Streamlined System for Purifying and Quantifying a Diverse Library of Compounds and the Effect of Compound Concentration Measurements on the Accurate Interpretation of Biological Assay Results

Popa‐Burke, Ioana; Issakova, Olga; Arroway, James D.; Bernasconi, Paul; Chen, Min; Coudurier, Louis; Galasinski, Scott C.; Jadhav, Ajit; Janzen, William P.; Lagasca, Dennis; Liu, Darren; Lewis, Roderic S.; Mohney, Robert P.; Sepetov, Nikolai F.; Sparkman, Darren A.; Hodge, C. Nicholas

doi:10.1021/ac0491859

Cited by 72 publications

(45 citation statements)

References 20 publications

(33 reference statements)

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“…Sample inconsistency, because of differences in compound preparation or stability, for example, is a particularly important consideration for traditional HTS, where actives are identified outside a defined threshold value. When this threshold is near the inflection point of the concentration-response curve, a small change in sample preparation could result in a compound no longer being classified as active, an important concern in traditional HTS (12). For example, resveratrol would be identified as active at 2.3 M in one sample but inactive in the other, when a 50% threshold is used (Fig.…”

Section: Resultsmentioning

confidence: 99%

Quantitative high-throughput screening: A titration-based approach that efficiently identifies biological activities in large chemical libraries

Inglese

Auld

Jadhav

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

723

1,048

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High-throughput screening (HTS) of chemical compounds to identify modulators of molecular targets is a mainstay of pharmaceutical development. Increasingly, HTS is being used to identify chemical probes of gene, pathway, and cell functions, with the ultimate goal of comprehensively delineating relationships between chemical structures and biological activities. Achieving this goal will require methodologies that efficiently generate pharmacological data from the primary screen and reliably profile the range of biological activities associated with large chemical libraries. Traditional HTS, which tests compounds at a single concentration, is not suited to this task, because HTS is burdened by frequent false positives and false negatives and requires extensive follow-up testing. We have developed a paradigm, quantitative HTS (qHTS), tested with the enzyme pyruvate kinase, to generate concentration-response curves for >60,000 compounds in a single experiment. We show that this method is precise, refractory to variations in sample preparation, and identifies compounds with a wide range of activities. Concentration-response curves were classified to rapidly identify pyruvate kinase activators and inhibitors with a variety of potencies and efficacies and elucidate structure-activity relationships directly from the primary screen. Comparison of qHTS with traditional single-concentration HTS revealed a high prevalence of false negatives in the single-point screen. This study demonstrates the feasibility of qHTS for accurately profiling every compound in large chemical libraries (>10 5 compounds). qHTS produces rich data sets that can be immediately mined for reliable biological activities, thereby providing a platform for chemical genomics and accelerating the identification of leads for drug discovery.1,536-well ͉ chemical genomics ͉ enzyme assay ͉ PubChem ͉ pyruvate kinase T he first description of biological effect versus chemical compound concentration was made by Paracelsus ca. 1534 and quantified by A. V. Hill in 1910 (1). The basis of these observations is that ligands affecting biological systems have optimal ranges of activity (EC 50 ), and give rise to concentration-effect relationships that can be complex, varying in potency, efficacy, and steepness of response. Far below an EC 50 , no effect may be seen (referred to as the no observable effect level or NOEL), and much above it, toxic or ''off-target'' effects may be observed. This well known behavior of chemical compounds in biological systems requires a specific dose of a compound to achieve a desired biological effect, whether in basic or clinical applications (2, 3).Historically, new compounds with medicinal qualities were discovered through laborious testing of samples using low-throughput assays including animal and isolated tissue models. In the early 1990s, the advent of combinatorial chemistry and commercial consolidation of small molecule collections resulted in a tremendous increase in compound numbers, requiring the development of high-throughput ...

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Section: Resultsmentioning

confidence: 99%

Quantitative high-throughput screening: A titration-based approach that efficiently identifies biological activities in large chemical libraries

Inglese

Auld

Jadhav

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

723

1,048

View full text Add to dashboard Cite

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“…In contrast, the increase at the lowest dilution step is suggestive of compound carryover from high to low concentration on contaminated tip surfaces. Furthermore, if the traditional aqueous serial dilution strategy is implemented, then to ensure accurate concentration information, concentration measurements should be made at each dilution step 18 for each compound in the library.…”

Section: Tip Reuse Studymentioning

confidence: 99%

Gradient, Contact-Free Volume Transfers Minimize Compound Loss in Dose-Response Experiments

Harris¹,

Olechno²,

Datwani³

et al. 2010

SLAS Discovery

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More accurate dose-response curves can be constructed by eliminating aqueous serial dilution of compounds. Traditional serial dilutions that use aqueous diluents can result in errors in dose-response values of up to 4 orders of magnitude for a significant percentage of a compound library. When DMSO is used as the diluent, the errors are reduced but not eliminated. The authors use acoustic drop ejection (ADE) to transfer different volumes of model library compounds, directly creating a concentration gradient series in the receiver assay plate. Sample losses and contamination associated with compound handling are therefore avoided or minimized, particularly in the case of less water-soluble compounds. ADE is particularly well suited for assay miniaturization, but gradient volume dispensing is not limited to miniaturized applications.

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“…Technology has advanced to enable confidence in compound delivery to the assay well, but as with any method, the effect of poor aqueous solubility or adsorption/partitioning to surfaces within the biological assay will still have an impact on HTS and accurate potency determination. 7 …”

Section: Discussionmentioning

confidence: 99%

Achieving Accurate Compound Concentration in Cell-Based Screening: Validation of Acoustic Droplet Ejection Technology

Grant¹,

Roberts²,

Pointon³

et al. 2009

SLAS Discovery

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Compound handling is a fundamental and critical step in compound screening throughout the drug discovery process. Although most compound-handling processes within compound management facilities use 100% DMSO solvent, conventional methods of manual or robotic liquid-handling systems in screening workflows often perform dilutions in aqueous solutions to maintain solvent tolerance of the biological assay. However, the use of aqueous media in these applications can lead to suboptimal data quality due to compound carryover or precipitation during the dilution steps. In cell-based assays, this effect is worsened by the unpredictable physical characteristics of compounds and the low DMSO tolerance within the assay. In some cases, the conventional approaches using manual or automated liquid handling resulted in variable IC 50 dose responses. This study examines the cause of this variability and evaluates the accuracy of screening data in these case studies. A number of liquid-handling options have been explored to address the issues and establish a generic compound-handling workflow to support cell-based screening across our screening functions. The authors discuss the validation of the Labcyte Echo reformatter as an effective noncontact solution for generic compound-handling applications against diverse compound classes using triple-quad liquid chromatography/mass spectrometry. The successful validation and implementation challenges of this technology for direct dosing onto cells in cell-based screening is discussed. (Journal of Biomolecular Screening 2009:452-459)

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Streamlined System for Purifying and Quantifying a Diverse Library of Compounds and the Effect of Compound Concentration Measurements on the Accurate Interpretation of Biological Assay Results

Cited by 72 publications

References 20 publications

Quantitative high-throughput screening: A titration-based approach that efficiently identifies biological activities in large chemical libraries

Quantitative high-throughput screening: A titration-based approach that efficiently identifies biological activities in large chemical libraries

Gradient, Contact-Free Volume Transfers Minimize Compound Loss in Dose-Response Experiments

Achieving Accurate Compound Concentration in Cell-Based Screening: Validation of Acoustic Droplet Ejection Technology

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