scholarly journals Development of a Simple Assay Method for Adenosine Deaminase via Enzymatic Formation of an Inosine-Tb3+ Complex

Sensors ◽  
2019 ◽  
Vol 19 (12) ◽  
pp. 2728
Author(s):  
Suji Lee ◽  
Heewon Park ◽  
Yeongcheol Ki ◽  
Hohjai Lee ◽  
Min Su Han
Keyword(s):  
Adenosine Deaminase ◽  
High Throughput Screening ◽  
Inhibition Efficiency ◽  
Lanthanide Complex ◽  
Human Diseases ◽  
Assay Method ◽  
Naked Eye ◽  
Enzymatic Formation ◽  
Strong Luminescence ◽  
Sensitization Efficiency

Adenosine deaminase (ADA), which catalyzes the irreversible deamination of adenosine to inosine, is related to various human diseases such as tuberculous peritonitis and leukemia. Therefore, the method used to detect ADA activity and screen the effectiveness of various inhibitor candidates has important implications for the diagnosis treatment for various human diseases. A simple and rapid assay method for ADA, based on the enzymatic formation of a luminescent lanthanide complex, is proposed in this study. Inosine, an enzymatic product of ADA with stronger sensitization efficiency for Tb3+ than adenosine, produced a strong luminescence by forming an inosine-Tb3+ complex, and it enabled the direct monitoring of ADA activity in real-time. By introducing only Tb3+ to adenosine and ADA in the buffer, the enhancement of luminescence enabled the detection of a low concentration of ADA (detection limit 1.6 U/L). Moreover, this method could accurately determine the inhibition efficiency (IC50) of the known ADA inhibitor, erhythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), and the inhibition of ADA could be confirmed by the naked eye. Considering its simplicity, this assay could be extended to the high-throughput screening of various ADA inhibitor candidates.

scholarly journals A sensitive, accurate, and high-throughput gluco-oligosaccharide oxidase based HRP colorimetric method for lytic polysaccharide monooxygenase activity assay

2021 ◽  
Author(s):  
Gang Liu
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Direct Method ◽  
Total Concentration ◽  
Colorimetric Method ◽  
Industrial Applications ◽  
Assay Method ◽  
Activity Assay ◽  
Lytic Polysaccharide Monooxygenase ◽  
Monooxygenase Activity

Abstract Background The AA9 (auxiliary activities) family of lytic polysaccharide monooxygenases (AA9 LPMOs) are ubiquitous and diverse group of enzymes amongst the fungal kingdom. They catalyze the oxidative cleavage of glycosidic bonds in lignocellulose and exhibit great potential for secondary biorefinery applications. Screening of AA9 LPMOs for desirable properties is crucial for biorefinery industrial applications. However, robust, high-throughput and direct method for AA9 LPMO activity assay, which is prerequisite for screening of LPMOs with excellent properties, is still lacking. Here, we have described a gluco-oligosaccharide oxidase (GOOX) based horseradish peroxidase (HRP) colorimetric method for AA9 LPMO activity assay. Results We cloned and expressed a GOOX gene from Sarocladium strictum in Trichoderma reesei, purified the recombinant SsGOOX, validated its properties, and set up a SsGOOX based HRP colorimetric method for cellobiose concentration assay. Then we expressed two AA9 LPMOs from Thielavia terrestris, TtAA9F and TtAA9G in T. reesei, purified the recombinant proteins, and analyzed their product profiles and regioselectivity towards phosphoric acid swollen cellulose (PASC). TtAA9F was characterized as a C1 type (class 1) LPMO, while TtAA9G was characterized as a C4 type (class 2) LPMO. Finally, the SsGOOX based HRP colorimetric method was used to quantify the total concentration of reducing lytic products from LPMO reaction, and consequently, the activities of both C1 and C4 types of LPMOs were analyzed. These LPMOs could be effectively analyzed with limits of detection (LoDs) lower than 30 nmol/L, and standard curves between A515 and LPMO concentrations with determination coefficients greater than 0.994 were obtained. Conclusions A novel, sensitive and accurate assay method that directly targets the main activity of both C1 and C4 type of AA9 LPMOs was established. This method is easy to use and could be performed on a microtiter plate ready for high-throughput screening of AA9 LPMOs with high properties.

scholarly journals High-Throughput Screening Method of Inhibitors that Block the Interaction between 2 Helical Regions of HIV-1 gp41

2005 ◽  
Vol 10 (1) ◽  
pp. 13-19 ◽  
Author(s):  
Bong-Suk Jin ◽  
Won-Kyu Lee ◽  
Kwangseog Ahn ◽  
Myung Kyu Lee ◽  
Yeon Gyu Yu
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Screening Method ◽  
Assay Method ◽  
Enzyme Linked Immunosorbent Assay ◽  
Compound Library ◽  
Elisa Method ◽  
Helical Bundle ◽  
Fusion Inhibitors ◽  
Hiv 1

The HIV-1 envelope glycoprotein transmembrane subunit, gp41, mediates the fusion of viral and target cell membranes. The 2 helical regions in the ectodomain of gp41, the N-helix and the C-helix, form a helical bundle complex that has been suggested as a fusion-active conformation. Previously, an enzyme-linked immunosorbent assay (ELISA) method had been established to measure the interaction of 2 helical regions of gp41. In this study, the ELISA method was modified to apply high-throughput screening (HTS) of an organic compound library. A few compounds had been identified to prevent the interaction between 2 helical regions of gp41, and they were further shown to inhibit the gp41-mediated viral infection. In addition, they specifically quenched the fluorescence of tryptophan in the N-helix region, indicating that these compounds bound to the N-helix rather than the C-helix of gp41. These results suggested that this assay method targeting gp41 could be used for HTS of HIV fusion inhibitors. ( Journal of Biomolecular Screening 2005:13-19)

scholarly journals LIGHTHOUSE illuminates therapeutics for a variety of diseases including COVID-19

2021 ◽  
Author(s):  
Hideyuki Shimizu ◽  
Manabu Kodama ◽  
Masaki Matsumoto ◽  
Yasuko Orba ◽  
Michihito Sasaki ◽  
...  
Keyword(s):  
Paradigm Shift ◽  
High Throughput Screening ◽  
Structural Information ◽  
Protein Structures ◽  
Therapeutic Targets ◽  
Basic Research ◽  
Human Diseases ◽  
Docking Simulations ◽  
Research Findings ◽  
Therapeutic Compounds

SUMMARYAlthough numerous promising therapeutic targets for human diseases have been discovered, most have not been successfully translated into clinical practice1. A bottleneck in the application of basic research findings to patients is the enormous cost, time, and effort required for high-throughput screening of potential drugs2 for given therapeutic targets. Recent advances in 3D docking simulations have not solved this problem, given that 3D protein structures with sufficient resolution are not always available and that they are computationally expensive to obtain. Here we have developed LIGHTHOUSE, a graph-based deep learning approach for discovery of the hidden principles underlying the association of small-molecule compounds with target proteins, and we present its validation by identifying potential therapeutic compounds for various human diseases. Without any 3D structural information for proteins or chemicals, LIGHTHOUSE estimates protein-compound scores that incorporate known evolutionary relations and available experimental data. It identified novel therapeutics for cancer, lifestyle-related disease, and bacterial infection. Moreover, LIGHTHOUSE predicted ethoxzolamide as a therapeutic for coronavirus disease 2019 (COVID-19), and this agent was indeed effective against alpha, beta, gamma, and delta variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that are rampant worldwide. Given that ethoxzolamide is already approved for several diseases, it could be rapidly deployed for the treatment of patients with COVID-19. We envision that LIGHTHOUSE will bring about a paradigm shift in translational medicine, providing a bridge from bench side to bedside.

scholarly journals Implications for human odor sensing revealed from the statistics of the odorant-receptor interactions

2018 ◽  
Author(s):  
Ji Hyun Bak ◽  
Seogjoo Jang ◽  
Changbong Hyeon
Keyword(s):  
High Throughput Screening ◽  
Odorant Receptor ◽  
Olfactory Receptors ◽  
Assay Method ◽  
Theoretic Approach ◽  
Screening Assay ◽  
Odor Perception ◽  
Activation Kinetics ◽  
High Throughput Screening Assay ◽  
Human Odor

Binding of odorants to olfactory receptors (ORs) elicits downstream chemical and neural signals, which are further processed to odor perception in the brain. Recently, Mainland et al. [Sci. data, (2015) 2:sdata20152] have measured ≳ 500 pairs of odorant-OR interaction by a high-throughput screening assay method, opening a new avenue to understanding the principles of human odor coding. Here, using a recently developed minimal model for OR activation kinetics [J. Phys. Chem. B (2017) 121, 1304–1311], we characterize the statistics of OR activation by odorants in terms of three empirical parameters: the half-maximum effective concentration EC50, the efficacy, and the basal activity. While the data size of odorants is still limited, the statistics offer meaningful information on the breadth and optimality of the tuning of human ORs to odorants, and allow us to relate the three parameters with the microscopic rate constants and binding affinities that define the OR activation kinetics. Despite the stochastic nature of the response expected at individual OR-odorant level, we assess that the confluence of signals in a neuron released from the multitude of ORs is effectively free of noise and deterministic with respect to changes in odorant concentration. Thus, setting a threshold to the fraction of activated OR copy number for neural spiking binarizes the electrophysiological signal of olfactory sensory neuron, thereby making an information theoretic approach a viable tool in studying the principles of odor perception.

scholarly journals A Novel High-Throughput Assay Enables the Direct Identification of Acyltransferases

Catalysts ◽  
2019 ◽  
Vol 9 (1) ◽  
pp. 64 ◽  
Author(s):  
Lukas Reisky ◽  
Vishnu S. T. Srinivasamurthy ◽  
Chris P. S. Badenhorst ◽  
Simon P. Godehard ◽  
Uwe T. Bornscheuer
Keyword(s):  
High Throughput ◽  
Dimethyl Carbonate ◽  
Enzyme Engineering ◽  
Aqueous Environment ◽  
Direct Identification ◽  
Activated Esters ◽  
Naked Eye ◽  
Fluorescence Measurements ◽  
Enzymatic Formation ◽  
High Throughput Assay

Acyltransferases are enzymes that are capable of catalyzing the transesterification of non-activated esters in an aqueous environment and therefore represent interesting catalysts for applications in various fields. However, only a few acyltransferases have been identified so far, which can be explained by the lack of a simple, broadly applicable high-throughput assay for the identification of these enzymes from large libraries. Here, we present the development of such an assay that is based on the enzymatic formation of oligocarbonates from dimethyl carbonate and 1,6-hexanediol. In contrast to the monomers used as substrates, the oligomers are not soluble in the aqueous environment and form a precipitate which is used to detect enzyme activity by the naked eye, by absorbance or by fluorescence measurements. With activity detected and thus confirmed for the enzymes Est8 and MsAcT, the assay enabled the first identification of acyltransferases that act on carbonates. It will thus allow for the discovery of further efficient acyltransferases or of more efficient variants via enzyme engineering.

Modern Computational Strategies for Designing Drugs to Curb Human Diseases: A Prospect

2019 ◽  
Vol 18 (31) ◽  
pp. 2702-2719 ◽  
Author(s):  
Khalid Bashir Dar ◽  
Aashiq Hussain Bhat ◽  
Shajrul Amin ◽  
Rabia Hamid ◽  
Suhail Anees ◽  
...  
Keyword(s):  
Clinical Trials ◽  
High Throughput Screening ◽  
Target Identification ◽  
Quantitative Structure Activity Relationship ◽  
Pharmacophore Modeling ◽  
Human Diseases ◽  
Other Information ◽  
Preclinical Trials ◽  
Dynamics Simulations ◽  
Effective Drugs

Drug discovery is an exhaustive and time-consuming process involving numerous stages like target identification, validation, lead optimization, preclinical trials, clinical trials and finally postmarketing vigilance for drug safety. The application of computer-aided drug designing (CADD) is an indispensable approach for developing safe and effective drugs. Previous methods based on combinatorial chemistry (CC) and high throughput screening (HTS) consumed a lot of time as well as expenditure. CADD based approaches including pharmacophore modeling (PM), molecular docking (MD), inverse docking, chemical similarity (CS), quantitative structure-activity relationship (QSAR), virtual screening (VS) and molecular dynamics simulations have been quite productive in predicting the therapeutic outcome of candidate drugs/compounds besides saving precious time. CADD tools exploit structural and other information available regarding the target (enzyme/receptor) and the ligands to identify the compounds with the ability to treat diseases notably cancer, neurodegenerative disorders, malaria, Ebola, HIV-AIDS and many more. Computational approaches have led to the discovery of many drugs that have passed preclinical and clinical trials and become novel therapeutics in the treatment of a variety of diseases. Some notable examples of CADD derived novel drugs include dorzolamide, saquinavir, ritonavir, indinavir, captopril and tirofiban. CADD plays important role in predicting absorption, distribution, metabolism, excretion and toxicity (ADME/T) of candidate drugs. Overall, CADD represents an effective and much-needed strategy for designing therapeutically effective drugs to combat human diseases.

scholarly journals Development and Validation of a Generic Fluorescent Methyltransferase Activity Assay Based on the Transcreener AMP/GMP Assay

2011 ◽  
Vol 17 (1) ◽  
pp. 59-70 ◽  
Author(s):  
Tony A. Klink ◽  
Matt Staeben ◽  
Kim Twesten ◽  
Andrew L. Kopp ◽  
Meera Kumar ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Drug Targets ◽  
Adenosine Monophosphate ◽  
Covalent Modification ◽  
Assay Method ◽  
Intact Protein ◽  
Detection Range ◽  
Enzyme Coupling ◽  
Assay Methods ◽  
Development And Validation

Methylation is a ubiquitous covalent modification used to control the function of diverse biomolecules including hormones, neurotransmitters, xenobiotics, proteins, nucleic acids, and lipids. Histone methyltransferases (HMTs) are currently of high interest as drug targets because of their role in epigenetic regulation; however, most HMT assay methods are either not amenable to a high-throughput screening (HTS) environment or are applicable to a limited number of enzymes. The authors developed a generic methyltransferase assay method using fluorescent immunodetection of adenosine monophosphate (AMP), which is formed from the MT reaction product S-adenosylhomocysteine in a dual-enzyme coupling step. The detection range of the assay; its suitability for HTS, including stability of reagents following dispensing and after addition to reactions; and the potential for interference from drug-like molecules was investigated. In addition, the use of the assay for measuring inhibitor potencies with peptide or intact protein substrates was examined through pilot screening with selected reference enzymes including HMT G9a. By combining a novel enzymatic coupling step with the well-characterized Transcreener AMP/GMP assay, the authors have developed a robust HTS assay for HMTs that should be broadly applicable to other types of methyltransferases as well.

Development of a High-Throughput Assay to Identify Inhibitors of ENPP1

2021 ◽  
pp. 247255522098232
Author(s):  
Meera Kumar ◽  
Robert G. Lowery
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Type I Interferon ◽  
Direct Detection ◽  
Innate Immune ◽  
Assay Method ◽  
Type I ◽  
Immune Response To Cancer ◽  
Z Value ◽  
High Throughput Assay

The innate immune response to cancer is initiated by cytosolic DNA, where it binds to cGAS and triggers type I interferon (IFN) expression via the STING receptor, leading to activation of tumor-specific T cells. Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) has been identified as the primary enzyme responsible for degrading cGAMP, and therefore it is under intense investigation as a therapeutic target for cancer immunotherapy. ENPP1 hydrolyzes cGAMP to produce AMP and GMP, and hydrolyzes ATP and other nucleotides to monophosphates and pyrophosphate. We developed a robust, high-throughput screening (HTS)-compatible enzymatic assay method for ENPP1 using the Transcreener AMP2/GMP2 Assay, a competitive fluorescence polarization (FP) immunoassay that enables direct detection of AMP and GMP in a homogenous format. The monoclonal antibody used in the Transcreener AMP2/GMP2 Assay showed more than 104-fold selectivity for AMP and GMP versus cGAMP, and 3000-fold selectivity for AMP over ATP, indicating that the assay can be used for detection at initial velocity with either substrate. A working concentration of 100 pM ENPP1 was determined as optimal with a 60 min reaction period, enabling screening with very low quantities of enzyme. A Z′ value of 0.72 was determined using ATP as substrate, indicating a high-quality assay. Consistent with previous studies, we found that ENPP1 preferred ATP as a substrate when compared with other nucleotides like GTP, ADP, and GDP. ENPP1 showed a 20-fold selectivity for 2′3′cGAMP compared with 2′3′c-diGMP and showed no activity with 3′3′c-diAMP. The Transcreener AMP2/GMP2 Assay should prove to be a valuable tool for the discovery of ENPP1 lead molecules.

scholarly journals Label-Free Time-Gated Luminescent Detection Method for the Nucleotides with Varying Phosphate Content

Sensors ◽  
2018 ◽  
Vol 18 (11) ◽  
pp. 3989
Author(s):  
Kari Kopra ◽  
Tanja Seppälä ◽  
Dana Rabara ◽  
Maria Abreu-Blanco ◽  
Sakari Kulmala ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Direct Detection ◽  
Cell Metabolism ◽  
Lanthanide Complex ◽  
Vital Role ◽  
Molecular Probe ◽  
Enzymatic Reactions ◽  
Label Free ◽  
Energy Economy ◽  
Light Harvesting Antenna

A new label-free molecular probe for luminescent nucleotide detection in neutral aqueous solution is presented. Phosphate-containing molecules, such as nucleotides possess vital role in cell metabolism, energy economy, and various signaling processes. Thus, the monitoring of nucleotide concentration and nucleotide related enzymatic reactions is of high importance. Two component lanthanide complex formed from Tb(III) ion carrier and light harvesting antenna, readily distinguishes nucleotides containing different number of phosphates and enable direct detection of enzymatic reactions converting nucleotide triphosphate (NTP) to nucleotide di/monophosphate or the opposite. Developed sensor enables the detection of enzymatic activity with a low nanomolar sensitivity, as highlighted with K-Ras and apyrase enzymes in their hydrolysis assays performed in a high throughput screening compatible 384-well plate format.

A Homogeneous Fluorescent Live-Cell Assay for Measuring 7-Transmembrane Receptor Activity and Agonist Functional Selectivity Through Beta-Arrestin Recruitment

2009 ◽  
Vol 14 (7) ◽  
pp. 798-810 ◽  
Author(s):  
Bonnie J. Hanson ◽  
Justin Wetter ◽  
Mark R. Bercher ◽  
Leisha Kopp ◽  
Maya Fuerstenau-Sharp ◽  
...  
Keyword(s):  
G Protein ◽  
High Throughput ◽  
High Throughput Screening ◽  
Second Messengers ◽  
Expression System ◽  
Live Cell ◽  
Assay Method ◽  
Protein Signaling ◽  
Functional Selectivity ◽  
7Tm Receptors

Seven-transmembrane (7TM) receptors play an essential role in the regulation of a wide variety of physiological processes, making them one of the top target classes for pharmaceuticals. 7TM receptor function is mediated and modulated through 2 primary processes: G-protein and beta-arrestin signaling. Classically, it has been recognized that these 2 processes can interact with one another during 7TM receptor desensitization, but it has more recently been recognized that these 2 processes can also act independently of one another and can activate parallel signaling pathways. As such, the methods used to interrogate 7TM receptor signaling, both from a biological and a pharmaceutical perspective, may need to be reevaluated and the question of whether functionally selective compounds (compounds that selectively activate one pathway over another) can be rationally developed must be raised. Although numerous high-throughput screening (HTS) compatible assays exist for studying second messengers arising from G-protein signaling, far fewer HTS compatible assays exist for studying beta-arrestin recruitment. The authors report on the Tango™ 7TM receptor assay technology, a high-throughput homogeneous assay method for monitoring beta-arrestin recruitment that uses a live-cell fluorescent readout. This assay format is broadly applicable to 7TM receptors, independent of G-protein coupling and, as such, has been used to produce assays for over 70 7TM receptor targets. The authors also show how flow cytometry can be used to select clones with desired pharmacological profiles and how an inducible expression system can increase the assay window for targets with high levels of constitutive activity. Finally, they demonstrate how the Tango™ system can be used in parallel with assays aimed at second-messenger signaling to enable functional selectivity studies. ( Journal of Biomolecular Screening 2009:798-810)

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