scholarly journals Probing the SAM Binding Site of SARS-CoV-2 Nsp14 In Vitro Using SAM Competitive Inhibitors Guides Developing Selective Bisubstrate Inhibitors

2021 ◽  
pp. 247255522110262
Author(s):  
Kanchan Devkota ◽  
Matthieu Schapira ◽  
Sumera Perveen ◽  
Aliakbar Khalili Yazdi ◽  
Fengling Li ◽  
...  
Keyword(s):  
Nonstructural Protein ◽  
Competitive Inhibitor ◽  
Human Immune System ◽  
Mtase Activity ◽  
Competitive Inhibitors ◽  
Human Immune ◽  
Rna Capping ◽  
Socioeconomic Consequences ◽  
Bisubstrate Inhibitors

The COVID-19 pandemic has clearly brought the healthcare systems worldwide to a breaking point, along with devastating socioeconomic consequences. The SARS-CoV-2 virus, which causes the disease, uses RNA capping to evade the human immune system. Nonstructural protein (nsp) 14 is one of the 16 nsps in SARS-CoV-2 and catalyzes the methylation of the viral RNA at N7-guanosine in the cap formation process. To discover small-molecule inhibitors of nsp14 methyltransferase (MTase) activity, we developed and employed a radiometric MTase assay to screen a library of 161 in-house synthesized S-adenosylmethionine (SAM) competitive MTase inhibitors and SAM analogs. Among six identified screening hits, SS148 inhibited nsp14 MTase activity with an IC50 value of 70 ± 6 nM and was selective against 20 human protein lysine MTases, indicating significant differences in SAM binding sites. Interestingly, DS0464 with an IC50 value of 1.1 ± 0.2 µM showed a bisubstrate competitive inhibitor mechanism of action. DS0464 was also selective against 28 out of 33 RNA, DNA, and protein MTases. The structure–activity relationship provided by these compounds should guide the optimization of selective bisubstrate nsp14 inhibitors and may provide a path toward a novel class of antivirals against COVID-19, and possibly other coronaviruses.

scholarly journals Probing the SAM Binding Site of SARS-CoV-2 nsp14 in vitro Using SAM Competitive Inhibitors Guides Developing Selective bi-substrate Inhibitors

2021 ◽  
Author(s):  
Kanchan Devkota ◽  
Matthieu Schapira ◽  
Sumera Perveen ◽  
Aliakbar Khalili Yazdi ◽  
Fengling Li ◽  
...  
Keyword(s):  
Binding Site ◽  
Rna Binding ◽  
Phenyl Group ◽  
Competitive Inhibitor ◽  
Structural Protein ◽  
Human Immune System ◽  
Rna Capping ◽  
Protein Methyltransferases ◽  
Socioeconomic Consequences

AbstractThe COVID-19 pandemic has clearly brought the healthcare systems world-wide to a breaking point along with devastating socioeconomic consequences. The SARS-CoV-2 virus which causes the disease uses RNA capping to evade the human immune system. Non-structural protein (nsp) 14 is one of the 16 nsps in SARS-CoV-2 and catalyzes the methylation of the viral RNA at N7-guanosine in the cap formation process. To discover small molecule inhibitors of nsp14 methyltransferase (MT) activity, we developed and employed a radiometric MT assay to screen a library of 161 in house synthesized S-adenosylmethionine (SAM) competitive methyltransferase inhibitors and SAM analogs. Among seven identified screening hits, SS148 inhibited nsp14 MT activity with an IC50 value of 70 ± 6 nM and was selective against 20 human protein lysine methyltransferases indicating significant differences in SAM binding sites. Interestingly, DS0464 with IC50 value of 1.1 ± 0.2 μM showed a bi-substrate competitive inhibitor mechanism of action. Modeling the binding of this compound to nsp14 suggests that the terminal phenyl group extends into the RNA binding site. DS0464 was also selective against 28 out of 33 RNA, DNA, and protein methyltransferases. The structure-activity relationship provided by these compounds should guide the optimization of selective bi-substrate nsp14 inhibitors and may provide a path towards a novel class of antivirals against COVID-19, and possibly other coronaviruses.

scholarly journals A High-Throughput RNA Displacement Assay for Screening SARS-CoV-2 nsp10-nsp16 Complex toward Developing Therapeutics for COVID-19

2021 ◽  
pp. 247255522098504
Author(s):  
Sumera Perveen ◽  
Aliakbar Khalili Yazdi ◽  
Kanchan Devkota ◽  
Fengling Li ◽  
Pegah Ghiabi ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Nonstructural Protein ◽  
Cost Effective ◽  
Titration Calorimetry ◽  
Human Immune System ◽  
High Throughput Method ◽  
Displacement Assay ◽  
Human Immune ◽  
Rna Capping

SARS-CoV-2, the coronavirus that causes COVID-19, evades the human immune system by capping its RNA. This process protects the viral RNA and is essential for its replication. Multiple viral proteins are involved in this RNA capping process, including the nonstructural protein 16 (nsp16), which is an S-adenosyl-l-methionine (SAM)-dependent 2′- O-methyltransferase. Nsp16 is significantly active when in complex with another nonstructural protein, nsp10, which plays a key role in its stability and activity. Here we report the development of a fluorescence polarization (FP)-based RNA displacement assay for nsp10-nsp16 complex in a 384-well format with a Z′ factor of 0.6, suitable for high-throughput screening. In this process, we purified the nsp10-nsp16 complex to higher than 95% purity and confirmed its binding to the methyl donor SAM, the product of the reaction, S-adenosyl-l-homocysteine (SAH), and a common methyltransferase inhibitor, sinefungin, using isothermal titration calorimetry (ITC). The assay was further validated by screening a library of 1124 drug-like compounds. This assay provides a cost-effective high-throughput method for screening the nsp10-nsp16 complex for RNA competitive inhibitors toward developing COVID-19 therapeutics.

scholarly journals A high throughput RNA displacement assay for screening SARS-CoV-2 nsp10-nsp16 complex towards developing therapeutics for COVID-19

2020 ◽  
Author(s):  
Sumera Perveen ◽  
Aliakbar Khalili Yazdi ◽  
Kanchan Devkota ◽  
Fengling Li ◽  
Pegah Ghiabi ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Nonstructural Protein ◽  
Cost Effective ◽  
Titration Calorimetry ◽  
Human Immune System ◽  
High Throughput Method ◽  
Displacement Assay ◽  
Human Immune ◽  
Rna Capping

AbstractSARS-CoV-2, the coronavirus that causes COVID-19, evades the human immune system by capping its RNA. This process protects the viral RNA and is essential for its replication. Multiple viral proteins are involved in this RNA capping process including the nonstructural protein 16 (nsp16) which is an S-adenosyl-L-methionine (SAM)-dependent 2’-O-methyltransferase. Nsp16 is significantly active when in complex with another nonstructural protein, nsp10, which plays a key role in its stability and activity. Here we report the development of a fluorescence polarization (FP)-based RNA displacement assay for nsp10-nsp16 complex in 384-well format with a Z′-Factor of 0.6, suitable for high throughput screening. In this process, we purified the nsp10-nsp16 complex to higher than 95% purity and confirmed its binding to the methyl donor SAM, product of the reaction, SAH, and a common methyltransferase inhibitor, sinefungin using Isothermal Titration Calorimetry (ITC). The assay was further validated by screening a library of 1124 drug-like compounds. This assay provides a cost-effective high throughput method for screening nsp10-nsp16 complex for RNA-competitive inhibitors towards developing COVID-19 therapeutics.

scholarly journals Mesenchymal Stem Cells in COVID-19: A Journey from Bench to Bedside

2020 ◽  
Vol 52 (1) ◽  
pp. 24-35
Author(s):  
Kamal Kant Sahu ◽  
Ahmad Daniyal Siddiqui ◽  
Jan Cerny
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Immune System ◽  
Therapeutic Option ◽  
Antiviral Agent ◽  
In Vitro Models ◽  
Human Immune System ◽  
Economic Sectors ◽  
Human Immune

Abstract The COVID-19 pandemic has led to a major setback in both the health and economic sectors across the globe. The scale of the problem is enormous because we still do not have any specific anti-SARS-CoV-2 antiviral agent or vaccine. The human immune system has never been exposed to this novel virus, so the viral interactions with the human immune system are completely naive. New approaches are being studied at various levels, including animal in vitro models and human-based studies, to contain the COVID-19 pandemic as soon as possible. Many drugs are being tested for repurposing, but so far only remdesivir has shown some positive benefits based on preliminary reports, but these results also need further confirmation via ongoing trials. Otherwise, no other agents have shown an impactful response against COVID-19. Recently, research exploring the therapeutic application of mesenchymal stem cells (MSCs) in critically ill patients suffering from COVID-19 has gained momentum. The patients belonging to this subset are most likely beyond the point where they could benefit from an antiviral therapy because most of their illness at this stage of disease is driven by inflammatory (over)response of the immune system. In this review, we discuss the potential of MSCs as a therapeutic option for patients with COVID-19, based on the encouraging results from the preliminary data showing improved outcomes in the progression of COVID-19 disease.

scholarly journals Inhibition of Hepatitis C Virus Replication by GS-6620, a PotentC-Nucleoside Monophosphate Prodrug

2014 ◽  
Vol 58 (4) ◽  
pp. 1930-1942 ◽  
Author(s):  
Joy Y. Feng ◽  
Guofeng Cheng ◽  
Jason Perry ◽  
Ona Barauskas ◽  
Yili Xu ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Nonstructural Protein ◽  
Competitive Inhibitor ◽  
High Dose ◽  
Diarrhea Virus ◽  
A Chain ◽  
Direct Acting ◽  
Hcv Ns5b

ABSTRACTAs a class, nucleotide inhibitors (NIs) of the hepatitis C virus (HCV) nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase offer advantages over other direct-acting antivirals, including properties, such as pangenotype activity, a high barrier to resistance, and reduced potential for drug-drug interactions. We studied thein vitropharmacology of a novelC-nucleoside adenosine analog monophosphate prodrug, GS-6620. It was found to be a potent and selective HCV inhibitor against HCV replicons of genotypes 1 to 6 and against an infectious genotype 2a virus (50% effective concentration [EC50], 0.048 to 0.68 μM). GS-6620 showed limited activities against other viruses, maintaining only some of its activity against the closely related bovine viral diarrhea virus (EC50, 1.5 μM). The active 5′-triphosphate metabolite of GS-6620 is a chain terminator of viral RNA synthesis and a competitive inhibitor of NS5B-catalyzed ATP incorporation, withKi/Kmvalues of 0.23 and 0.18 for HCV NS5B genotypes 1b and 2a, respectively. With its unique dual substitutions of 1′-CN and 2′-C-Me on the ribose ring, the active triphosphate metabolite was found to have enhanced selectivity for the HCV NS5B polymerase over host RNA polymerases. GS-6620 demonstrated a high barrier to resistancein vitro. Prolonged passaging resulted in the selection of the S282T mutation in NS5B that was found to be resistant in both cellular and enzymatic assays (>30-fold). Consistent with itsin vitroprofile, GS-6620 exhibited the potential for potent anti-HCV activity in a proof-of-concept clinical trial, but its utility was limited by the requirement of high dose levels and pharmacokinetic and pharmacodynamic variability.

scholarly journals Immunomodulatory Effects of Edible and Medicinal Mushrooms and Their Bioactive Immunoregulatory Products

2020 ◽  
Vol 6 (4) ◽  
pp. 269
Author(s):  
Shuang Zhao ◽  
Qi Gao ◽  
Chengbo Rong ◽  
Shouxian Wang ◽  
Zhekun Zhao ◽  
...  
Keyword(s):  
Bioactive Compounds ◽  
Immune Modulation ◽  
Essential Amino Acids ◽  
Current Status ◽  
Human Immune System ◽  
Mushroom Species ◽  
Medicinal Mushrooms ◽  
Human Immune

Mushrooms have been valued as food and health supplements by humans for centuries. They are rich in dietary fiber, essential amino acids, minerals, and many bioactive compounds, especially those related to human immune system functions. Mushrooms contain diverse immunoregulatory compounds such as terpenes and terpenoids, lectins, fungal immunomodulatory proteins (FIPs) and polysaccharides. The distributions of these compounds differ among mushroom species and their potent immune modulation activities vary depending on their core structures and fraction composition chemical modifications. Here we review the current status of clinical studies on immunomodulatory activities of mushrooms and mushroom products. The potential mechanisms for their activities both in vitro and in vivo were summarized. We describe the approaches that have been used in the development and application of bioactive compounds extracted from mushrooms. These developments have led to the commercialization of a large number of mushroom products. Finally, we discuss the problems in pharmacological applications of mushrooms and mushroom products and highlight a few areas that should be improved before immunomodulatory compounds from mushrooms can be widely used as therapeutic agents.

scholarly journals Structure–function analysis of the nsp14 N7–guanine methyltransferase reveals an essential role in Betacoronavirus replication

2021 ◽  
Vol 118 (49) ◽  
pp. e2108709118
Author(s):  
Natacha S. Ogando ◽  
Priscila El Kazzi ◽  
Jessika C. Zevenhoven-Dobbe ◽  
Brenda W. Bontes ◽  
Alice Decombe ◽  
...  
Keyword(s):  
Function Analysis ◽  
Nonstructural Protein ◽  
Site Directed Mutagenesis ◽  
Messenger Rnas ◽  
Mtase Activity ◽  
Mrna Capping ◽  
Activity Spectrum ◽  
Immune Sensing ◽  
Key Residues

As coronaviruses (CoVs) replicate in the host cell cytoplasm, they rely on their own capping machinery to ensure the efficient translation of their messenger RNAs (mRNAs), protect them from degradation by cellular 5′ exoribonucleases (ExoNs), and escape innate immune sensing. The CoV nonstructural protein 14 (nsp14) is a bifunctional replicase subunit harboring an N-terminal 3′-to-5′ ExoN domain and a C-terminal (N7-guanine)–methyltransferase (N7-MTase) domain that is presumably involved in viral mRNA capping. Here, we aimed to integrate structural, biochemical, and virological data to assess the importance of conserved N7-MTase residues for nsp14’s enzymatic activities and virus viability. We revisited the crystal structure of severe acute respiratory syndrome (SARS)–CoV nsp14 to perform an in silico comparative analysis between betacoronaviruses. We identified several residues likely involved in the formation of the N7-MTase catalytic pocket, which presents a fold distinct from the Rossmann fold observed in most known MTases. Next, for SARS-CoV and Middle East respiratory syndrome CoV, site-directed mutagenesis of selected residues was used to assess their importance for in vitro enzymatic activity. Most of the engineered mutations abolished N7-MTase activity, while not affecting nsp14-ExoN activity. Upon reverse engineering of these mutations into different betacoronavirus genomes, we identified two substitutions (R310A and F426A in SARS-CoV nsp14) abrogating virus viability and one mutation (H424A) yielding a crippled phenotype across all viruses tested. Our results identify the N7-MTase as a critical enzyme for betacoronavirus replication and define key residues of its catalytic pocket that can be targeted to design inhibitors with a potential pan-coronaviral activity spectrum.

scholarly journals Immune Organs and Immune Cells on a Chip: An Overview of Biomedical Applications

Micromachines ◽  
2020 ◽  
Vol 11 (9) ◽  
pp. 849 ◽  
Author(s):  
Margaretha Morsink ◽  
Niels Willemen ◽  
Jeroen Leijten ◽  
Ruchi Bansal ◽  
Su Shin
Keyword(s):  
Immune System ◽  
Human Immune System ◽  
Immune Organs ◽  
Future Challenges ◽  
Human Immune ◽  
Conventional Models ◽  
On Chip ◽  
And Behavior

Understanding the immune system is of great importance for the development of drugs and the design of medical implants. Traditionally, two-dimensional static cultures have been used to investigate the immune system in vitro, while animal models have been used to study the immune system’s function and behavior in vivo. However, these conventional models do not fully emulate the complexity of the human immune system or the human in vivo microenvironment. Consequently, many promising preclinical findings have not been reproduced in human clinical trials. Organ-on-a-chip platforms can provide a solution to bridge this gap by offering human micro-(patho)physiological systems in which the immune system can be studied. This review provides an overview of the existing immune-organs-on-a-chip platforms, with a special emphasis on interorgan communication. In addition, future challenges to develop a comprehensive immune system-on-chip model are discussed.

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