scholarly journals Inhibition of glycogen synthase kinase-3-beta (GSK3β) blocks nucleocapsid phosphorylation and SARS-CoV-2 replication

Author(s):  
Tirosh Shapira ◽  
Celine Rens ◽  
Virginia Pichler ◽  
William Rees ◽  
Theodor Steiner ◽  
...  

Abstract GSK3β has been proposed to have an essential role in Coronaviridae infection. Screening of a targeted library of GSK3β inhibitors against SARS-CoV-2 and HCoV-229E resulted in identification of high proportion of active compounds with low toxicity to host cells. A select lead compound, T-1686568, showed dose-dependent activity against SARS-CoV-2 transcription, translation and viral particle release in multiple cell lines and primary organoids. A protein kinase substrate profiling assay combined with western blot analysis showed that SARS-CoV-2 nucleocapsid is phosphorylated by GSK3β on S180/S184, S190/S194 and T198 which have already been primed in the adjacent phospho-sites S188, T198 and S206 respectively. Inhibition by T-1686568 resulted in reduction of the S1 Spike protein levels, an accumulation of the Nucleocapsid (N) protein and maintenance of the non-structural (NSP2) level in infected Huh-7.5.1 cells, indicating that N phosphorylation might serve as a critical precursor for processing and release of mature viruses.

2013 ◽  
Vol 2013 ◽  
pp. 1-13 ◽  
Author(s):  
Deivid C. Soares ◽  
Nathalya A. Portella ◽  
Mônica Freiman de S. Ramos ◽  
Antonio C. Siani ◽  
Elvira M. Saraiva

This study investigated the leishmanicidal activity againstLeishmania amazonensisof four commercial oils fromCopaiferaspp. named as C1, C2, C3, and C4, the sesquiterpene and diterpene pools obtained from distilling C4, and isolatedβ-caryophyllene (CAR). Copaiba oils chemical compositions were analyzed by gas chromatography and correlated with biological activities. Diterpenes-rich oils C2 and C3 showed antipromastigote activity. Sesquiterpenes-rich C1 and C4, and isolated CAR presented a dose-dependent activity against intracellular amastigotes, with IC50sof 2.9 µg/mL, 2.3 µg/mL, and 1.3 µg/mL (6.4 µM), respectively. Based on the highest antiamastigote activity and the low toxicity to the host cells, C4 was steamdistillated to separate pools of sesquiterpenes and diterpenes. Both pools were less active againstL. amazonensisand more toxic for the macrophages than the whole C4 oil. The leishmanicidal activity of C3 and C4 oils, as well as C4 fractions and CAR, appears to be independent of nitric oxide production by macrophages. This study pointed outβ-caryophyllene as an effective antileishmanial compound and also to its role as potential chemical marker in copaiba oils or fractions derived thereof, aiming further development of this rainforest raw material for leishmaniasis therapy.


Plants ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 346
Author(s):  
Caitlin W. Lehman ◽  
Kylene Kehn-Hall ◽  
Megha Aggarwal ◽  
Nicole R. Bracci ◽  
Han-Chi Pan ◽  
...  

The host proteins Protein Kinase B (AKT) and glycogen synthase kinase-3 (GSK-3) are associated with multiple neurodegenerative disorders. They are also important for the replication of Venezuelan equine encephalitis virus (VEEV), thereby making the AKT/GSK-3 pathway an attractive target for developing anti-VEEV therapeutics. Resveratrol, a natural phytochemical, has been shown to substantially inhibit the AKT pathway. Therefore, we attempted to explore whether it exerts any antiviral activity against VEEV. In this study, we utilized green fluorescent protein (GFP)- and luciferase-encoding recombinant VEEV to determine the cytotoxicity and antiviral efficacy via luciferase reporter assays, flow cytometry, and immunofluorescent assays. Our results indicate that resveratrol treatment is capable of inhibiting VEEV replication, resulting in increased viability of Vero and U87MG cells as well as reduced virion production and viral RNA contents within host cells for at least 48 h with a single treatment. Furthermore, the suppression of apoptotic signaling adaptors, caspase-3, caspase-7, and annexin V may also be implicated in resveratrol-mediated antiviral activity. We found that decreased phosphorylation of the AKT/GSK-3 pathway, mediated by resveratrol, can be triggered during the early stages of VEEV infection, suggesting that resveratrol disrupts the viral replication cycle and consequently promotes cell survival. Finally, molecular docking and dynamics simulation studies revealed that resveratrol can directly bind to VEEV glycoproteins, which may interfere with virus attachment and entry. In conclusion, our results suggest that resveratrol exerts inhibitory activity against VEEV infection and upon further modification could be a useful compound to study in neuroprotective research and veterinary sciences.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Dandan Shan ◽  
Joseph M. Johnson ◽  
Syrena C. Fernandes ◽  
Hannah Suib ◽  
Soyoon Hwang ◽  
...  

AbstractThe COVID-19 pandemic continues to have an unprecedented impact on societies and economies worldwide. There remains an ongoing need for high-performance SARS-CoV-2 tests which may be broadly deployed for infection monitoring. Here we report a highly sensitive single molecule array (Simoa) immunoassay in development for detection of SARS-CoV-2 nucleocapsid protein (N-protein) in venous and capillary blood and saliva. In all matrices in the studies conducted to date we observe >98% negative percent agreement and >90% positive percent agreement with molecular testing for days 1–7 in symptomatic, asymptomatic, and pre-symptomatic PCR+ individuals. N-protein load decreases as anti-SARS-CoV-2 spike-IgG increases, and N-protein levels correlate with RT-PCR Ct-values in saliva, and between matched saliva and capillary blood samples. This Simoa SARS-CoV-2 N-protein assay effectively detects SARS-CoV-2 infection via measurement of antigen levels in blood or saliva, using non-invasive, swab-independent collection methods, offering potential for at home and point of care sample collection.


2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii13-ii13
Author(s):  
Wangxian Gu ◽  
Guoqing Wan ◽  
Yanjun Zheng ◽  
Xintong Yang ◽  
Peng Zhang ◽  
...  

Abstract Diacylglycerol kinase (DGK) is a lipid kinase that catalyzes the phosphorylation of diacylglycerol (DAG) to produce phosphatidic acid (PA), which uses ATP as a phosphate donor. Diacylglycerol kinases ζ(DGKζ) is characterized as specific type IV due to its myristoylated alanine-rich C-kinase substrate (MARCKS), ankyrin, and PDZ binding domain. Similar to other DGKs, DGKζ is also reported to be abnormally expressed in human colorectal cancer cells, and it is indispensable for the proliferation of cancer cells. However, its implications in human glioblastoma (GBM) is largely unknown. Both the mRNA and protein levels of DGKζ were significantly higher in GBM tissues than in precancerous lesions. Knockdown of DGKζ inhibited GBM cell proliferation, cell cycle and promoted apoptosis of GBM cells. Moreover, down-regulation of DGKζ markedly reduced in vitro colony formation and in vivo tumorigenic capability. Furthermore, we confirmed that DGKζ was the downstream target of miR-34a. The expression level of DGKζ was negatively correlated with miR-34a in GBM tissues. Overexpression of DGKζ reversed the tumor suppressive roles of miR-34a in GBM cells. Taken together, DGKζ can act as a potential prognostic biomarker for GBM patients and promote the growth of GBM cells was regulated by miR-34a, and it may represent a promising therapeutic target for patients with GBM.


2020 ◽  
Vol 8 (2) ◽  
pp. 164 ◽  
Author(s):  
Xifang Zhu ◽  
Yaqi Dong ◽  
Eric Baranowski ◽  
Xixi Li ◽  
Gang Zhao ◽  
...  

Molecules contributing to microbial cytoadhesion are important virulence factors. In Mycoplasma bovis, a minimal bacterium but an important cattle pathogen, binding to host cells is emerging as a complex process involving a broad range of surface-exposed structures. Here, a new cytoadhesin of M. bovis was identified by producing a collection of individual knock-out mutants and evaluating their binding to embryonic bovine lung cells. The cytoadhesive-properties of this surface-exposed protein, which is encoded by Mbov_0503 in strain HB0801, were demonstrated at both the mycoplasma cell and protein levels using confocal microscopy and ELISA. Although Mbov_0503 disruption was only associated in M. bovis with a partial reduction of its binding capacity, this moderate effect was sufficient to affect M. bovis interaction with the host-cell tight junctions, and to reduce the translocation of this mycoplasma across epithelial cell monolayers. Besides demonstrating the capacity of M. bovis to disrupt tight junctions, these results identified novel properties associated with cytoadhesin that might contribute to virulence and host colonization. These findings provide new insights into the complex interplay taking place between wall-less mycoplasmas and the host-cell surface.


Foods ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 382
Author(s):  
Laura Arru ◽  
Francesca Mussi ◽  
Luca Forti ◽  
Annamaria Buschini

The Mediterranean-style diet is rich in fruit and vegetables and has a great impact on the prevention of major chronic diseases, such as cardiovascular diseases and cancer. In this work we investigated the ability of spinach extracts obtained by different extraction methods and of the single main components of the phytocomplex, alone or mixed, to modulate proliferation, antioxidant defense, and genotoxicity of HT29 human colorectal cells. Spinach extracts show dose-dependent activity, increasing the level of intracellular endogenous reactive oxygen species (ROS) when tested at higher doses. In the presence of oxidative stress, the activity is related to the oxidizing agent involved (H2O2 or menadione) and by the extraction method. The single components of the phytocomplex, alone or mixed, do not alter the intracellular endogenous level of ROS but again, in the presence of an oxidative insult, the modulation of antioxidant defense depends on the oxidizing agent used. The application of the phytocomplex extracts seem to be more effective than the application of the single phytocomplex components.


2019 ◽  
Vol 2019 ◽  
pp. 1-6 ◽  
Author(s):  
Éva Áy ◽  
Attila Hunyadi ◽  
Mária Mezei ◽  
János Minárovits ◽  
Judit Hohmann

Here we report the evaluation of the antiretroviral effect of two flavonoid 7-O-glucosides, herbacitrin (1) and gossypitrin (2), together with quercetin (3), a well-studied flavonol. Antiviral activity of the flavonoids was assessed by analyzing HIV-1 p24 core protein levels in the supernatants of HIV-1 infected MT-4 and MT-2 cell cultures. The compounds showed mild to weak cytotoxic activities on the host cells; herbacitrin was the strongest in this regard (CC50=27.8 and 63.64 μM on MT-4 and MT-2 cells, respectively). In nontoxic concentrations, herbacitrin and quercetin reduced HIV-1 replication, whereas gossypitrin was ineffective. Herbacitrin was found to inhibit reverse transcriptase at 21.5 μM, while it was a more potent integrase inhibitor already active at 2.15 μM. Therefore, our observations suggest that herbacitrin exerts antiretroviral activity through simultaneously acting on these two targets of HIV-1 and that integrase inhibition might play a major role in this activity.


2015 ◽  
Vol 35 (8) ◽  
pp. 1390-1400 ◽  
Author(s):  
Nancy Yu ◽  
Michael Kakunda ◽  
Victoria Pham ◽  
Jennie R. Lill ◽  
Pan Du ◽  
...  

The Wnt/β-catenin pathway causes accumulation of β-catenin in the cytoplasm and its subsequent translocation into the nucleus to initiate the transcription of the target genes. Without Wnt stimulation, β-catenin forms a complex with axin (axis inhibitor), adenomatous polyposis coli (APC), casein kinase 1α (CK1α), and glycogen synthase kinase 3β (GSK3β) and undergoes phosphorylation-dependent ubiquitination. Phosphatases, such as protein phosphatase 2A (PP2A), interestingly, also are components of this degradation complex; therefore, a balance must be reached between phosphorylation and dephosphorylation. How this balance is regulated is largely unknown. Here we show that a heat shock protein, HSP105, is a previously unidentified component of the β-catenin degradation complex. HSP105 is required for Wnt signaling, since depletion of HSP105 compromises β-catenin accumulation and target gene transcription upon Wnt stimulation. Mechanistically, HSP105 depletion disrupts the integration of PP2A into the β-catenin degradation complex, favoring the hyperphosphorylation and degradation of β-catenin. HSP105 is overexpressed in many types of tumors, correlating with increased nuclear β-catenin protein levels and Wnt target gene upregulation. Furthermore, overexpression of HSP105 is a prognostic biomarker that correlates with poor overall survival in breast cancer patients as well as melanoma patients participating in the BRIM2 clinical study.


Author(s):  
Xiaoling Wu ◽  
Zhiqin Yang ◽  
Huimin Dang ◽  
Huixia Peng ◽  
Zhijun Dai

Baicalein, a flavonoid derived from the root of Scutellaria baicalensis, has been reported to possess multiple pharmacological activities, such as anticancer and anti-inflammatory properties. This study investigated the effect of baicalein in cervical cancer cells. Cell growth curve and MTT assay were performed and revealed that baicalein inhibited the proliferation of SiHa and HeLa cells in a dose-dependent manner. We further found that baicalein arrested the cell cycle of SiHa and HeLa cells at the G0/G1 phase by suppressing the expression of cyclin D1 through the downregulation of phosphorylated protein kinase B (p-AKT) and phosphorylated glycogen synthase kinase 3β (p-GSK3β) according to FACS assays and Western blotting. Moreover, when CHIR-99021, a GSK3β inhibitor, was added to baicalein-treated SiHa cells, the expression of cyclin D1 was recovered, and cell proliferation was promoted. In conclusion, these data indicated that baicalein suspended the cell cycle at the G0/G1 phase via the downregulation of cyclin D1 through the AKT‐GSK3β signaling pathway and further inhibited the proliferation of SiHa and HeLa cervical cancer cells.


2021 ◽  
Vol 3 (Supplement_6) ◽  
pp. vi6-vi6
Author(s):  
Takashi Fujii ◽  
Shun Yamamuro ◽  
Masamichi Takahashi ◽  
Akihide Kondo ◽  
Yoshitaka Narita ◽  
...  

Abstract The therapeutic outcome of glioblastomas (GBMs) is still very poor. Therefore, invention of novel therapeutic methods against GBM cases is considered urgent. The antitumor effects of naturally-derived compounds are attracting attention recently, and therapeutic efficacy of curcumin, a plant-derived compound previously used for multiple purpose, has been indicated in many cancer systems; however, clinical application of curcumin is considered difficult because of its poor bioavailability (under 1 %). Curcumin monoglucuronide (CMG), a water-soluble prodrug of curcumin recently developed for overcoming this weakness, has been demonstrated excellent antitumor effects for several malignancies in vitro and in vivo; therefore, we investigated the effects of CMG against GBM cells. CMG induced cell death of human GBM cells lines (T98G, U251MG, and U87MG) by dose dependent manner by triggering multiple forms of cell death such as apoptosis and perthanatos. Immunoblotting of CMG-treated GBM cell lysates demonstrated activation of multiple cell death signaling. Furthermore, immunodeficiency mice harboring intracerebral U87MG cell xenografts systemically treated by CMG showed significantly prolonged survival compared with control mice. These results suggest CMG would be a novel therapeutic agent against GBM cases.


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