scholarly journals The NSP14/NSP10 RNA repair complex as a Pan-coronavirus therapeutic target

2021 ◽  
Author(s):  
Gergely Rona ◽  
Andras Zeke ◽  
Bearach Miwatani-Minter ◽  
Maren de Vries ◽  
Ramanjit Kaur ◽  
...  
Keyword(s):  
Therapeutic Target ◽  
Human Population ◽  
Model Compound ◽  
Antiviral Effect ◽  
Cellular Systems ◽  
Proof Of Concept ◽  
Human Coronavirus ◽  
Rna Repair ◽  
In Silico Modeling

AbstractThe risk of zoonotic coronavirus spillover into the human population, as highlighted by the SARS-CoV-2 pandemic, demands the development of pan-coronavirus antivirals. The efficacy of existing antiviral ribonucleoside/ribonucleotide analogs, such as remdesivir, is decreased by the viral proofreading exonuclease NSP14-NSP10 complex. Here, using a novel assay and in silico modeling and screening, we identified NSP14-NSP10 inhibitors that increase remdesivir’s potency. A model compound, sofalcone, both inhibits the exonuclease activity of SARS-CoV-2, SARS-CoV, and MERS-CoV in vitro, and synergistically enhances the antiviral effect of remdesivir, suppressing the replication of SARS-CoV-2 and the related human coronavirus OC43. The validation of top hits from our primary screenings using cellular systems provides proof-of-concept for the NSP14 complex as a therapeutic target.

scholarly journals In vitro evaluation of the activity of terpenes and cannabidiol against Human Coronavirus E229

2021 ◽  
Author(s):  
Lior Chatow ◽  
Adi Nudel ◽  
Iris Nesher ◽  
David Hayo Hemo ◽  
Perri Rozenberg ◽  
...  
Keyword(s):  
Antiviral Effect ◽  
Host Cells ◽  
Lung Fibroblasts ◽  
Human Coronavirus ◽  
Cell Viability Assay ◽  
Viability Assay ◽  
A Cell ◽  
Quantitative Results ◽  
Better Than

AbstractThe activity of a new, terpene-based formulation, code-named NT-VRL-1, against Human Coronavirus (HCoV) strain 229E was evaluated in human lung fibroblasts (MRC-5 cells), with and without the addition of cannabidiol (CBD). The tested formulation exhibited an antiviral effect when it was pre-incubated with the host cells prior to virus infection. The combination of NT-VRL-1 with CBD potentiated the antiviral effect better than the positive controls pyrazofurin and glycyrrhizin. There was a strong correlation between the quantitative results from a cell-viability assay and the cytopathic effect seen under the microscope after 72 h. To the best of our knowledge, this is the first report of activity of a combination of terpenes and CBD against a coronavirus.

scholarly journals In Vitro Evaluation of the Activity of Terpenes and Cannabidiol against Human Coronavirus E229

Life ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 290
Author(s):  
Lior Chatow ◽  
Adi Nudel ◽  
Iris Nesher ◽  
David Hayo Hemo ◽  
Perri Rozenberg ◽  
...  
Keyword(s):  
Antiviral Effect ◽  
Host Cells ◽  
Lung Fibroblasts ◽  
Human Coronavirus ◽  
Cell Viability Assay ◽  
Viability Assay ◽  
A Cell ◽  
Quantitative Results ◽  
Better Than

The activity of a new, terpene-based formulation, code-named NT-VRL-1, against Human Coronavirus (HCoV) strain 229E was evaluated in human lung fibroblasts (MRC-5 cells), with and without the addition of cannabidiol (CBD). The main constituents in the terpene formulation used for the experiment were beta caryophyllene, eucalyptol, and citral. The tested formulation exhibited an antiviral effect when it was pre-incubated with the host cells prior to virus infection. The combination of NT-VRL-1 with CBD potentiated the antiviral effect better than the positive controls pyrazofurin and glycyrrhizin. There was a strong correlation between the quantitative results from a cell-viability assay and the cytopathic effect seen under the microscope after 72 h. To the best of our knowledge, this is the first report of activity of a combination of terpenes and CBD against a coronavirus.

scholarly journals A high-throughput screening method for evolving a demethylase enzyme with improved and new functionalities

2020 ◽  
Author(s):  
Yuru Wang ◽  
Christopher D Katanski ◽  
Christopher Watkins ◽  
Jessica N Pan ◽  
Qing Dai ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Screening Method ◽  
Targeted Mutagenesis ◽  
Rna Repair ◽  
Dna And Rna ◽  
Modified Dna ◽  
Dna Substrates ◽  
Trna Sequencing

Abstract AlkB is a DNA/RNA repair enzyme that removes base alkylations such as N1-methyladenosine (m1A) or N3-methylcytosine (m3C) from DNA and RNA. The AlkB enzyme has been used as a critical tool to facilitate tRNA sequencing and identification of mRNA modifications. As a tool, AlkB mutants with better reactivity and new functionalities are highly desired; however, previous identification of such AlkB mutants was based on the classical approach of targeted mutagenesis. Here, we introduce a high-throughput screening method to evaluate libraries of AlkB variants for demethylation activity on RNA and DNA substrates. This method is based on a fluorogenic RNA aptamer with an internal modified RNA/DNA residue which can block reverse transcription or introduce mutations leading to loss of fluorescence inherent in the cDNA product. Demethylation by an AlkB variant eliminates the blockage or mutation thereby restores the fluorescence signals. We applied our screening method to sites D135 and R210 in the Escherichia coli AlkB protein and identified a variant with improved activity beyond a previously known hyperactive mutant toward N1-methylguanosine (m1G) in RNA. We also applied our method to O6-methylguanosine (O6mG) modified DNA substrates and identified candidate AlkB variants with demethylating activity. Our study provides a high-throughput screening method for in vitro evolution of any demethylase enzyme.

scholarly journals HDAC4 promotes nasopharyngeal carcinoma progression and serves as a therapeutic target

2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Chun Cheng ◽  
Jun Yang ◽  
Si-Wei Li ◽  
Guofu Huang ◽  
Chenxi Li ◽  
...  
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Tumor Growth ◽  
Therapeutic Target ◽  
Histone Deacetylases ◽  
Migration And Invasion ◽  
Poor Overall Survival ◽  
Mesenchymal Transition ◽  
E Cadherin

AbstractHistone deacetylases (HDACs) are involved in tumor progression, and some have been successfully targeted for cancer therapy. The expression of histone deacetylase 4 (HDAC4), a class IIa HDAC, was upregulated in our previous microarray screen. However, the role of HDAC4 dysregulation and mechanisms underlying tumor growth and metastasis in nasopharyngeal carcinoma (NPC) remain elusive. Here, we first confirmed that the HDAC4 levels in primary and metastatic NPC tissues were significantly increased compared with those in normal nasopharyngeal epithelial tissues and found that high HDAC4 expression predicted a poor overall survival (OS) and progression-free survival (PFS). Functionally, HDAC4 accelerated cell cycle G1/S transition and induced the epithelial-to-mesenchymal transition to promote NPC cell proliferation, migration, and invasion in vitro, as well as tumor growth and lung metastasis in vivo. Intriguingly, knockdown of N-CoR abolished the effects of HDAC4 on the invasion and migration abilities of NPC cells. Mechanistically, HDAC3/4 binds to the E-cadherin promoter to repress E-cadherin transcription. We also showed that the HDAC4 inhibitor tasquinimod suppresses tumor growth in NPC. Thus, HDAC4 may be a potential diagnostic marker and therapeutic target in patients with NPC.

scholarly journals Direct antivirals working against the novel coronavirus: azithromycin (DAWn-AZITHRO), a randomized, multicenter, open-label, adaptive, proof-of-concept clinical trial of new antivirals working against SARS-CoV-2—azithromycin trial

Trials ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Iwein Gyselinck ◽  
◽  
Laurens Liesenborghs ◽  
Ewout Landeloos ◽  
Ann Belmans ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Standard Of Care ◽  
Ordinal Scale ◽  
Cytokine Signaling ◽  
Nasopharyngeal Swab ◽  
The Novel ◽  
Proof Of Concept ◽  
Open Label ◽  
Novel Coronavirus

Abstract Background The rapid emergence and the high disease burden of the novel coronavirus SARS-CoV-2 have created a medical need for readily available drugs that can decrease viral replication or blunt the hyperinflammatory state leading to severe COVID-19 disease. Azithromycin is a macrolide antibiotic, known for its immunomodulatory properties. It has shown antiviral effect specifically against SARS-CoV-2 in vitro and acts on cytokine signaling pathways that have been implicated in COVID-19. Methods DAWn-AZITHRO is a randomized, open-label, phase 2 proof-of-concept, multicenter clinical trial, evaluating the safety and efficacy of azithromycin for treating hospitalized patients with COVID-19. It is part of a series of trials testing promising interventions for COVID-19, running in parallel and grouped under the name DAWn-studies. Patients hospitalized on dedicated COVID wards are eligible for study inclusion when they are symptomatic (i.e., clinical or radiological signs) and have been diagnosed with COVID-19 within the last 72 h through PCR (nasopharyngeal swab or bronchoalveolar lavage) or chest CT scan showing typical features of COVID-19 and without alternate diagnosis. Patients are block-randomized (9 patients) with a 2:1 allocation to receive azithromycin plus standard of care versus standard of care alone. Standard of care is mostly supportive, but may comprise hydroxychloroquine, up to the treating physician’s discretion and depending on local policy and national health regulations. The treatment group receives azithromycin qd 500 mg during the first 5 consecutive days after inclusion. The trial will include 284 patients and recruits from 15 centers across Belgium. The primary outcome is time from admission (day 0) to life discharge or to sustained clinical improvement, defined as an improvement of two points on the WHO 7-category ordinal scale sustained for at least 3 days. Discussion The trial investigates the urgent and still unmet global need for drugs that may impact the disease course of COVID-19. It will either provide support or else justify the discouragement of the current widespread, uncontrolled use of azithromycin in patients with COVID-19. The analogous design of other parallel trials of the DAWN consortium will amplify the chance of identifying successful treatment strategies and allow comparison of treatment effects within an identical clinical context. Trial registration EU Clinical trials register EudraCT Nb 2020-001614-38. Registered on 22 April 2020

scholarly journals TRAF3 mediates neuronal apoptosis in early brain injury following subarachnoid hemorrhage via targeting TAK1-dependent MAPKs and NF-κB pathways

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yan Zhou ◽  
Tao Tao ◽  
Guangjie Liu ◽  
Xuan Gao ◽  
Yongyue Gao ◽  
...  
Keyword(s):  
Subarachnoid Hemorrhage ◽  
Brain Injury ◽  
Therapeutic Target ◽  
Cortical Neurons ◽  
Neuronal Apoptosis ◽  
Early Brain Injury ◽  
Neurological Deficits ◽  
Human Brain Tissue

AbstractNeuronal apoptosis has an important role in early brain injury (EBI) following subarachnoid hemorrhage (SAH). TRAF3 was reported as a promising therapeutic target for stroke management, which covered several neuronal apoptosis signaling cascades. Hence, the present study is aimed to determine whether downregulation of TRAF3 could be neuroprotective in SAH-induced EBI. An in vivo SAH model in mice was established by endovascular perforation. Meanwhile, primary cultured cortical neurons of mice treated with oxygen hemoglobin were applied to mimic SAH in vitro. Our results demonstrated that TRAF3 protein expression increased and expressed in neurons both in vivo and in vitro SAH models. TRAF3 siRNA reversed neuronal loss and improved neurological deficits in SAH mice, and reduced cell death in SAH primary neurons. Mechanistically, we found that TRAF3 directly binds to TAK1 and potentiates phosphorylation and activation of TAK1, which further enhances the activation of NF-κB and MAPKs pathways to induce neuronal apoptosis. Importantly, TRAF3 expression was elevated following SAH in human brain tissue and was mainly expressed in neurons. Taken together, our study demonstrates that TRAF3 is an upstream regulator of MAPKs and NF-κB pathways in SAH-induced EBI via its interaction with and activation of TAK1. Furthermore, the TRAF3 may serve as a novel therapeutic target in SAH-induced EBI.

scholarly journals SMAD1 as a biomarker and potential therapeutic target in drug-resistant multiple myeloma

2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Jian Wu ◽  
Min Zhang ◽  
Omar Faruq ◽  
Eldad Zacksenhaus ◽  
Wenming Chen ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Therapeutic Target ◽  
Biological Activities ◽  
Xenograft Model ◽  
Myeloma Cell ◽  
Soft Agar ◽  
Drug Resistant ◽  
Migration Assay

Abstract Background SMAD1, a central mediator in TGF-β signaling, is involved in a broad range of biological activities including cell growth, apoptosis, development and immune response, and is implicated in diverse type of malignancies. Whether SMAD1 plays an important role in multiple myeloma (MM) pathogenesis and can serve as a therapeutic target are largely unknown. Methods Myeloma cell lines and primary MM samples were used. Cell culture, cytotoxicity and apoptosis assay, siRNA transfection, Western blot, RT-PCR, Soft-agar colony formation, and migration assay, Chromatin immunoprecipitation (Chip), animal xenograft model studies and statistical analysis were applied in this study. Results We demonstrate that SMAD1 is highly expressed in myeloma cells of MM patients with advanced stages or relapsed disease, and is associated with significantly shorter progression-free and overall survivals. Mechanistically, we show that SMAD1 is required for TGFβ-mediated proliferation in MM via an ID1/p21/p27 pathway. TGF-β also enhanced TNFα-Induced protein 8 (TNFAIP8) expression and inhibited apoptosis through SMAD1-mediated induction of NF-κB1. Accordingly, depletion of SMAD1 led to downregulation of NF-κB1 and TNFAIP8, resulting in caspase-8-induced apoptosis. In turn, inhibition of NF-κB1 suppressed SMAD1 and ID1 expression uncovering an autoregulatory loop. Dorsomorphin (DM), a SMAD1 inhibitor, exerted a dose-dependent cytotoxic effect on drug-resistant MM cells with minimal cytotoxicity to normal hematopoietic cells, and further synergized with the proteasomal-inhibitor bortezomib to effectively kill drug-resistant MM cells in vitro and in a myeloma xenograft model. Conclusions This study identifies SMAD1 regulation of NF-κB1/TNFAIP8 and ID1-p21/p27 as critical axes of MM drug resistance and provides a potentially new therapeutic strategy to treat drug resistance MM through targeted inhibition of SMAD1.

A Sensitive Method for Assay of Mixed-Function Oxygenase (p-450 complex) in Cell Culture

1988 ◽  
Vol 15 (3) ◽  
pp. 219-223
Author(s):  
Jørgen Clausen ◽  
Søren Achim Nielsen
Keyword(s):  
Human Lymphocytes ◽  
Cellular Systems ◽  
Metabolic Effects ◽  
Assay Method ◽  
Related Family ◽  
Oxygenase Activity ◽  
Metabolic Transformation ◽  
Mixed Function Oxygenase

The mixed-function oxygenase system involved in the metabolism of drugs and xenobiotics has been extensively studied in various animal species and in various organs (1). It is now apparent that in humans the p-450 complex is one representative of a related family, expressed by 13 c-DNA genes showing approximately 36% similarity between the different subfamilies (2). In order to compare the in vivo and in vitro metabolic effects of drugs and xenobiotics, the induction capabilities of the mixed-function oxygenase must be known. The most sensitive non-isotopic assay system for determination of mixed-function oxygenase activity is the method of Nebert & Gelboin (3,4), which is based on the metabolic transformation of benzo-(a)-pyrene to its fluorescent hydroxyl derivatives (5). However, the levels of the mixed-function oxygenase enzymes in different cellular systems show great variations, with the highest activities in liver cells. Therefore, in order to use human lymphocytes and other cellular systems with low mixed-function oxygenase activities, the assay method for determining oxygenase activity must have the highest possible sensitivity. The present communication is devoted to a study aimed at increasing the sensitivity of Nebert & Gelboin's methods for assay of mixed-function oxygenase subfamilies using benzo-(a)-pyrene as a substrate.

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