  
Ke Li ◽  
Y-x Cao ◽  
S-m Jiao ◽  
G-h Du ◽  
Y-g Du ◽  
...  
Keyword(s):  
Linkage Analysis ◽  
Monosaccharide Composition ◽  
Structure Study ◽  
Nuclear Magnetic Resonance Analysis ◽  
Astragali Radix ◽  
Immune Activity ◽  
Molecular Weights ◽  
Screening Experiments

Saccharides are the most abundant substance with the strongest immunological activity in Astragali Radix (AR). However, systematic structure study and immunoactivity screening of polysaccharides with different molecular weights (Mw) in AR have yet to be conducted. In this study, Astragalus polysaccharides (APSs) were divided into three fragments of different Mw values, >2,000 kDa (APS-Ⅰ), about 10 kDa (APS-Ⅱ), and about 300 Da (APS-Ⅲ), by using ultrafiltration for the first time. The structural differences of the three products were determined on the basis of monosaccharide composition, FT-IR spectrum, linkage analysis, and nuclear magnetic resonance analysis. Cellular immune activity experiments in vitro and cyclophosphamide immunosuppression animal model experiments in vivo for nonspecific and specific immunoactivity screening were applied to identify the most immunogenic fragment in APSs. Linkage analysis results showed that APS-Ⅰ, APS-Ⅱ, and APS-Ⅲ have different attachment sites of monosaccharide residues. Immune screening experiments indicated that the Mw of the APSs influenced their activity, and APS-Ⅱ had the strongest immunoenhancing activity among the products. This research may serve as a reference for further study on APSs with different structures and immune activities, and as a guidance for the quality control of APSs and the development of new APS products.

scholarly journals The adaptive potential of a survival artist: characterization of thein vitrointeractions ofToxoplasma gondiitachyzoites with di-cationic compounds in human fibroblast cell cultures

Parasitology ◽  
2011 ◽  
Vol 139 (2) ◽  
pp. 208-220 ◽  
Author(s):  
CHRISTIAN KROPF ◽  
KARIM DEBACHE ◽  
CHRISTOPH RAMPA ◽  
FABIENNE BARNA ◽  
MICHELLE SCHORER ◽  
...  
Keyword(s):  
Drug Exposure ◽  
Environmental Changes ◽  
Negative Impact ◽  
Fibroblast Cell ◽  
Drug Candidate ◽  
Selective Toxicity ◽  
Cationic Compounds ◽  
The Impact

SUMMARYThe impact of di-cationic pentamidine-analogues againstToxoplama gondii(Rh- and Me49-background) was investigated. The 72 h-growth assays showed that the arylimidamide DB750 inhibited the proliferation of tachyzoites ofT. gondii RhandT. gondii Me49with an IC50of 0·11 and 0·13μm, respectively. Pre-incubation of fibroblast monolayers with 1μmDB750 for 12 h and subsequent culture in the absence of the drug also resulted in a pronounced inhibiton of parasite proliferation. However, upon 5–6 days of drug exposure,T. gondiitachyzoites adapted to the compound and resumed proliferation up to a concentration of 1·2μm. Out of a set of 32 di-cationic compounds screened forin vitroactivity againstT. gondii,the arylimidamide DB745, exhibiting an IC50of 0·03μmand favourable selective toxicity was chosen for further studies. DB745 also inhibited the proliferation of DB750-adaptedT. gondii(IC50=0·07μm). In contrast to DB750, DB745 also had a profound negative impact on extracellular non-adaptedT. gondiitachyzoites, but not on DB750-adaptedT. gondii. Adaptation ofT. gondiito DB745 (up to a concentration of 0·46μm) was much more difficult to achieve and feasible only over a period of 110 days. In cultures infected with DB750-adaptedT. gondiiseemingly intact parasites could occasionally be detected by TEM. This illustrates the astonishing capacity ofT. gondiitachyzoites to adapt to environmental changes, at least underin vitroconditions, and suggests that DB745 could be an interesting drug candidate for further assessments in appropriatein vivomodels.

scholarly journals Molecular Structure Study on the Polyelectrolyte Properties of Actin Filaments

2022 ◽  
Author(s):  
Santiago M Bedoya ◽  
Marcelo Marucho
Keyword(s):  
Molecular Structure ◽  
Actin Filaments ◽  
Surrounding Medium ◽  
Electrical Potential ◽  
Finite Size ◽  
Electrolyte Solutions ◽  
Structure Study ◽  
Structure Model ◽  
The Impact

An accurate characterization of the polyelectrolyte properties of actin filaments might provide a deeper understanding of the fundamental mechanisms governing the intracellular ionic wave packet propagation in neurons. Infinitely long cylindrical models for actin filaments and approximate electrochemical theories for the electrolyte solutions were recently used to characterize these properties in in-vitro and intracellular conditions. This article uses a molecular structure model for actin filaments to investigate the impact of roughness and finite size on the mean electrical potential, ionic density distributions, currents, and conductivities. We solved the electrochemical theories numerically without further approximations. Our findings bring new insights into the electrochemical interactions between a filament′s irregular surface charge density and the surrounding medium. The irregular shape of the filament structure model generated pockets, or hot spots, where the current density reached higher or lower magnitudes than those in neighboring areas throughout the filament surface. It also revealed the formation of a well-defined asymmetric electrical double layer with a thickness larger than that commonly used for symmetric models.

scholarly journals Exploiting Mannuronan C-5 Epimerases in Commercial Alginate Production

Marine Drugs ◽  
2020 ◽  
Vol 18 (11) ◽  
pp. 565
Author(s):  
Anne Tøndervik ◽  
Olav A. Aarstad ◽  
Randi Aune ◽  
Susan Maleki ◽  
Philip D. Rye ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Brown Algae ◽  
Amperometric Detection ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Proton Nuclear Magnetic Resonance ◽  
Drug Candidate ◽  
Low Molecular Weight ◽  
Laminaria Hyperborea ◽  
Mannuronic Acid

Alginates are one of the major polysaccharide constituents of marine brown algae in commercial manufacturing. However, the content and composition of alginates differ according to the distinct parts of these macroalgae and have a direct impact on the concentration of guluronate and subsequent commercial value of the final product. The Azotobacter vinelandii mannuronan C-5 epimerases AlgE1 and AlgE4 were used to determine their potential value in tailoring the production of high guluronate low-molecular-weight alginates from two sources of high mannuronic acid alginates, the naturally occurring harvested brown algae (Ascophyllum nodosum, Durvillea potatorum, Laminaria hyperborea and Lessonia nigrescens) and a pure mannuronic acid alginate derived from fermented production of the mutant strain of Pseudomonas fluorescens NCIMB 10,525. The mannuronan C-5 epimerases used in this study increased the content of guluronate from 32% up to 81% in both the harvested seaweed and bacterial fermented alginate sources. The guluronate-rich alginate oligomers subsequently derived from these two different sources showed structural identity as determined by proton nuclear magnetic resonance (1H NMR), high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and size-exclusion chromatography with online multi-angle static laser light scattering (SEC-MALS). Functional identity was determined by minimum inhibitory concentration (MIC) assays with selected bacteria and antibiotics using the previously documented low-molecular-weight guluronate enriched alginate OligoG CF-5/20 as a comparator. The alginates produced using either source showed similar antibiotic potentiation effects to the drug candidate OligoG CF-5/20 currently in development as a mucolytic and anti-biofilm agent. These findings clearly illustrate the value of using epimerases to provide an alternative production route for novel low-molecular-weight alginates.

scholarly journals Towards High-Throughput Chemobehavioural Phenomics in Neuropsychiatric Drug Discovery

Marine Drugs ◽  
2019 ◽  
Vol 17 (6) ◽  
pp. 340 ◽  
Author(s):  
Jason Henry ◽  
Donald Wlodkowic
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Therapeutic Potential ◽  
Innovative Methods ◽  
Approaches To Study ◽  
The Central Nervous System ◽  
Psychiatric Conditions ◽  
The Impact ◽  
Screening Approaches

Identifying novel marine-derived neuroactive chemicals with therapeutic potential is difficult due to inherent complexities of the central nervous system (CNS), our limited understanding of the molecular foundations of neuro-psychiatric conditions, as well as the limited applications of effective high-throughput screening models that recapitulate functionalities of the intact CNS. Furthermore, nearly all neuro-modulating chemicals exhibit poorly characterized pleiotropic activities often referred to as polypharmacology. The latter renders conventional target-based in vitro screening approaches very difficult to accomplish. In this context, chemobehavioural phenotyping using innovative small organism models such as planarians and zebrafish represent powerful and highly integrative approaches to study the impact of new chemicals on central and peripheral nervous systems. In contrast to in vitro bioassays aimed predominantly at identification of chemicals acting on single targets, phenotypic chemobehavioural analysis allows for complex multi-target interactions to occur in combination with studies of polypharmacological effects of chemicals in a context of functional and intact milieu of the whole organism. In this review, we will outline recent advances in high-throughput chemobehavioural phenotyping and provide a future outlook on how those innovative methods can be utilized for rapidly screening and characterizing marine-derived compounds with prospective applications in neuropharmacology and psychosomatic medicine.

scholarly journals Inhibition of SARS-CoV-2 viral entry in vitro upon blocking N- and O-glycan elaboration

2020 ◽  
Author(s):  
Qi Yang ◽  
Thomas A. Hughes ◽  
Anju Kelkar ◽  
Xinheng Yu ◽  
Kai Cheng ◽  
...  
Keyword(s):  
Viral Entry ◽  
Multiple Roles ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Mechanistic Studies ◽  
Post Translational Modification ◽  
Minor Contribution ◽  
Chemical Inhibitors ◽  
The Impact

ABSTRACTThe Spike protein of SARS-CoV-2, its receptor binding domain (RBD), and its primary receptor ACE2 are extensively glycosylated. The impact of this post-translational modification on viral entry is yet unestablished. We expressed different glycoforms of the Spike-protein and ACE2 in CRISPR-Cas9 glycoengineered cells, and developed corresponding SARS-CoV-2 pseudovirus. We observed that N- and O-glycans had only minor contribution to Spike-ACE2 binding. However, these carbohydrates played a major role in regulating viral entry. Blocking N-glycan biosynthesis at the oligomannose stage using both genetic approaches and the small molecule kifunensine dramatically reduced viral entry into ACE2 expressing HEK293T cells. Blocking O-glycan elaboration also partially blocked viral entry. Mechanistic studies suggest multiple roles for glycans during viral entry. Among them, inhibition of N-glycan biosynthesis enhanced Spike-protein proteolysis. This could reduce RBD presentation on virus, lowering binding to host ACE2 and decreasing viral entry. Overall, chemical inhibitors of glycosylation may be evaluated for COVID-19.

scholarly journals Immunogenicity of trimetric spike protein associated to Poly(I:C) plus Alum

2021 ◽  
Author(s):  
Julio dos Santos ◽  
Luan Firmino Cruz ◽  
Alessandra Marcia da Fonseca-Martins ◽  
Diogo Oliveira Maciel ◽  
Gustavo Guadagnini Perez ◽  
...  
Keyword(s):  
B Cells ◽  
Vaccination Strategy ◽  
Lavage Fluid ◽  
Neutralization Assay ◽  
Spike Protein ◽  
Poly I:C ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cellular Immune ◽  
The Impact

The SARS-CoV-2 pandemic has had a social and economic impact worldwide, and vaccination is an efficient strategy for diminishing those damages. New adjuvant formulations are required for the high vaccine demands, especially adjuvant formulations that induce a Th1 phenotype. Herein we assess a vaccination strategy using a combination of Alum and polyinosinic:polycytidylic acid (Poly(I:C)) adjuvants plus the SARS-CoV-2 spike protein in a prefusion trimeric conformation by an intradermal (ID) route. We found high levels of IgG anti-spike antibodies in the serum by enzyme linked immunosorbent assay (ELISA) and high neutralizing titers against SARS-CoV-2 in vitro by neutralization assay, after one or two boosts. By evaluating the production of IgG subtypes, as expected, we found that formulations containing Poly(I:C) induced IgG2a whereas Alum did not. The combination of these two adjuvants induced high levels of both IgG1 and IgG2a. In addition, cellular immune responses of CD4+ and CD8+ T cells producing interferon-gamma were equivalent, demonstrating that the Alum + Poly(I:C) combination supported a Th1 profile. Based on the high neutralizing titers, we evaluated B cells in the germinal centers, which are specific for receptor-binding domain (RBD) and spike, and observed that more positive B cells were induced upon the Alum + Poly(I:C) combination. Moreover, these B cells produced antibodies against both RBD and non-RBD sites. We also studied the impact of this vaccination preparation (S Ptn with Alum + Poly(I:C)) in the lungs of mice challenged with inactivated SARS-CoV-2 virus. We found a production of IgG, but not IgA, and a reduction in neutrophil recruitment in the bronchoalveolar lavage fluid (BALF) of mice, suggesting that our immunization scheme reduced lung inflammation. Altogether, our data suggest that Alum and Poly(I:C) together is a possible adjuvant combination for vaccines against SARS-CoV-2 by the intradermal route.

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