DNA-linked inhibitor antibody assay (DIANA) as a new method for screening influenza neuraminidase inhibitors

Influenza neuraminidase is responsible for the escape of new viral particles from the infected cell surface. Several neuraminidase inhibitors are used clinically to treat patients or stockpiled for emergencies. However, the increasing development of viral resistance against approved inhibitors has underscored the need for the development of new antivirals effective against resistant influenza strains. A facile, sensitive, and inexpensive screening method would help achieve this goal. Recently, we described a multiwell plate-based DNA-linked inhibitor antibody assay (DIANA). This highly sensitive method can quantify femtomolar concentrations of enzymes. DIANA also has been applied to high-throughput enzyme inhibitor screening, allowing the evaluation of inhibition constants from a single inhibitor concentration. Here, we report the design, synthesis, and structural characterization of a tamiphosphor derivative linked to a reporter DNA oligonucleotide for the development of a DIANA-type assay to screen potential influenza neuraminidase inhibitors. The neuraminidase is first captured by an immobilized antibody, and the test compound competes for binding to the enzyme with the oligo-linked detection probe, which is then quantified by qPCR. We validated this novel assay by comparing it with the standard fluorometric assay and demonstrated its usefulness for sensitive neuraminidase detection as well as high-throughput screening of potential new neuraminidase inhibitors.

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Rapid, sensitive and high-throughput screening method for detection of SARS-CoV-2 antibodies by bio layer interferometry

10.1101/2020.08.15.20175851 ◽

2020 ◽

Author(s):

Sudarshan Reddy Lokireddy ◽

Sridhar Rao Kunchala ◽

Ranga Pratyusha Godavarthy ◽

Venkata Sri Krishna Kona ◽

Laxmaiah Avula ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Screening Method ◽

Antibody Detection ◽

Therapeutic Monitoring ◽

Antibody Testing ◽

Specific Detection ◽

Antibody Assay ◽

Antiviral Therapies ◽

Antibody Titers

Present pandemic scenario, there exists an unmet global need for the development of a rapid and sensitive method for the detection of SARS-CoV-2 infection. The available options for identification of SARS-CoV-2 infection are detection of viral RNA by qRT-PCR, Antigen or Antibody testing by serological methods. Even though many kits available commercially but none of them are rapid, sensitive and high throughput. OnCovid total antibody assay is a diagnostic method developed by us uses the principle of bio-layer Interferometry to detect IgM, IgA and IgG antibodies against SARS-CoV-2 antigens. This method overcomes many of the limitations normally faced in antibody detection by other methods and offers a superior platform for a rapid, sensitive and specific detection of SARS-CoV-2 infection. The test is economical, and the results can be obtained in as short as 30 seconds per test. In addition to its standalone use in early diagnosis of SARS-CoV-2, OnCovid total antibody assay can be used to therapeutic monitoring of antiviral therapies used in clinical management and to estimate the antibody titers during convalescent plasma donation.

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Identification of Novel Carbonic Anhydrase IX Inhibitors Using High-Throughput Screening of Pooled Compound Libraries by DNA-Linked Inhibitor Antibody Assay (DIANA)

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555220918836 ◽

2020 ◽

Vol 25 (9) ◽

pp. 1026-1037

Author(s):

Jan Tykvart ◽

Václav Navrátil ◽

Michael Kugler ◽

Pavel Šácha ◽

Jiří Schimer ◽

...

Keyword(s):

Carbonic Anhydrase ◽

High Throughput ◽

High Throughput Screening ◽

Enzyme Inhibitor ◽

False Positives ◽

Carbonic Anhydrase Ix ◽

High Signal ◽

Antibody Assay ◽

Compound Libraries ◽

Enzyme Detection

The DNA-linked inhibitor antibody assay (DIANA) has been recently validated for ultrasensitive enzyme detection and for quantitative evaluation of enzyme inhibitor potency. Here we present its adaptation for high-throughput screening of human carbonic anhydrase IX (CAIX), a promising drug and diagnostic target. We tested DIANA’s performance by screening a unique compound collection of 2816 compounds consisting of lead-like small molecules synthesized at the Institute of Organic Chemistry and Biochemistry (IOCB) Prague (“IOCB library”). Additionally, to test the robustness of the assay and its potential for upscaling, we screened a pooled version of the IOCB library. The results from the pooled screening were in agreement with the initial nonpooled screen with no lost hits and no false positives, which shows DIANA’s potential to screen more than 100,000 compounds per day. All DIANA screens showed a high signal-to-noise ratio with a Z′ factor of >0.89. The DIANA screen identified 13 compounds with Ki values equal to or better than 10 µM. All retested hits were active also in an orthogonal enzymatic assay showing zero false positives. However, further biophysical validation of identified hits revealed that the inhibition activity of several hits was caused by a single highly potent CAIX inhibitor, being present as a minor impurity. This finding eventually led us to the identification of three novel CAIX inhibitors from the screen. We confirmed the validity of these compounds by elucidating their mode of binding into the CAIX active site by x-ray crystallography.

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Novel cell-free high-throughput screening method for pharmacological tools targeting K+ channels

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1602815113 ◽

2016 ◽

Vol 113 (20) ◽

pp. 5748-5753 ◽

Cited By ~ 43

Author(s):

Zhenwei Su ◽

Emily C. Brown ◽

Weiwei Wang ◽

Roderick MacKinnon

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Low Cost ◽

Control Cell ◽

Ion Homeostasis ◽

Screening Method ◽

Biological Research ◽

Cell Excitability ◽

Highly Sensitive ◽

Neuronal Disorders

K+ channels, a superfamily of ∼80 members, control cell excitability, ion homeostasis, and many forms of cell signaling. Their malfunctions cause numerous diseases including neuronal disorders, cardiac arrhythmia, diabetes, and asthma. Here we present a novel liposome flux assay (LFA) that is applicable to most K+ channels. It is robust, low cost, and high throughput. Using LFA, we performed small molecule screens on three different K+ channels and identified new activators and inhibitors for biological research on channel function and for medicinal development. We further engineered a hERG (human ether-à-go-go-related gene) channel, which, when used in LFA, provides a highly sensitive (zero false negatives on 50 hERG-sensitive drugs) and highly specific (zero false positives on 50 hERG-insensitive drugs), low-cost hERG safety assay.

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Directed Evolution of P450 Fatty Acid Decarboxylases via High-Throughput Screening Towards Improved Catalytic Activity

10.26434/chemrxiv.9791162 ◽

2019 ◽

Author(s):

Huifang Xu ◽

Weinan Liang ◽

Linlin Ning ◽

Yuanyuan Jiang ◽

Wenxia Yang ◽

...

Keyword(s):

Catalytic Activity ◽

Fatty Acid ◽

Directed Evolution ◽

High Throughput ◽

High Throughput Screening ◽

Colorimetric Detection ◽

Screening Method ◽

Random Mutagenesis ◽

Direct Production ◽

Consumption Activity

P450 fatty acid decarboxylases (FADCs) have recently been attracting considerable attention owing to their one-step direct production of industrially important 1-alkenes from biologically abundant feedstock free fatty acids under mild conditions. However, attempts to improve the catalytic activity of FADCs have met with little success. Protein engineering has been limited to selected residues and small mutant libraries due to lack of an effective high-throughput screening (HTS) method. Here, we devise a catalase-deficient Escherichia coli host strain and report an HTS approach based on colorimetric detection of H2O2-consumption activity of FADCs. Directed evolution enabled by this method has led to effective identification for the first time of improved FADC variants for medium-chain 1-alkene production from both DNA shuffling and random mutagenesis libraries. Advantageously, this screening method can be extended to other enzymes that stoichiometrically utilize H2O2 as co-substrate.

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Development of a Novel High-Throughput Screen and Identification of Small-Molecule Inhibitors of the Gα–RGS17 Protein–Protein Interaction Using AlphaScreen

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057111410427 ◽

2011 ◽

Vol 16 (8) ◽

pp. 869-877 ◽

Cited By ~ 13

Author(s):

Duncan I. Mackie ◽

David L. Roman

Keyword(s):

Small Molecule ◽

High Throughput ◽

Protein Interaction ◽

High Throughput Screening ◽

Drug Targets ◽

Screening Method ◽

Fold Increase ◽

Rgs Proteins ◽

High Throughput Screen ◽

Protein Protein Interaction

In this study, the authors used AlphaScreen technology to develop a high-throughput screening method for interrogating small-molecule libraries for inhibitors of the Gαo–RGS17 interaction. RGS17 is implicated in the growth, proliferation, metastasis, and the migration of prostate and lung cancers. RGS17 is upregulated in lung and prostate tumors up to a 13-fold increase over patient-matched normal tissues. Studies show RGS17 knockdown inhibits colony formation and decreases tumorigenesis in nude mice. The screen in this study uses a measurement of the Gαo–RGS17 protein–protein interaction, with an excellent Z score exceeding 0.73, a signal-to-noise ratio >70, and a screening time of 1100 compounds per hour. The authors screened the NCI Diversity Set II and determined 35 initial hits, of which 16 were confirmed after screening against controls. The 16 compounds exhibited IC50 <10 µM in dose–response experiments. Four exhibited IC50 values <6 µM while inhibiting the Gαo–RGS17 interaction >50% when compared to a biotinylated glutathione-S-transferase control. This report describes the first high-throughput screen for RGS17 inhibitors, as well as a novel paradigm adaptable to many other RGS proteins, which are emerging as attractive drug targets for modulating G-protein-coupled receptor signaling.

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

Nucleic Acids Research ◽

10.1093/nar/gkaa1213 ◽

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.

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A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A

International Journal of Molecular Sciences ◽

10.3390/ijms22063041 ◽

2021 ◽

Vol 22 (6) ◽

pp. 3041

Author(s):

Gheorghita Menghiu ◽

Vasile Ostafe ◽

Radivoje Prodanović ◽

Rainer Fischer ◽

Raluca Ostafe

Keyword(s):

High Throughput ◽

Cell Sorting ◽

High Throughput Screening ◽

Screening Method ◽

Enzymatic Reaction ◽

Hydrolytic Enzymes ◽

Screening System ◽

Fluorescence Activated Cell Sorting ◽

Fungal Cell ◽

Chitinase A

Chitinases catalyze the degradation of chitin, a polymer of N-acetylglucosamine found in crustacean shells, insect cuticles, and fungal cell walls. There is great interest in the development of improved chitinases to address the environmental burden of chitin waste from the food processing industry as well as the potential medical, agricultural, and industrial uses of partially deacetylated chitin (chitosan) and its products (chito-oligosaccharides). The depolymerization of chitin can be achieved using chemical and physical treatments, but an enzymatic process would be more environmentally friendly and more sustainable. However, chitinases are slow-acting enzymes, limiting their biotechnological exploitation, although this can be overcome by molecular evolution approaches to enhance the features required for specific applications. The two main goals of this study were the development of a high-throughput screening system for chitinase activity (which could be extrapolated to other hydrolytic enzymes), and the deployment of this new method to select improved chitinase variants. We therefore cloned and expressed the Bacillus licheniformis DSM8785 chitinase A (chiA) gene in Escherichia coli BL21 (DE3) cells and generated a mutant library by error-prone PCR. We then developed a screening method based on fluorescence-activated cell sorting (FACS) using the model substrate 4-methylumbelliferyl β-d-N,N′,N″-triacetyl chitotrioside to identify improved enzymes. We prevented cross-talk between emulsion compartments caused by the hydrophobicity of 4-methylumbelliferone, the fluorescent product of the enzymatic reaction, by incorporating cyclodextrins into the aqueous phases. We also addressed the toxicity of long-term chiA expression in E. coli by limiting the reaction time. We identified 12 mutants containing 2–8 mutations per gene resulting in up to twofold higher activity than wild-type ChiA.

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