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 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 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 Development of a High-Throughput Screening Assay to Identify Inhibitors of the SARS-CoV-2 Guanine-N7-Methyltransferase Using RapidFire Mass Spectrometry

2021 ◽  
pp. 247255522110006
Author(s):  
Lesley-Anne Pearson ◽  
Charlotte J. Green ◽  
De Lin ◽  
Alain-Pierre Petit ◽  
David W. Gray ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Mutational Analysis ◽  
Nonstructural Protein ◽  
Translation Efficiency ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Starting Point ◽  
Approved Drugs ◽  
High Throughput Assay

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) represents a significant threat to human health. Despite its similarity to related coronaviruses, there are currently no specific treatments for COVID-19 infection, and therefore there is an urgent need to develop therapies for this and future coronavirus outbreaks. Formation of the cap at the 5′ end of viral RNA has been shown to help coronaviruses evade host defenses. Nonstructural protein 14 (nsp14) is responsible for N7-methylation of the cap guanosine in coronaviruses. This enzyme is highly conserved among coronaviruses and is a bifunctional protein with both N7-methyltransferase and 3′-5′ exonuclease activities that distinguish nsp14 from its human equivalent. Mutational analysis of SARS-CoV nsp14 highlighted its role in viral replication and translation efficiency of the viral genome. In this paper, we describe the characterization and development of a high-throughput assay for nsp14 utilizing RapidFire technology. The assay has been used to screen a library of 1771 Food and Drug Administration (FDA)-approved drugs. From this, we have validated nitazoxanide as a selective inhibitor of the methyltransferase activity of nsp14. Although modestly active, this compound could serve as a starting point for further optimization.

scholarly journals A High-Throughput Screening Strategy to Identify Protein-Protein Interaction Inhibitors That Block the Fanconi Anemia DNA Repair Pathway

2016 ◽  
Vol 21 (6) ◽  
pp. 626-633 ◽  
Author(s):  
Andrew F. Voter ◽  
Kelly A. Manthei ◽  
James L. Keck
Keyword(s):  
Dna Repair ◽  
High Throughput ◽  
Fanconi Anemia ◽  
Fluorescence Polarization ◽  
High Throughput Screening ◽  
Titration Calorimetry ◽  
Common Mechanism ◽  
Physical Interaction ◽  
Repair Pathway ◽  
Dna Crosslinking

Induction of the Fanconi anemia (FA) DNA repair pathway is a common mechanism by which tumors evolve resistance to DNA crosslinking chemotherapies. Proper execution of the FA pathway requires interaction between the FA complementation group M protein (FANCM) and the RecQ-mediated genome instability protein (RMI) complex, and mutations that disrupt FANCM/RMI interactions sensitize cells to DNA crosslinking agents. Inhibitors that block FANCM/RMI complex formation could be useful therapeutics for resensitizing tumors that have acquired chemotherapeutic resistance. To identify such inhibitors, we have developed and validated high-throughput fluorescence polarization and proximity assays that are sensitive to inhibitors that disrupt interactions between the RMI complex and its binding site on FANCM (a peptide referred to as MM2). A pilot screen of 74,807 small molecules was performed using the fluorescence polarization assay. Hits from the primary screen were further tested using the proximity assay, and an orthogonal proximity assay was used to assess inhibitor selectivity. Direct physical interaction between the RMI complex and the most selective inhibitor identified through the screening process was measured by surface plasmon resonance and isothermal titration calorimetry. Observation of direct binding by this small molecule validates the screening protocol.

scholarly journals Long-Term Storage of Compound Solutions for High-Throughput Screening by Using a Novel 1536-Well Microplate

2009 ◽  
Vol 14 (5) ◽  
pp. 492-498 ◽  
Author(s):  
Martin Joseph Pfeifer ◽  
Guenther Scheel
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Cost Effective ◽  
The Novel ◽  
Long Term Storage ◽  
Plate Design ◽  
Minor Losses ◽  
Storage Times ◽  
Term Storage

This report describes the features and the performance of a new and significantly improved 1536-well microplate design. The design allows for simple, automation-friendly, and cost-effective storage of compound solutions for high-throughput screening. The plate design is based on Society for Biomolecular Sciences standards for microplates and can be molded from polystyrene or cycloolefin copolymer, thus making the plate suitable for use with acoustic dispensing as well as other conventional liquid dispensing in the nanoliter range. For a 9:1 DMSO/water mix as solvent, the novel plate design has shown to perform over 4 months with only minor losses in solvent. Thus, this novel plate design creates the basis for further reductions in compound storage volumes and allows for an increase in the storage times for microliter volumes for up to a year or more. The high protection against solvent evaporation is also visible for aqueous solutions, thus allowing for reduced edge effects during screening campaigns.

scholarly journals A High-Throughput Screening Triage Workflow to Authenticate a Novel Series of PFKFB3 Inhibitors

2017 ◽  
Vol 23 (1) ◽  
pp. 11-22
Author(s):  
Stephen A. St-Gallay ◽  
Neil Bennett ◽  
Susan E. Critchlow ◽  
Nicola Curtis ◽  
Gareth Davies ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Isothermal Calorimetry ◽  
Binding Mode ◽  
Carbonyl Oxygen ◽  
Enzyme Concentration ◽  
Titration Calorimetry ◽  
X Ray ◽  
X Ray Crystallography ◽  
The Hill

A high-throughput screen (HTS) of human 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) resulted in several series of compounds with the potential for further optimization. Informatics was used to identify active chemotypes with lead-like profiles and remove compounds that commonly occurred as actives in other HTS screens. The activities were confirmed with IC50 measurements from two orthogonal assay technologies, and further analysis of the Hill slopes and comparison of the ratio of IC50 values at 10 times the enzyme concentration were used to identify artifact compounds. Several series of compounds were rejected as they had both high slopes and poor ratios. A small number of compounds representing the different leading series were assessed using isothermal titration calorimetry, and the X-ray crystal structure of the complex with PFKFB3 was solved. The orthogonal assay technology and isothermal calorimetry were demonstrated to be unreliable in identifying false-positive compounds in this case. Presented here is the discovery of the dihydropyrrolopyrimidinone series of compounds as active and novel inhibitors of PFKFB3, shown by X-ray crystallography to bind to the adenosine triphosphate site. The crystal structures of this series also reveal it is possible to flip the binding mode of the compounds, and the alternative orientation can be driven by a sigma-hole interaction between an aromatic chlorine atom and a backbone carbonyl oxygen. These novel inhibitors will enable studies to explore the role of PFKFB3 in driving the glycolytic phenotype of tumors.

scholarly journals Discovery of Acid-Stable Oxygen Evolution Catalysts : High-throughput Computational Screening of Equimolar Bimetallic Oxides

2020 ◽  
Author(s):  
Seoin Back ◽  
Kevin Tran ◽  
Zachary Ulissi
Keyword(s):  
Oxygen Evolution ◽  
High Throughput ◽  
High Throughput Screening ◽  
Noble Metals ◽  
Cost Effective ◽  
Oxide Catalysts ◽  
Computational Screening ◽  
Energy Conversion Systems ◽  
Acid Stable ◽  
Bimetallic Oxides

Discovering acid-stable, cost-effective and active catalysts for oxygen evolution reaction (OER) is critical since this reaction is bottlenecking many electrochemical energy conversion systems. Current systems use extremely expensive iridium oxide catalysts. Identifying Ir-free or catalysts with reduced Ir-composition has been suggested as goals, but no systematic strategy to discover such catalysts has been reported. In this work, we performed high-throughput computational screening to investigate bimetalic oxide catalysts with space groups derived from those of IrO$_x$, identified promising OER catalysts predicted to satisfy all the desired properties: Co-Ir, Fe-Ir and Mo-Ir bimetallic oxides. We find that for the given crystal structures explored, it is essential to include noble metals to maintain the acid-stability, although one-to-one mixing of noble and non-noble metal oxides could keep the materials survive under the acidic conditions. Based on the calculated results, we provide insights to efficiently perform future high-throughput screening to discover catalysts with desirable properties.

A scalable filtration method for high throughput screening based on cell deformability

Lab on a Chip ◽  
2019 ◽  
Vol 19 (2) ◽  
pp. 343-357 ◽  
Author(s):  
Navjot Kaur Gill ◽  
Chau Ly ◽  
Kendra D. Nyberg ◽  
Linus Lee ◽  
Dongping Qi ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Cell Deformability ◽  
Filtration Method ◽  
High Throughput Method

We demonstrate a scalable, high throughput method to screen cells based on their ability to deform through micron-scale gaps.

scholarly journals Sorting out antibiotics' mechanisms of action: a double fluorescent protein reporter for high throughput screening of ribosome and DNA biosynthesis inhibitors

2016 ◽  
pp. AAC.02117-16 ◽  
Author(s):  
Ilya A. Osterman ◽  
Ekaterina S. Komarova ◽  
Dmitry I. Shiryaev ◽  
Ilya A. Korniltsev ◽  
Irina M. Khven ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Protein Gene ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Cost Effective ◽  
High Signal ◽  
Red Fluorescent Protein ◽  
Reporter System ◽  
Ribosome Stalling

In order to accelerate drug discovery, a simple, reliable and cost-effective system for high-throughput identification of a potential antibiotic mechanism of action is required. To facilitate such screening of new antibiotics, we created a double reporter system for not only antimicrobial activity detection, but also for simultaneous sorting of potential antimicrobials into those that cause ribosome stalling, and others that induce SOS response due to DNA damage. In this reporter system the red fluorescent protein generfpwas placed under the control of the SOS-induciblesulApromoter. The far-red fluorescent protein genekatushka2Swas inserted downstream the tryptophan attenuator where two tryptophan codons were replaced by alanine codons, with simultaneous replacement of the complementary part of the attenuator, to preserve the ability to form secondary structures that influence transcription termination. This genetically modified attenuator makes possible Katushka2S expression only upon exposure to any ribosome stalling compounds. The application of red and far-red fluorescent proteins provides a high signal-to-background ratio without any need in enzymatic substrates for detection of the reporter activity. This reporter was shown to be efficient in high-throughput screening of both synthetic and natural chemicals.

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