The goal of the PubChem BioAssay system is to provide services to make the screening data of this large scale collection easily accessible to the public, and provide data analysis tools to facilitate the utilization of this valuable information. To this end, information content of PubChem can be accessed through the NCBI Entrez system. One can search, in Entrez's PubChem Compound database, a compound with a chemical synonym, and subsequently link to a list of screening experiments involving the given compound via the ‘BioAssays’ link (1). One can also find, in Entrez's PubChem BioAssay database, all bioassay tests for a specific target by querying the name of the protein target. The Entrez ‘Limits’ facility allows one to construct a specific query based on one's research need. Furthermore, PubChem provides a set of web-based tools to integrate the chemical and biological activity information, and support the navigation and in-depth data analysis that facilitates identification of chemical probes and biological interesting targets contained within PubChem databases. For these tools, PubChem implements a queuing mechanism to balance web requests.

The BioAssay information system provides access to review the deposited bioassay record in detail. It also provides exploratory data analysis tools by using the ‘summary’ biological test results. Screening descriptions and test results in PubChem BioAssay database are diverse and assay-specific. PubChem allows depositions of as many readouts as it needs to be, and provides tools to search, select and retrieve such deposited information. Despite this flexibility, PubChem requires a summary result for each tested chemical sample for defining the bioactivity outcome and bioactivity score. PubChem bioactivity outcome summary includes five categories, e.g. chemical probe, active, inactive, inconclusive and unspecified. For the dose–response screening test, the primary endpoint, such as IC50, is denoted as ‘active concentration’ summary, and needs to be reported in micromolar units. Such summary results allow one to classify and rank hits of a screening test. More importantly, having a bioactivity outcome summary for each tested sample allows PubChem to provide tools for rapid comparative analysis across multiple screening reports for given chemical samples. PubChem has integrated the bioactivity analysis tools (Figure 1) using these summary results to provide a comprehensive review of biological tests, compare biological activity data from multiple screenings, and explore structure–activity relationship.

Depending on users’ goals, the summary of bioactivity can be switched between compound-centric and substance-centric views. If centered on substance descriptions, this particular tool provides a summary view of all available biological tests and the respective bioactivity outcome contributed by a single organization. If the substances are deposited by the MLSMR (NIH Molecular Libraries Small Molecule Repository), they can be tested by multiple screening centers within the NIH Molecular Library Program. If centered on compound descriptions, the service provides a comprehensive summary view of biological activity by aggregating all screening data across multiple contributors for the unique chemical structures. Given the difference in scope between compounds and substances, we will consider only the case of compounds in more detail below.

The BioActivity Summary tool can be invoked from any BioAssay Summary page. In this case, the active compounds of the bioassay will become the default focus. For example, in the ‘Cathepsin B inhibitor Series SAR Study’ assay, AID 523(http://pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?aid=523), 10 compounds are identified as potential cathepsin B inhibitors (Figure 2). One may perform the BioActivity Summary analysis for the active compounds using the ‘BioActivity Summary’ link under the ‘BioActive Compounds’ section (Figure 2). The result of such analysis is shown in Figure 3. These 10 active compounds are tested in over 300 assays, deposited in PubChem at the time of this writing, including AID 523. In the summary table, assays are sorted by the count of active compounds. Each assay is summarized, one per line, with the count of active, inactive and tested compounds. The assay name, bioactivity outcome method type, e.g. primary versus confirmatory, and protein target specification give a summary of what screenings are performed for the compounds under analysis. More importantly, one can see that some of the compounds that are active in AID 523 also show activity in a number of other assays. The confirmatory assays ranked near the top of the table include assay targets from several other protease-related proteins, such as West Nile Virus NS2bNS3 proteinase, Factor XIa, kallikrein-related peptidase 5, Cathepsin G and Factor XIIa. This suggests that some of these 10 compounds are nonspecific inhibitors for the Cathepsin B target and demonstrate cross reactivity against a group of biologically related proteins.

The BioActivity Summary service provides several powerful selection-revise features that enable one to rapidly revise the focus of the analysis by modifying the set of selected compounds and assays. As depicted in Figure 3, one may choose to expand the current set of compounds to include all tested compounds or active compounds within a given set of bioassays, or expand the compounds to include those having similar 2D structures to the ones in the current set. Alternatively, one may choose to limit the compounds to only those found active in the assay subset. Similarly, the selected assay set can be expanded to include those that are active or tested for a given set of compounds. Additionally, bioassays with similar bioactivity profiles and bioassays with similar protein target sequence can be added to selection. For example, to focus on a subset of the input compounds that are active in one or more of the selected assays, user would click the ‘Select Active’ link in the ‘Revise Compound Selection’ section to exclude the less interesting compounds, or to explore additional assay screens where the given compounds are considered active by using the ‘Add Active’ link in the ‘Revise BioAssay Selection’ section. One may also choose to focus on confirmatory assays or assays with specific molecular targets using the filtering features provided in the ‘Other Filters’ pop-up menu.

One of the common entry points for accessing the BioActivity Summary tool is from a single PubChem compound summary record. Invoking the BioActivity Summary tool from a compound summary record will readily generate an overview of all biological screenings performed for that compound. From the BioActivity Summary page users can expand the analysis by including compound similar in 2D chemical structures via ‘Add Similar Compounds’ link in the ‘Revise Compound Selection’ section. This operation adds compounds with significant 2D structural similarity and allows users to collect and examine the bioactivity data among tested analogs. A further request of all bioassays tested for the analog series using the ‘Add Tested’ link in the ‘Revise BioAssay Selection’ section may reveal additional important screenings where the structural analogs are tested. Subsequent analysis using the Structure–Activity Analysis tool (to be described) enables further evaluation on the SAR and bioactivity profile of such analog series.

Other entry points include NCBI Entrez ‘DocSum’ reports for PubChem substance, compound and bioassay records, where the BioActivity Summary tool can be invoked for each individual record as well as for the entire data set resulted from an Entrez search. This can be done by using the explicit ‘BioActivity Analysis’ link, or clicking the double six-member ring icon from the ‘Tools’ area. For example, one may start, in Entrez's PubChem Compound database, with a compound submitted to PubChem by a journal article reporting specific enzyme inhibitors. To verify the discussed inhibition activity of the enzyme inhibitor, one can compare the reported bioactivity information to the biological tests deposited in PubChem. Alternatively, one may start a structure search with a given substructure using the service provided at http://pubchem.ncbi.nlm.nih.gov/search/search.cgi, and launch the BioActivity Summary tool for the resulting compound set to the link described above. In another case, one may search PubChem BioAssay database for all available screening tests for a particular target, then use the BioActivity Summary tool to examine the bioactivity outcomes from each screening experiment, compare the hit list and compile a library of bioactive compounds for the target. Users can also choose to access this analysis tool through the common gateway of PubChem BioActivity Analysis Service provided at: http://pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?p=bioactivity. From this entry point, the dataset of assay, substance/compound, which are subject to the analysis, can be specified by entering an ID list, providing a text file contains the IDs (comma separated, or one ID per line), or referring to an Entrez search history. This entry point provides the flexibility to focus the analysis on a well defined compound and assay dataset, for example, to evaluate the toxicity properties for the compounds of one's interest using only toxicity profiling assays available at PubChem. In the case that only compound input is specified, a summary for all available biological test results will be provided.

In addition to providing a bioactivity overview, this service serves as the starting point in the bioactivity analysis process, which leads users to further analysis using the Structure–Activity Analysis tool and Data Table tool which will be described in the following sections. PubChem Data Table tool supports the retrieval of assay data from multiple depositions. Prior to such analysis, multiple assays can be specified using the checkbox on the page provided with the BioActivity Summary tool. The results of the BioActivity Summary analysis are saved on a temporary server, and will be available only for a limited period of time, usually 48 hours. The status of this analysis, however, can be saved through the ‘Save View’ feature to facilitate scientific communication. Analysis can be resumed by importing the status file through the web server at http://pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?p=qfile under the common gateway of PubChem BioActivity Analysis Service.

The results are presented through the use of an interactive heatmap display. With this web-based service, a group of compounds and assays may be clustered using various means. Chemical structures may be clustered based on 2D structure similarity (as measured by Tanimoto score using the PubChem dictionary-based fingerprint [ftp://ftp.ncbi.nlm.nih.gov/pubchem/specifications/pubchem_fingerprints.txt]) or biological response, as measured by reported bioactivity outcome, bioactivity score, or active concentration (e.g. IC50, EC50, AC50, etc.). Assays can be clustered based on similarity in biological response of the compound set, similarity in sequence of assay targets, or similarity provided by depositors. Some of the similarity data (e.g. protein target similarity) are pre-computed to obtain optimal analysis performance.

‘Revise Selection’ features, similarly to those in the BioActivity Summary tool, are provided to define and modify the focus of the analysis. These features can be accessed by clicking on the ‘+’ sign shown on the left of the Revise Selection section (Figure 4). Furthermore, facilities for further analyzing the clustering results and navigating between various PubChem tools, including the Entrez search system, are provided and may be accessed throughout the heatmap display. For example, as shown in Figure 4, in the dendrogram display, one may click on a blue circle attached to a node of a compound cluster to invoke a feature menu, and to display the sub-tree, prune the sub-tree, or add compounds similar to those contained in a sub-tree. Users can also retrieve chemical structure similarity score matrix used in the clustering and send the selected compounds to one of PubChem tools or to Entrez system. Using similar functionalities associated with assay cluster, one may retrieve assay target similarity score matrix, or revise assay selection to include the various types of related bioassays. Users can also perform a number of operations on a combined group of assays and compounds. To define such a subset, one can zoom in the heatmap display by clicking on two cells in the heatmap. The operations available include sending the compounds in the sub-cluster to the ‘Structure Clustering’ service to visualize the chemical structure classes, sending assays, or compounds to the Entrez system to, for example, check the availability of protein 3D structure complex or look for information on biological mechanism using linked PubMed articles, etc. Using this feature, one can further compare multiple test results in details by retrieving all readouts using the Data Table tool (to be described).

A common entry point for the Structure–Activity Analysis service is from the previously described BioActivity Summary service, which can be used to narrow down the compound and assay set, thus to prepare an appropriate input for the SAR study. This tool can also be accessed from the BioAssay Summary service for a given bioassay record to analyze the set of active compounds in a single assay through identification of chemical structure clusters that exhibit similar biological response. In this particular application, one may want to take advantage of the integrated tools to further expand the assay scope, for example, to combine the screening results for related targets using the ‘Add Related BioAssays’ functionality provided in the Revise Selection section, and attempt to search compounds demonstrating high selectivity towards a particular target. This service can be also accessed through the common gateway of the PubChem BioActivity Analysis Service at http://pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?p=heat, where assays, substances or compounds can be flexibly specified based on one's research need.

Results from such structure–activity analysis, including image, data table and similarity matrix can be exported in respective formats including the Graph Modelling Language format for the dendrograms. Similarly to the BioActivity Summary service, the results of this analysis are saved temporarily, and can be accessed for only a limited period of time, usually 48 hours. Users can use the ‘Save View’ button to save the analysis in a status file and use this file to communicate the results with collaborators, who can open the status file at http://pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?p=qfile to see the exact same analysis page at a later time.

Test results are shown in tabular format (Figure 5). Each row in the data table displays a chemical structure, SID, CID, bioactivity outcome, score and associated assay readouts. Bioactivity outcome is highlighted using icon (rectangle shape), with ‘active’ outcome colored in red and ‘inactive’ outcome colored in blue. Each CID is linked to the respective PubChem compound summary server, while SID(s), which can be multiple with the merged data presentation from the ‘compound view’ (see description below), is linked to the substance ‘DocSum’ report in Entrez. Cross references for a specific test result, such as the GenBank accession number for the protein target of a compound, or PubMed ID of an article where the activity information is extracted, can be reported along with assay readouts in the data table. In such case, they are hyperlinked to the respective Entrez records.

‘Concise Data’ view, including bioactivity outcome, score and active concentration if provided, is shown in the data table as the default for rapid review, while complete results including all provided readouts can be obtained upon request using the ‘Data Table, Complete’ link. Data table can be sorted flexibly based on SID/CID, bioactivity score, or a user-selected readout. Data table can be paginated vertically for navigating the test results for different chemical samples, and can be paginated horizontally in the case of multiple assay data retrieval, for navigating the test results from different bioassay depositions. These features are necessary to support the analysis of screening results within PubChem, which sometimes contain hundreds of thousands of chemical samples, and a few hundreds of readout fields.

Assay results can be downloaded in multiple formats including CSV, XML and ASN.1. Chemical structure data can be downloaded as SD files, as well as in many other data formats, such as SMILES, InCHI, XML and ASN.1. Given the large data volume, when downloading test results simultaneously for multiple bioassay records, only the data fields explicitly shown on the current data table can be downloaded at each time. The PubChem BioAssay system currently supports the retrieval and display of readout fields for up to 20 assay records at one time. If users want to request the next 20 bioassay records, they can use the pagination functionality to retrieve and download additional test results. No such limitation is set for data rows, e.g. this service allows downloading all shown test result fields for the entire set of chemical samples.