Keywords and definitions
Accuracy (of measurement)
Closeness of the agreement between the result of a measurement and a true value of the property being measured.
Note 1: Accuracy is a qualitative concept.
Note 2: The term “precision” should not be used for accuracy
Active (screen active)
A sample that produces a response or signal above a defined threshold at a tested concentration in a single assay or screen. Note: When the properties and identity of an active or hit are confirmed by subsequent experiment it becomes a confirmed active. See also inactive.
Graphical representation of the number of samples present in each activity range.
Note: Often shown as a frequency distribution graph, it provides an overview of the screening results and typically allows the determination of the overall background signal and threshold for selection of actives.
Minimum activity that defines actives in a primary screen. It is usually expressed as percentage of inhibition or stimulation. This would typically be calculated as test sample values >3x robust standard deviation of control samples.
Observation, effect, or result that is inaccurate because it is produced by the methodology used in scientific investigation or by experimental error. See also false positive, false negative.
Test run to determine pharmacologic response, often in the presence of a test substance. Assays are typically miniaturized and run in high throughput to identify inhibitors or activators as a first step in drug discovery. Assays may be run with isolated cellular components or using whole cells, with a variety of readouts. See also assay development, assay miniaturization.
Design and optimization process by which an assay is made ready for routine screening. Success criteria include expected responses to known standards, reproducibility, robustness and cost. The specifics of the criteria are set by institutions for screening and by the FDA for clinical use.
Experimental design aimed at decreasing the reaction volume of an assay and consequently the amount and cost of reagents. Examples: Adaptation of assays from test tube or 96 well format to high-density microplates (e.g. 1536 wells).
Experiments conducted to verify that the output measurement of the assay is consistently reflective of the activity against the target. Results are compared internally over multiple runs and externally (when available) to existing literature parameters such as Kd, Ki, Km, or EC50.
Magnitude of a signal produced in an assay or screen in the absence of a test substance. Also, signal detected from an assay in the absence of target activity; often equivalent to negative control.
Preclinical studies (medicinal chemistry, pharmacology, drug metabolism and pharmacokinetics) are performed on leads to identify a lead candidate compound. Several activities, such as pharmacology, in vivo efficacy, drug metabolism and pharmacokinetics, and experimental toxicology are performed on the lead candidate compound. This data package forms the basis of a candidate selection, after which the drug candidate is further subject to additional preclinical development activities. These preclinical activities provide the basis for an Investigational New Drug (IND) application to the FDA for permission to initiate clinical testing in humans.
Chemical Probe (tool compound)
A compound (or chemical series) that has sufficient potency, selectivity, cell permeability and bioavailability to be used for the validation of a biological hypothesis when used in cellular or animal models of disease.
Informatics technologies that integrate chemical data with analytic and molecular design tools. Cheminformatics applications provide access to compound-related information, including chemical structure, properties, structure-activity relationships.
Compound Collection (compound library, e.g. HTS collection)
Set of chemicals that has been assembled and annotated for easy storage and retrieval and that is available for screening. Generally consists of compounds synthesized by combinatorial or standard synthetic methods, purchased from commercial sources or of samples of natural products either as pure samples or as mixtures.
Testing a set of (often structurally related) molecules against a series of assays with the aim of providing insight into the pharmacological structure-activity relationships of chemical series.
A standard of comparison by which experimental results are evaluated. A control differs from the experiment in a single variable, and enables the assessment of significance of experimental results.
Dose Response (concentration response)
Figure quantifying the effect of a compound in an assay at varying concentrations. Note: This experiment provides information about the potency and efficacy of the tested compound. Classical thermodynamics of a 1:1 interaction between compound and target generally results in a hyperbolic increase of the assay signal upon linear increase in compound concentration. Thus, compound concentration responses are usually determined using logarithmic serial dilutions, e.g. 10 µmol/L, 1 µmol/L, 100 nmol/L, 10nmol/L, 1 nmol/L. Related terms: Ki, Kd, EC50, IC50.
Dose Response Screen
A screen in which confirmed actives are re-test against the same assay format, but where dose response curves (typically consisting of 11 doses of a 3-fold dilution series starting at 50 uM in duplicate) are generated.
Encoded Library Technology (ELT)
Combines the power of affinity based enrichment with combinatorial chemistry to enable the rapid selection of hits from small molecule libraries containing up to several billion compounds. Each ELT library molecule carries a unique DNA tag which encodes the chemical composition of the small molecule. Libraries are "selected" against protein targets by affinity based methods. Enriched binders are isolated, sequenced, and translated back into their corresponding chemical structures. The hit compounds are then synthesized without the DNA tag and assayed for activity against the target.
Effective Concentration (EC)
Concentration of a substance that causes a defined magnitude of response in a given system. EC50 is the median concentration that causes 50% of the maximal response. This usually refers to an agonist in a receptor system. The value of this quantity may result from either an increase or a decrease in a biological function.
Assay result in which a sample known to be active does not produce either the expected signal or a signal above the activity threshold.
Note: A false negative can occur when an assay lacks appropriate discriminatory power, when the threshold is inappropriately set, or as a result of mistaken identity of the test sample. A false negative can also occur due to specific compound properties such as poor solubility, high non-specific protein binding, or interference with the assay signal.
Assay result in which a sample known to be inactive produces a signal or response above the activity threshold.
Note: A false positive can occur when an assay lacks appropriate discriminatory power, when the threshold is inappropriately set, as a result of certain physical properties of the substance
(e.g., a fluorescent compound in a fluorescence intensity assay, aggregation), or as a result of mistaken identity of the substance.
Assay in which the biological or physiological activity of the target is measured. Example: An assay in which an agonist stimulates, in receptor-transfected cells, the production of a second messenger that is detected with a fluorescence readout.
Assay in which the response is detected only after the physical separation by methods such as filtration or centrifugation of one or more assay components. See also heterogeneous binding assay and homogeneous assay.
High-Content Screening Assay
Assay that produces multiple biological readouts.
Note: Most commonly used in relation to the mathematical (quantitative) analysis of an image acquired using an automated microscope. Analysis algorithms are used to quantify cellular parameters (e.g. number, motility, neurite outgrowth, size, shape) and subcellular events (e.g. receptor internalization, protein translocation, protein expression nuclei shape).
High-Throughput Screening (HTS)
A method in which a large number of compounds (in the case of GSK, ~2 million) are tested against optimized assays in a relatively short period of time. Typically, these assays are carried out in microplates in 1536-well format, using automated or robotic technologies.
Sample that produces confirmed activity above the hit threshold in an assay and whose structural identity has been confirmed. A substance becomes a hit when the properties of an active are confirmed by elimination of false positive results and artifacts.
Note: In the past, the terms confirmed hit, true hit and confirmed active were used with this meaning.
An experimental process by which samples which are shown to be active from a screen are demonstrated to be confirmed hits via demonstration that their pharmacological activity is due to a productive mechanism driven by a confirmed chemical structure.
Assay in which all reagents and reactants are of a uniform phase (typically, liquid phase) and assay response is detected without the need for physical separation of assay components. See also heterogeneous assay.
IC50 (Inhibition Concentration 50)
The concentration of an enzyme inhibitor or receptor antagonist that reduces the enzyme activity or agonist response by 50%.
Note: IC50 values are influenced by experimental conditions (substrate or agonist concentration—which should be specified). Related terms: EC50, inhibition constant, Ki.
Sample that, at the tested concentration, does not produce a response above the hit threshold in an assay.
Note: A sample may also be designated as inactive when attempts to confirm an active fail.
See also artifact, false positive, hit.
The best exemplar of a chemical series that has a pharmacological or biological activity and has suitable chemical properties to act as a starting point for further chemical optimization. Typically, a lead shows appropriate activity at the target, demonstrates activity in a relevant cell-based assay and has a tractable structure-activity relationship (SAR) with potential to achieve the required activity, selectivity, physicochemical characteristics, patentability and bioavailability of a candidate quality molecule.
Process that is targeted toward the generation of at least one compound series patentable that meets the requirements for progression to lead optimization. It typically encompasses the steps from the detection of initial activity (via high throughput screening and other lead finding activities) through hit confirmation and hit-to-lead activities.
Process in which the drug-like properties of an initial lead or lead series are improved. Typically, biological activity will be enhanced, in vivo efficacy will be demonstrated and compounds with a physicochemical, pharmacological and toxicological profile consistent with progression to the clinic will be identified.
A series of structurally related compounds that have pharmacological or biological activity and has suitable chemical properties to act as a starting point for further chemical optimization. Typically, a lead series shows appropriate activity at the target, demonstrates activity in a relevant cell-based assay and has a tractable structure-activity relationship (SAR) with potential to achieve the required activity, selectivity, physicochemical characteristics and bioavailability of a candidate quality molecule.
Medicinal chemistry is the chemistry discipline concerned with the design, development and synthesis of pharmaceutical drugs. The discipline combines expertise from chemistry and pharmacology to identify, develop and synthesize chemical agents that have a therapeutic use and to evaluate the properties of existing drugs.
Initial screen applied to assess the activity of a collection of compounds and identify hits or actives against a biological target of interest. This screen identifies actives from a library. See also secondary screen.
Primary Screening Assay
This is the assay format that will need to support HIT identification, typically up to 3 million wells for a full HTS.
An assay that monitors observable physical or biochemical changes within a cell that are not dependent upon a specific target. Such assays are typically designed to be as disease-relevant as possible, for example using primary or stem-cell derived materials.
Molecular properties concerned with, or relating to physical chemistry of molecules. Physico-chemical influences a wide range of molecular properties such as pKa, solubility, and lipophilicity.
Dose of drug required to produce a specific effect of given intensity as compared to a standard reference. Potency is a comparative rather than an absolute expression of drug activity. Drug potency depends on both affinity and efficacy. Thus, two agonists can be equipotent, but have different intrinsic efficacies with compensating differences in affinity. More potent compounds have lower IC50 or EC50 values implying that less is needed for an effect.
Closeness of agreement between independent test results obtained by applying the experimental procedure under stipulated conditions. The smaller the random part of the experimental errors that affect the results, the more precise the procedure. A measure of precision (or imprecision) is the standard deviation.
Process by which all of the biological materials (e.g. target proteins, substrate, ligands, cell lines) required to run an assay/screen are developed and/or procured. See also assay development.
Process by which all of the biological materials (e.g. target proteins, substrate, ligands, cell lines) required to run a large-scale screen are developed and/or procured. For example, an HTS campaign requires sufficient material to run around 3 million assay wells.
Closeness of agreement between independent results obtained with the same method on identical test material but in distinct experiments (different operators, different apparatus, different laboratories, and/or after different intervals of time). The measure of reproducibility is the standard deviation qualified with the term “reproducibility” as reproducibility standard deviation. In some contexts, reproducibility may be defined as the value below which the absolute difference between two single test results on identical material obtained under the above conditions may be expected to lie with a specified probability.
Automated device that performs tasks (i.e., screen functions) that would normally be performed by a human.
Note: A robot in laboratory automation is usually used to move microplates in an automated assay procedure and usually consists of either a robotic arm with at least three degrees of freedom or a plate gripper.
Operation or series of operations that contributes to the validation of screening results by establishing the acceptable limits of performance of internal controls. Examples: Validation of liquid handling devices and plate readers, determination of the Z' factor and use of assay controls, and post-experiment controls.
Note: Results of a screen are validated only after a set of quality controls has been performed.
Screen (Screening Campaign)
The execution, analysis, and interpretation of a large number of assays to evaluate the activity of a collection of samples against a target, pathway or in a phenotypic screen.
Assay conditions, as determined by assay validation, are performed in the chosen plate format with an acceptable signal to background ratio as described by the Z' factor. Validation typically requires testing a number of samples a minimum of three times and allows for the estimation of false positives, false negatives and the selection of statistically significant thresholds.
Screen applied to independently confirm actives from the primary screen.
Note: A secondary screen may employ an assay that differs in type from the primary screen (biochemical assay vs. cell based assay), or it may be of the same type with different readout.
Assay used to determine the relative potency of active or lead compounds towards an alternative target. A selectivity assay (or panel of assays) may include targets of the same family or unrelated targets.
Tool Compound (chemical probe)
A compound (or chemical series) that has sufficient potency, selectivity, cell permeability, and bioavailability to be used for the validation of a biological hypothesis when used in cellular or animal models of disease.
Number of results that can be generated in a given timeframe. In high-throughput screening (HTS), throughput is often defined by the number of assay samples that can be processed in a day (e.g. 50,000 samples per day). Related terms: high throughput, ultra-high throughput.
Dimensionless statistical parameter that is used extensively in biomolecular screening. The Z' factor is a characteristic of an assay without the intervention of test compounds. It provides a practical index of the quality and reliability of the assay, and can be used as a guide to assay development and optimization where the standard deviations of the high and low controls and μc+ and μc- are the means of the high and low controls, respectively. The useful range of Z' values is from 0 (very poor) to +1 (excellent). In most cases, a Z' factor greater than 0.6 is required for an assay to be accepted for HTS.
All definitions are from internal GSK processes or adapted from:
- Steinmetz et al. 2009, http://www.biomedcentral.com/1471-2377/9/S1/S2)
- Pure Appl. Chem., ASAP Article doi:10.1351/PAC-REC-09-05-03 © 2011 IUPAC, Publication date (Web): 7 February 2011
- Society for Laboratory Automation and Screening