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

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.

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

2018 ◽  
Vol 475 (23) ◽  
pp. 3847-3860 ◽  
Author(s):  
Milan Kožíšek ◽  
Václav Navrátil ◽  
Kateřina Rojíková ◽  
Jana Pokorná ◽  
Carlos Berenguer Albiñana ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Enzyme Inhibitor ◽  
Screening Method ◽  
Neuraminidase Inhibitors ◽  
Antibody Assay ◽  
Design Synthesis ◽  
Viral Particles ◽  
Highly Sensitive ◽  
Detection Probe

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.

Inhibition Profiling of Urease and Carbonic Anhydrase II by High- Throughput Screening and Molecular Docking Studies of Structurally Diverse Organic Compounds

2020 ◽  
Vol 17 ◽  
Author(s):  
Majid Ali ◽  
Syed Majid Bukhari ◽  
Asma Zaidi ◽  
Farhan A. Khan ◽  
Umer Rashid ◽  
...  
Keyword(s):  
Molecular Docking ◽  
Carbonic Anhydrase ◽  
Metal Complexes ◽  
Organic Compounds ◽  
High Throughput ◽  
High Throughput Screening ◽  
Docking Studies ◽  
Carbonic Anhydrase Ii ◽  
Molecular Docking Studies ◽  
Ic50 Values

Background:: Structurally diverse organic compounds and available drugs were screened against urease and carbonic anhydrase II in a formulation acceptable for high-throughput screening. Objective: The study was conducted to find out potential inhibitors of urease and carbonic anhydrase II. Methods:: Quantification of the possible HITs was carried out by determining their IC50 values. Results and Discussion:: of several screened compounds including derivatives of oxadiazole, coumarins, chromane-2, 4- diones and metal complexes of cysteine-omeprazole showed promising inhibitory activities with IC50 ranging from 47 μM to 412 μM against the urease. The interactions of active compounds with active sites of enzymes were investigated through molecular docking studies which revealed that (R)-1-(4-amino-4-(5-(thiophen-2-yl)-1,3,4-oxadiazol-2-yl) butyl) guanidine possessing IC50 of 47 μM, interacts with one of the nickel metal atom of urease besides further interactions as predictable hydrogen bonds with KCX490, Asp633, His492, His407 and His409 along with Ala440 and 636. Bi-ligand metal complexes of 4-aminoantipyrine based Schiff bases showed activation of urease with AC50 ranging from 68 μM to 112 μM. Almost 21 compounds with varying functional groups including pyrimidines, oxadiazoles, imidazoles, hydrazides and tin based compounds were active carbonic anhydrase II inhibitors presenting 98 μM to 390 μM IC50 values. Several N-substituted sulfonamide derivatives were inactive against carbonic anhydrase II. Conclusion:: Among all the screened compounds, highly active inhibitor of carbonic anhydrase II was (4-(3- hydroxyphenyl)-6-phenyl-2-thioxo-1,2,3,4-tetrahydropyrimidin-5-yl)phenyl) methanone with IC50 of 98.0 μM. This particular compound showed metallic interaction with Zn ion of carbonic anhydrase II through hydroxyl group of phenyl ring.

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

2018 ◽  
Vol 23 (7) ◽  
pp. 697-707 ◽  
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.

scholarly journals Drug discovery from Nature: automated high-quality sample preparation

2000 ◽  
Vol 22 (5) ◽  
pp. 149-157 ◽  
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.

scholarly journals Colloidal Aggregate Detection by Rapid Fluorescence Measurement of Liquid Surface Curvature Changes in Multiwell Plates

2007 ◽  
Vol 12 (7) ◽  
pp. 966-971 ◽  
Author(s):  
Lifeng Cai ◽  
Miriam Gochin
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Dynamic Range ◽  
Liquid Surface ◽  
Enzyme Inhibitor ◽  
Fluorescence Emission ◽  
Fluorescence Measurement ◽  
Screening Assay ◽  
Colloidal Aggregate ◽  
Multiwell Plates

A simple fluorescence method is reported for the detection of colloidal aggregate formation in solution, with specific applications to determine the critical micelle concentration (CMC) of surfactants and detect small-molecule promiscuous inhibitors. The method exploits the meniscus curvature changes in high-density multiwell plates associated with colloidal changes in solution. The shape of the meniscus has a significant effect on fluorescence intensity when detected using a top-read fluorescence plate reader because of the effect of total internal reflection on fluorescence emission through a curved liquid surface. A dynamic range of 60% is calculated and observed and is measured with a relative sensitivity of 2%. Facile determination of the CMC of a variety of surfactants is demonstrated, as well as a screening assay for aggregate forming properties of small drug-like compounds, a common cause of promiscuous inhibition in high-throughput screening (HTS) enzyme inhibitor assays. Our preliminary results show a potential HTS assay with Z′ factor of 0.76, with good separation between aggregating and nonaggregating small molecules. The method combines the high sensitivity and universality of classic surface tension methods with simplicity and high-throughput determination, enabling facile detection of molecular interactions involving a change in liquid or solid surface character. ( Journal of Biomolecular Screening 2007:966-971)

scholarly journals Multidimensional Profiling of CSF1R Screening Hits and Inhibitors

2011 ◽  
Vol 16 (9) ◽  
pp. 1007-1017 ◽  
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.

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

2001 ◽  
Vol 6 (6) ◽  
pp. 429-440 ◽  
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.

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

2005 ◽  
Vol 10 (4) ◽  
pp. 374-382 ◽  
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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