Establishment of Novel High-Standard Chemiluminescent Assay for NTPase in Two Protozoans and Its High-Throughput Screening

Toxoplasma gondii is a major protozoan parasite and infects human and many other warm-blooded animals. The infection leads to Toxoplasmosis, a serious issue in AIDS patients, organ transplant recipients and pregnant women. Neospora caninum, another type of protozoa, is closely related to Toxoplasma gondii. Infections of the protozoa in animals also causes serious diseases such as Encephalomyelitis and Myositis-Polyradiculitis in dogs or abortion in cows. Both Toxoplasma gondii and Neospora caninum have similar nucleoside triphosphate hydrolases (NTPase), NcNTPase and TgNTPase-I in Neospora caninum and Toxoplasma gondii, respectively. These possibly play important roles in propagation and survival. Thus, we targeted the enzymes for drug discovery and tried to establish a novel high-standard assay by a combination of original biochemical enzyme assay and fluorescent assay to determine ADP content. We then validated whether or not it can be applied to high-throughput screening (HTS). Then, it fulfilled criterion to carry out HTS in both of the enzymes. In order to identify small molecules having inhibitory effects on the protozoan enzyme, we also performed HTS using two synthetic compound libraries and an extract library derived from marine bacteria and then, identified 19 compounds and 6 extracts. Nagasaki University collected many extracts from over 18,000 marine bacteria found in local Omura bay, and continues to compile an extensive collection of synthetic compounds from numerous drug libraries established by Japanese chemists.

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Drug discovery from Nature: automated high-quality sample preparation

Journal of Automated Methods and Management in Chemistry ◽

10.1155/s1463924600000249 ◽

2000 ◽

Vol 22 (5) ◽

pp. 149-157 ◽

Cited By ~ 12

Author(s):

Ralf Thiericke

Keyword(s):

Drug Discovery ◽

Sample Preparation ◽

High Throughput ◽

High Throughput Screening ◽

Solid Phase ◽

Structural Diversity ◽

Synthetic Compound ◽

Natural Origin ◽

Screening Programs ◽

Compound Libraries

Secondary metabolites from plants, animals and microorganisms have been proven to be an outstanding source for new and innovative drugs and show a striking structural diversity that supplements chemically synthesized compounds or libraries in drug discovery programs. Unfortunately, extracts from natural sources are usually complex mixtures of compounds:: often generated in time consuming and for the most part manual processes. As quality and quantity of the provided samples play a pivotal role in the success of high-throughput screening programs this poses serious problems. In order to make samples of natural origin competitive with synthetic compound libraries, we devised a novel, automated sample preparation procedure based on solid-phase extraction (SPE). By making use of a modified Zymark RapidTrace®SPE workstation an easy-to-handle and effective fractionation method has been developed which allows the generation of highquality samples from natural origin, fulfilling the requirements of an integration into high-throughput screening programs.

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Natural Products in High Throughput Screening: Automated High-Quality Sample Preparation

CrossRef Listing of Deleted DOIs ◽

10.1177/108705719900400104 ◽

1999 ◽

Vol 4 (1) ◽

pp. 15-25 ◽

Cited By ~ 35

Author(s):

Ingrid Schmid ◽

Isabel Sattler ◽

Susanne Grabley ◽

Ralf Thiericke

Keyword(s):

Drug Discovery ◽

Sample Preparation ◽

High Throughput ◽

High Throughput Screening ◽

Solid Phase ◽

Structural Diversity ◽

High Quality ◽

Synthetic Compound ◽

Natural Origin ◽

Compound Libraries

At present, compound libraries from combinatorial chemistry are the major source for high throughput screening (HTS) programs in drug discovery. On the other hand, nature has been proven to be an outstanding source for new and innovative drugs. Secondary metabolites from plants, animals, and microorganisms show a striking structural diversity that supplements chemically synthesized compounds or libraries in drug discovery programs. Unfortunately, extracts from natural sources are usually complex mixtures of compounds, often generated in time-consuming and, for the most part, manual processes. Because quality and quantity of the provided samples play a pivotal role in the success of HTS programs, this poses serious problems. In order to make samples of natural origin competitive with synthetic compound libraries, we devised a novel, automated sample preparation procedure based on solid-phase extraction (SPE). By making use of modified Zymark (Hopkinton, MA) RapidTrace® SPE workstations, we developed an easy-to-handle and effective fractionation method that generates high-quality samples from natural origin, fulfilling the requirements for an integration in high throughput drug discovery programs.

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A Fully Automated High-Throughput Flow Cytometry Screening System Enabling Phenotypic Drug Discovery

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555218773086 ◽

2018 ◽

Vol 23 (7) ◽

pp. 697-707 ◽

Cited By ~ 2

Author(s):

John Joslin ◽

James Gilligan ◽

Paul Anderson ◽

Catherine Garcia ◽

Orzala Sharif ◽

...

Keyword(s):

Flow Cytometry ◽

Drug Discovery ◽

High Throughput ◽

High Throughput Screening ◽

Screening System ◽

Preclinical Development ◽

Drug Identification ◽

Therapeutic Setting ◽

Compound Libraries ◽

Automated Platform

The goal of high-throughput screening is to enable screening of compound libraries in an automated manner to identify quality starting points for optimization. This often involves screening a large diversity of compounds in an assay that preserves a connection to the disease pathology. Phenotypic screening is a powerful tool for drug identification, in that assays can be run without prior understanding of the target and with primary cells that closely mimic the therapeutic setting. Advanced automation and high-content imaging have enabled many complex assays, but these are still relatively slow and low throughput. To address this limitation, we have developed an automated workflow that is dedicated to processing complex phenotypic assays for flow cytometry. The system can achieve a throughput of 50,000 wells per day, resulting in a fully automated platform that enables robust phenotypic drug discovery. Over the past 5 years, this screening system has been used for a variety of drug discovery programs, across many disease areas, with many molecules advancing quickly into preclinical development and into the clinic. This report will highlight a diversity of approaches that automated flow cytometry has enabled for phenotypic drug discovery.

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Developing an Automated Enzyme Immunoassay for High-Throughput Screening of Bovine Serum Antibodies to Neospora Caninum

JALA Journal of the Association for Laboratory Automation ◽

10.1016/s1535-5535-04-00241-2 ◽

2003 ◽

Vol 8 (1) ◽

pp. 46-50

Author(s):

John T. Y. Wu ◽

Sally Dreger ◽

Eva Y. W. Chow ◽

Evelyn E. Bowlby ◽

Lester S. Y. Wong

Keyword(s):

Enzyme Immunoassay ◽

High Throughput ◽

Bovine Serum ◽

High Throughput Screening ◽

Neospora Caninum ◽

Enzyme Linked Immunosorbent Assay ◽

Predictive Values ◽

Automated Assay ◽

Robotic Workstation ◽

Elisa Data

An enzyme-linked immunosorbent assay (ELISA) for Neospora caninum antibodies was automated with a robotic workstation, the Beckman Coulter Biomek 2000, to screen 200 bovine sera. Comparing these results with manually run ELISA data, a 95.92% agreement (K = 0.9592) between the two assays was obtained. The automated assay was specific and sensitive with excellent positive and negative predictive values. The results were repeatable and reproducible. The automation flexibility was high and the operation complexity was minimal. High-throughput screening (HTS) for bovine antibodies to Neospora caninum was achieved. The assay was developed according to the internationally recognized ISO17025 standard requirements.

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Abstract 5155: RapidFire MS/MS enables both rapid evaluation of multiple histone methyltransferases and label-free high throughput screening of targeted compound libraries

10.1158/1538-7445.am2014-5155 ◽

2014 ◽

Author(s):

Patrick J. Bingham ◽

Karen Maegley ◽

Cody T. Krivacic

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Rapid Evaluation ◽

Label Free ◽

Histone Methyltransferases ◽

Compound Libraries

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Multidimensional Profiling of CSF1R Screening Hits and Inhibitors

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057111418113 ◽

2011 ◽

Vol 16 (9) ◽

pp. 1007-1017 ◽

Cited By ~ 16

Author(s):

Joost C. M. Uitdehaag ◽

Cecile M. Sünnen ◽

Antoon M. van Doornmalen ◽

Nikki de Rouw ◽

Arthur Oubrie ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Active Form ◽

Dissociation Rate ◽

The Past ◽

Compound Libraries ◽

Competitive Kinetics ◽

Homogeneous Assay ◽

High Throughput Assay ◽

Stimulating Factor

Over the past years, improvements in high-throughput screening (HTS) technology and compound libraries have resulted in a dramatic increase in the amounts of good-quality screening hits, and there is a growing need for follow-on hit profiling assays with medium throughput to further triage hits. Here the authors present such assays for the colony-stimulating factor 1 receptor (CSF1R, Fms), including tests for cellular activity and a homogeneous assay to measure affinity for inactive CSF1R. They also present a high-throughput assay to measure target residence time, which is based on competitive binding kinetics. To better fit koff rates, they present a modified mathematical model for competitive kinetics. In all assays, they profiled eight reference inhibitors (imatinib, sorafenib, sunitinib, tandutinib, dasatinib, GW2580, Ki20227, and J&J’s pyrido[2,3-d]pyrimidin-5-one). Using the known biochemical selectivities of these inhibitors, which can be quantified using metrics such as the selectivity entropy, the authors have determined which assay readout best predicts hit selectivity. Their profiling shows surprisingly that imatinib has a preference for the active form of CSF1R and that Ki20227 has an unusually slow target dissociation rate. This confirms that follow-on hit profiling is essential to ensure that the best hits are selected for lead optimization.

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High-Density Miniaturized Thermal Shift Assays as a General Strategy for Drug Discovery

CrossRef Listing of Deleted DOIs ◽

10.1177/108705710100600609 ◽

2001 ◽

Vol 6 (6) ◽

pp. 429-440 ◽

Cited By ~ 573

Author(s):

Michael W. Pantoliano ◽

Eugene C. Petrella ◽

Joseph D. Kwasnoski ◽

Victor S. Lobanov ◽

James Myslik ◽

...

Keyword(s):

Drug Discovery ◽

Ligand Binding ◽

High Throughput ◽

High Throughput Screening ◽

Screening Strategy ◽

General Strategy ◽

General Applicability ◽

High Throughput Drug Screening ◽

Compound Libraries ◽

Thermal Shift

More general and universally applicable drug discovery assay technologies are needed in order to keep pace with the recent advances in combinatorial chemistry and genomics-based target generation. Ligand-induced conformational stabilization of proteins is a well-understood phenomenon in which substrates, inhibitors, cofactors, and even other proteins provide enhanced stability to proteins on binding. This phenomenon is based on the energetic coupling of the ligand-binding and protein-melting reactions. In an attempt to harness these biophysical properties for drug discovery, fully automated instrumentation was designed and implemented to perform miniaturized fluorescence-based thermal shift assays in a microplate format for the high throughput screening of compound libraries. Validation of this process and instrumentation was achieved by investigating ligand binding to more than 100 protein targets. The general applicability of the thermal shift screening strategy was found to be an important advantage because it circumvents the need to design and retool new assays with each new therapeutic target. Moreover, the miniaturized thermal shift assay methodology does not require any prior knowledge of a therapeutic target's function, making it ideally suited for the quantitative high throughput drug screening and evaluation of targets derived from genomics.

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High-Throughput Screening with HyperCyt® Flow Cytometry to Detect Small Molecule Formylpeptide Receptor Ligands

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057105274532 ◽

2005 ◽

Vol 10 (4) ◽

pp. 374-382 ◽

Cited By ~ 53

Author(s):

Susan M. Young ◽

Cristian Bologa ◽

Eric R. Prossnitz ◽

Tudor I. Oprea ◽

Larry A. Sklar ◽

...

Keyword(s):

Flow Cytometry ◽

High Throughput ◽

High Throughput Screening ◽

Receptor Ligands ◽

Chemical Library ◽

Assay Sensitivity ◽

Compound Libraries ◽

Analysis System ◽

G Protein Coupled ◽

Formylpeptide Receptor

High-throughput flow cytometry (HTFC), enabled by faster automated sample processing, represents a promising high- content approach for compound library screening. HyperCyt® is a recently developed automated HTFC analysis system by which cell samples are rapidly aspirated from microplate wells and delivered to the flow cytometer. The formylpeptide receptor (FPR) family of G protein–coupled receptors contributes to the localization and activation of tissue-damaging leukocytes at sites of chronic inflammation. Here, the authors describe development and application of an HTFC screening approach to detect potential anti-inflammatory compounds that block ligand binding to FPR. Using a homogeneous no-wash assay, samples were routinely processed at 1.5 s/well (~2500 cells analyzed/sample), allowing a 96-well plate to be processed in less than 2.5 min. Assay sensitivity and accuracy were validated by detection of a previously documented active compound with relatively low FPR affinity (sulfinpyrazone, inhibition constant [Ki]=14 μM) from among a collection of 880 compounds in the Prestwick Chemical Library. The HyperCyt® system was therefore demonstrated to be a robust, sensitive, and highly quantitative method with which to screen lead compound libraries in a 96-well format.

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A Cell-Based Ultra-High-Throughput Screening Assay for Identifying Inhibitors of D-Amino Acid Oxidase

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057106288181 ◽

2006 ◽

Vol 11 (5) ◽

pp. 481-487 ◽

Cited By ~ 37

Author(s):

Philip E. Brandish ◽

Chi-Sung Chiu ◽

Jonathan Schneeweis ◽

Nicholas J. Brandon ◽

Clare L. Leech ◽

...

Keyword(s):

Amino Acid ◽

High Throughput ◽

High Throughput Screening ◽

Hormone Receptors ◽

Chinese Hamster ◽

Acid Oxidase ◽

Amino Acid Oxidase ◽

Compound Libraries ◽

High Throughput Screening Assay ◽

Artificial Environment

Enzymes are often considered less “druggable” targets than ligand-regulated proteins such as G-protein-coupled receptors, ion channels, or other hormone receptors. Reasons for this include cellular location (intracellular vs. cell surface), typically lower affinities for the binding of small molecules compared to ligand-specific receptors, and binding (catalytic) sites that are often charged or highly polar. A practical drawback to the discovery of compounds targeting enzymes is that screening of compound libraries is typically carried out in cell-free activity assays using purified protein in an inherently artificial environment. Cell-based assays, although often arduous to design for enzyme targets, are the preferred discovery tool for the screening of large compound libraries. The authors have recently described a novel cell-based approach to screening for inhibitors of a phosphatase enzyme and now report on the development and implementation of a homogeneous 3456-well plate assay for D-amino acid oxidase (DAO). Human DAO was stably expressed in Chinese hamster ovary (CHO) cells, and its activity was measured as the amount of hydrogen peroxide detected in the growth medium following feeding the cells with D-serine. In less than 12 weeks, the authors proved the concept in 96-and then 384-well formats, miniaturized the assay to the 3456-well (nanoplate) scale, and screened a library containing more than 1 million compounds. They have identified several cell-permeable inhibitors of DAO from this cell-based high-throughput screening, which provided the discovery program with a few novel and attractive lead structures.

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