PAINS: Relevance to Tool Compound Discovery and Fragment-Based Screening

2013 ◽  
Vol 66 (12) ◽  
pp. 1483 ◽  
Author(s):  
Jonathan B. Baell ◽  
Lori Ferrins ◽  
Hendrik Falk ◽  
George Nikolakopoulos
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Medicinal Chemistry ◽  
Great Caution ◽  
Starting Point ◽  
Very High ◽  
Assay Interference

Pan assay interference compounds (PAINS) are readily discovered in any bioassay and can appear to give selective and optimisable hits. The most common PAINS can be readily recognised by their structure. However, there are compounds that closely resemble PAINS that are not specifically recognised by the PAINS filters. In addition, highly reactive compounds are not encoded for in the PAINS filters because they were excluded from the high-throughput screening (HTS) library used to develop the filters and so were never present to provide indicting data. A compounding complication in the area is that very occasionally a PAINS compound may serve as a viable starting point for progression. Despite such an occasional example, the literature is littered with an overwhelming number of examples of compounds that fail to progress and were probably not optimisable in the first place, nor useful tool compounds. Thus it is with great caution and diligence that compounds possessing a known PAINS core should be progressed through to medicinal chemistry optimisation, if at all, as the chances are very high that the hits will be found to be non-progressable, often after a significant waste of resources.

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.

Development of a β-Lactamase Reporter Gene Assay for Metabotropic Glutamate Receptor 1 by Using Coexpression of Glutamate Transporter

2010 ◽  
Vol 15 (2) ◽  
pp. 148-158 ◽  
Author(s):  
Gentaroh Suzuki ◽  
Hiroshi Kawamoto ◽  
Hisashi Ohta
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Metabotropic Glutamate Receptor ◽  
Chinese Hamster ◽  
High Sensitivity ◽  
Reporter Assay ◽  
Activated T Cells ◽  
Basal Activity ◽  
Gene Assay ◽  
Very High

mGluR1 antagonists have been postulated to be novel CNS drugs, including antipsychotics. Toward this end, the authors developed a β-lactamase reporter assay to identify mGluR1 antagonists. β-Lactamase has several interesting features for high-throughput screening, including very high sensitivity and less well-to-well variation than other reporter enzymes. mGluR1-expressing Chinese hamster ovary (CHO) cells with the β-lactamase gene under control of the nuclear factor of activated T cells (NFAT) promoter (CHO-NFAT-bla-hmGluR1b) exhibited very high basal activity, resulting in an inadequate signal-to-basal (S/B) ratio. Coexpression of glutamate/aspartate transporter (GLAST) with mGluR1 in the cell line (CHO-NFAT-bla-hmGluR1b-GLAST) dramatically decreased basal activity and improved the S/B ratio (from 2- to 20-fold). The contribution of GLAST to lowering basal activity and increasing the S/B ratio was validated by the expression level of GLAST mRNA and by a GLAST inhibitor. Antagonistic activities of known mGluR1 antagonists in the β-lactamase reporter assay were comparable with those in the conventional Ca2+ mobilization assay. The Z′ factor of the β-lactamase reporter assay was 0.89 under optimized conditions. Taken together, the β-lactamase reporter assay with CHO-NFAT-bla-hmGluR1b-GLAST could be a novel high-throughput assay for mGluR1 antagonist screening. This is the first description of a successful β-lactamase reporter assay among all mGluR subtypes.

scholarly journals Stability of Screening Compounds in Wet DMSO

2008 ◽  
Vol 13 (10) ◽  
pp. 999-1006 ◽  
Author(s):  
Caroline Engeloch ◽  
Ulrich Schopfer ◽  
Ingo Muckenschnabel ◽  
Francois Le Goff ◽  
Hervé Mees ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Medicinal Chemistry ◽  
Storage Conditions ◽  
Pragmatic Approach ◽  
Analytical Quality ◽  
The Past ◽  
Effect Of Water ◽  
The Impact

The impact of storage conditions on compound stability and compound solubility has been debated intensely over the past 5 years. At Novartis, the authors decided to opt for a storage concept that can be considered controversial because they are using a DMSO/water (90/10) mixture as standard solvent. To assess the effect of water in DMSO stocks on compound stability, the authors monitored the purity of a subset of 1404 compounds from ongoing medicinal chemistry projects over several months. The study demonstrated that 85% of the compounds were stable in wet DMSO over a 2-year period at 4 °C. This result validates the storage concept developed at Novartis as a pragmatic approach that takes advantage of the benefits of DMSO/water mixtures while mediating the disadvantages. In addition, the authors describe how purity data collected over the course of the chemical validation of high-throughput screening actives are used to improve the analytical quality of the Novartis screening deck. ( Journal of Biomolecular Screening 2008:999-1006)

scholarly journals Using the BioAssay Ontology for Analyzing High-Throughput Screening Data

2014 ◽  
Vol 20 (3) ◽  
pp. 402-415 ◽  
Author(s):  
Linda Zander Balderud ◽  
David Murray ◽  
Niklas Larsson ◽  
Uma Vempati ◽  
Stephan C. Schürer ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Method Comparison ◽  
Formyl Peptide Receptor ◽  
The Public ◽  
Starting Point ◽  
Technology Gaps ◽  
Formyl Peptide ◽  
Assay Methods ◽  
Hit Identification

High-throughput screening (HTS) is the main starting point for hit identification in drug discovery programs. This has led to a rapid increase of available screening data both within pharmaceutical companies and the public domain. We have used the BioAssay Ontology (BAO) 2.0 for assay annotation within AstraZeneca to enable comparison with external HTS methods. The annotated assays have been analyzed to identify technology gaps, evaluate new methods, verify active hits, and compare compound activity between in-house and PubChem assays. As an example, the binding of a fluorescent ligand to formyl peptide receptor 1 (FPR1, involved in inflammation, for example) in an in-house HTS was measured by fluorescence intensity. In total, 155 active compounds were also tested in an external ligand binding flow cytometry assay, a method not used for in-house HTS detection. Twelve percent of the 155 compounds were found active in both assays. By the annotation of assay protocols using BAO terms, internal and external assays can easily be identified and method comparison facilitated. They can be used to evaluate the effectiveness of different assay methods, design appropriate confirmatory and counterassays, and analyze the activity of compounds for identification of technology artifacts.

scholarly journals A Bioluminogenic HDAC Activity Assay

2011 ◽  
Vol 16 (10) ◽  
pp. 1227-1235 ◽  
Author(s):  
Francoise Halley ◽  
Jeanette Reinshagen ◽  
Bernhard Ellinger ◽  
Markus Wolf ◽  
Andrew L. Niles ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Chemical Diversity ◽  
Signal Stability ◽  
Stability Robustness ◽  
Pharmacological Response ◽  
Acetylation State ◽  
Single Addition ◽  
Validation Experiments ◽  
Assay Interference

Histone deacetylase (HDAC) enzymes modify the acetylation state of histones and other important proteins. Aberrant HDAC enzyme function has been implicated in many diseases, and the discovery and development of drugs targeting these enzymes is becoming increasingly important. In this article, the authors report the evaluation of homogeneous, single-addition, bioluminogenic HDAC enzyme activity assays that offer less assay interference by compounds in comparison to fluorescence-based formats. The authors assessed the key operational assay properties including sensitivity, scalability, reproducibility, signal stability, robustness (Z′), DMSO tolerance, and pharmacological response to standard inhibitors against HDAC-1, HDAC-3/NcoR2, HDAC-6, and SIRT-1 enzymes. These assays were successfully miniaturized to a 10 µL assay volume, and their suitability for high-throughput screening was tested in validation experiments using 640 drugs approved by the Food and Drug Administration and the Hypha Discovery MycoDiverse natural products library, which is a collection of 10 049 extracts and fractions from fermentations of higher fungi and contains compounds that are of low molecular weight and wide chemical diversity. Both of these screening campaigns confirmed that the bioluminogenic assay was high-throughput screening compatible and yielded acceptable performance in confirmation, counter, and compound/extract and fraction concentration-response assays.

SuFEx-Enabled High-Throughput Medicinal Chemistry

2019 ◽  
Author(s):  
Seiya Kitamura ◽  
Qinheng Zheng ◽  
Jordan L. Woehl ◽  
angelo solan ◽  
Emily Chen ◽  
...  
Keyword(s):  
Click Chemistry ◽  
Small Molecule ◽  
High Throughput ◽  
Cysteine Protease ◽  
High Throughput Screening ◽  
Medicinal Chemistry ◽  
Biologically Active ◽  
Drug Candidates ◽  
Biological Probes ◽  
Small Molecule Probes

<p>Optimization of small-molecule probes or drugs is a lengthy, challenging and resource-intensive process. Lack of automation and reliance on skilled medicinal chemists is cumbersome in both academic and industrial settings. Here, we demonstrate a high-throughput hit-to-lead process based on the biocompatible SuFEx click chemistry. A modest high-throughput screening hit against a bacterial cysteine protease SpeB was modified with a SuFExable iminosulfur oxydifluoride [RN=S(O)F2] motif, rapidly diversified into 460 analogs in overnight reactions, and the products directly screened to yield drug-like inhibitors with 300-fold higher potency. We showed that the improved molecule is drug-like and biologically active in a bacteria-host coculture. Since these reactions can be performed on a picomole scale to conserve reagents, we anticipate our methodology can accelerate the development of robust biological probes and drug candidates.</p>

scholarly journals Discovery of a Novel General Anesthetic Chemotype Using High-throughput Screening

2015 ◽  
Vol 122 (2) ◽  
pp. 325-333 ◽  
Author(s):  
Andrew R. McKinstry-Wu ◽  
Weiming Bu ◽  
Ganesha Rai ◽  
Wendy A. Lea ◽  
Brian P. Weiser ◽  
...  
Keyword(s):  
Isothermal Titration Calorimetry ◽  
High Throughput ◽  
High Throughput Screening ◽  
Medicinal Chemistry ◽  
Titration Calorimetry ◽  
Anesthetic Activity ◽  
High Throughput Screen ◽  
High Potency ◽  
Low Toxicity ◽  
Anesthetic Potency

Abstract Background: The development of novel anesthetics has historically been a process of combined serendipity and empiricism, with most recent new anesthetics developed via modification of existing anesthetic structures. Methods: Using a novel high-throughput screen employing the fluorescent anesthetic 1-aminoanthracene and apoferritin as a surrogate for on-pathway anesthetic protein target(s), we screened a 350,000 compound library for competition with 1-aminoanthracene–apoferritin binding. Hit compounds meeting structural criteria had their binding affinities for apoferritin quantified with isothermal titration calorimetry and were tested for γ-aminobutyric acid type A receptor binding using a flunitrazepam binding assay. Chemotypes with a strong presence in the top 700 and exhibiting activity via isothermal titration calorimetry were selected for medicinal chemistry optimization including testing for anesthetic potency and toxicity in an in vivo Xenopus laevis tadpole assay. Compounds with low toxicity and high potency were tested for anesthetic potency in mice. Results: From an initial chemical library of more than 350,000 compounds, we identified 2,600 compounds that potently inhibited 1-aminoanthracene binding to apoferritin. A subset of compounds chosen by structural criteria (700) was successfully reconfirmed using the initial assay. Based on a strong presence in both the initial and secondary screens the 6-phenylpyridazin-3(2H)-one chemotype was assessed for anesthetic activity in tadpoles. Medicinal chemistry efforts identified four compounds with high potency and low toxicity in tadpoles, two were found to be effective novel anesthetics in mice. Conclusion: The authors demonstrate the first use of a high-throughput screen to successfully identify a novel anesthetic chemotype and show mammalian anesthetic activity for members of that chemotype.

scholarly journals Identification of Activators of Human Fumarate Hydratase by Quantitative High-Throughput Screening

2019 ◽  
Vol 25 (1) ◽  
pp. 43-56
Author(s):  
Hu Zhu ◽  
Olivia W. Lee ◽  
Pranav Shah ◽  
Ajit Jadhav ◽  
Xin Xu ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Cell Cancer ◽  
Krebs Cycle ◽  
Fumarate Hydratase ◽  
Metabolic Enzyme ◽  
Oxidation Activity ◽  
Drug Candidates ◽  
Starting Point

Fumarate hydratase (FH) is a metabolic enzyme that is part of the Krebs cycle and reversibly catalyzes the hydration of fumarate to malate. Mutations of the FH gene have been associated with fumarate hydratase deficiency (FHD), hereditary leiomyomatosis and renal cell cancer (HLRCC), and other diseases. Currently, there are no high-quality small-molecule probes for studying human FH. To address this, we developed a quantitative high-throughput screening (qHTS) FH assay and screened a total of 57,037 compounds from in-house libraries in dose–response. While no inhibitors of FH were confirmed, a series of phenyl-pyrrolo-pyrimidine-diones were identified as activators of human FH. These compounds were not substrates of FH, were inactive in a malate dehydrogenase counterscreen, and showed no detectable reduction–oxidation activity. The binding of two compounds from the series to human FH was confirmed by microscale thermophoresis. The low hit rate in this screening campaign confirmed that FH is a “tough target” to modulate, and the small-molecule activators of human FH reported here may serve as a starting point for further optimization and development into cellular probes of human FH and potential drug candidates.

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