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Reporting Data from High-Throughput Screening of Small-Molecule Libraries

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Reporting Data from High-Throughput Screening of Small-Molecule Libraries COMMENTARY Reporting data from high-throughput screening of small-molecule libraries James Inglese, Caroline E Shamu & R Kiplin Guy Publications reporting results of small-molecule screens are becoming more common as academic researchers increasingly make use of high-throughput screening (HTS) facilities. However, no standards have been formally established for reporting small-molecule screening data, and often key information important for the evaluation and interpretation of results is omitted in published HTS protocols. Here, we propose concise guidelines for reporting small-molecule HTS data. High-throughput screening (HTS) is becoming providing a basis for evaluation, comparison in the assay. Controls are used to determine a routine method for identifying probes used in and replication of small-molecule screens. We an assay ‘window’ and validate the biological chemical biology. The flexibility of the process have divided small-molecule screen protocol response. It is often desirable to provide an has allowed numerous and disparate areas of information into five categories: the assay, the indication as to the efficacy of the controls. biology to engage with an equally diverse palate library, the HTS process, the post-HTS analy- For example, here is an assay strategy descrip- of chemistry. However, with this diversity, the sis of data and compound structures, and the tion that might be written for a fluorescence many components associated with HTS—the screen results (Fig. 1). Within each category, polarization assay: bioassay, the assay format, the nature of the we describe key pieces of information that are library, reagent and sample delivery meth- important for interpretation and replication This screen was carried out to identify compounds ods, the detection instrumentation, the level of a screen (Table 1). that disrupt formation of the X–Y protein com- plex. The assay uses fluorescence polarization to of automation, and the data analysis algo- monitor binding of a fluorescein-labeled peptide, rithms—all conspire to make HTS one of the Assay X36* (derived from the protein X extracellular least standardized processes used in academia Assays fall into three general types: isolated domain), to full-length protein Y. Screening posi- today. Nonetheless, there exists commonality molecular target assays, cell-free multicompo- tives are compounds that block binding of X36* in HTS that allows reporting of data describing nent assays, and cell- or organism-based assays. to protein Y. Addition of unlabeled X36 peptide fundamental parameters of the assay, screen, Assays on purified enzymes such as proteases (5 µM) completely blocks binding of X36* in this library, and outcome that are useful for gain- or kinases, and assays on activities associated assay and is used as a positive control. DMSO ing insight into these HTS processes and for with cell extracts, membranes or reconstituted alone is the best negative control for this assay comparing results between screens. signaling cascades, are representative examples during HTS. Here we suggest data and descriptive of the first two assay types. Cellular assays can information to include in HTS protocols in be subdivided into ‘reporter gene’–type assays For some assays (in particular for cell-free manuscripts and databases that should aid in and phenotypic assays that measure outputs assays), it is possible to get quantitative mea- resulting from intact cellular processes. surements of the dynamic range and sensitivity A description of the logic behind the assay, of the assay. When available, this information is James Inglese is at the US National Institutes of including positive and negative control con- useful to help evaluate the quality of an assay. Health Chemical Genomics Center, National ditions, is critical to understanding how For example, this information might be pre- Human Genome Institute, National Institutes library compounds will be scored as active sented for a fluorescence polarization assay of Health, 9800 Medical Center Drive, Bethesda, in the assay. This description provides con- as “This assay was linear in the range of 60 Maryland 20892-3370, USA; Caroline E. Shamu text for the assay’s sensitivity to specific types mP units (the polarization observed for free is at the ICCB-Longwood Screening Facility, of interference. Positive controls are condi- peptide) to 170 mP units (the polarization Harvard Medical School, 250 Longwood Ave., tions (for example, small-molecule addition, observed for fully bound peptide) with an error Boston, Massachusetts 02115, USA; and R. RNA interference knockdown, or mutations) of ± 5 mP units. Under screening conditions, Kiplin Guy is in the Department of Chemical that produce the same result in the assay as we estimate that differences of 5 to 10% of pep- Biology and Therapeutics, St. Jude Children’s a desired active compound. Negative con- tide bound could be reliably distinguished.” Research Hospital, North Lauderdale, Memphis, trols are usually ‘vehicle’-only conditions (for The sources of all reagents used in the screen Tennessee 38105, USA. example, DMSO) or, where appropriate, small should be documented. Catalog and batch e-mail: [email protected] molecules demonstrated to have no activity numbers (if relevant) for all commercially 438 VOLUME 3 NUMBER 8 AUGUST 2007 NATURE CHEMICAL BIOLOGY COMMENTARY . Inhibitors? e S µM) ( uctur 0 N 5 F Compound no Str IC N N F H Fluorescence F N FC(F)(F)C(C=C1)=CC2=C1SC3=C(N2CCCN4CCN(C)CC4)C=CC=C3 Number of 1 0.06 H compounds N N HN HN O Well position N H N N Inhibitors? COC1=CC=C(NC2=C3CCNC2)C3=C1 2 1.9 Cl O HN O Plate number ClC1=CC2=C(OC(C=CC=C3)=C3N=C2N4CCNCC4)C=C1 H H O Br N N S 3 0.3 Number of O O compounds Reporter HN ' O Z Well position O=C(CS(C1=CC=C(C(C)=O)C=C1)(=O)=O)NC2CCCCC2 Post-HTS Assay Library HTS process Results analysis Figure 1 The flow of materials and data from assay to reported results in HTS. available components should be listed. should be provided. For natural products, the dispenser tip). Intraplate controls are essen- Descriptions of how all noncommercial nature of the samples should be described tial to the establishment of the assay window reagents were generated or obtained should be (that is, are the samples purified and struc- (as described above), and over the course of a provided. Availability of proprietary reagents tures determined, or are extracts being used?). screen they permit the analysis of the unifor- should be described. It is helpful if the amounts Finally, information should be provided about mity of the biological response. The following of key assay reagents required to screen a library the quality control procedures used to acquire two examples from Davis et al.1 illustrate how of a defined size in a specified assay format are and maintain the library, and about the source this information can be described: mentioned explicitly. of the library. If available a web link can be A clear summary of the assay protocol included providing additional library infor- Interplate controls: plates containing vehicle only should be provided for each screen. The mation. When files detailing the composition (in place of test compounds) were uniformly distributed throughout the screen at ten-plate instruments used to perform each step should of a library are provided, where possible they intervals to monitor systematic variation in be indicated. This can be written in paragraph should include structure and unique/vendor background. form; for example, “Assay plates were filled with ID information. Example: 1,000 HeLa cells per well in 30 µl of medium Intraplate controls: columns 1–4 of the 1,536- (Matrix Wellmate plate filler) and incubated The library screened consisted of 50,000 com- well plates were used for arraying of controls. Columns 1 and 2 contained a 16-point dose- in a Liconic STX200 incubator for 16 hours pounds arrayed in 384-well plates as single com- pounds at 10 mM in DMSO (additional detail response curve of MG132, with each concentra- before compound addition....” Presenting HTS describing the library may be obtained from the tion present in duplicate. In column 3, the top assay protocols in table form is also a good links included in the supplementary informa- 24 wells contained doxycycline only (for use as option, especially for more complex protocols. tion). The quality of all compounds was assured normalization to the minimum signal), and the Table 2 shows an example adapted from Davis by the vendor as greater than 90% pure, with bottom 8 wells contained assay medium alone et al.1. provided quality control data; this was verified for use as a reference for induction by doxycy- internally on 5% random sampling. The library cline. In column 4, the top 24 wells contained Library was screened at a constant 1:1,000 dilution, with a the highest concentration of MG132 (for use as In order to allow others to evaluate the nature 10-µM final concentration of compound in each a 100% activation reference), and the bottom 8 of the results and to replicate work in the well (0.1% DMSO). Supplementary information: wells again contained assay medium alone. future, it is essential to provide sufficient http://www.msdiscovery.com/spectrum.html for library .xls or .sdf files. knowledge regarding the nature of the library The number of assay plates and screen that has been screened. First, the constituency duration are useful metrics that should be of the library should be described (for instance, HTS process incorporated into a description of the HTS. peptide, natural product, known drugs), with Though most HTS laboratories use microtiter Reagent and compound dispensing systems, corresponding detail provided about the type plate–based platforms, the following descrip- detectors (type, model, settings), and output of compounds (for instance, core scaffolds or tive information should serve as a general mode (end point, kinetic, and so on) should privileged structures) represented.
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