Since 2009, the Tox21 project has screened ∼8500 chemicals in more than 70 high-throughput assays, generating upward of 100 million data points, with all data publicly available through partner websites at the United States Environmental Protection Agency (EPA), National Center for Advancing Translational Sciences (NCATS), and National Toxicology Program (NTP). Underpinning this public effort is the largest compound library ever constructed specifically for improving understanding of the chemical basis of toxicity across research and regulatory domains. Each Tox21 federal partner brought specialized resources and capabilities to the partnership, including three approximately equal-sized compound libraries. All Tox21 data generated to date have resulted from a confluence of ideas, technologies, and expertise used to design, screen, and analyze the Tox21 10K library. The different programmatic objectives of the partners led to three distinct, overlapping compound libraries that, when combined, not only covered a diversity of chemical structures, use-categories, and properties but also incorporated many types of compound replicates. The history of development of the Tox21 "10K" chemical library and data workflows implemented to ensure quality chemical annotations and allow for various reproducibility assessments are described. Cheminformatics profiling demonstrates how the three partner libraries complement one another to expand the reach of each individual library, as reflected in coverage of regulatory lists, predicted toxicity end points, and physicochemical properties. ToxPrint chemotypes (CTs) and enrichment approaches further demonstrate how the combined partner libraries amplify structure−activity patterns that would otherwise not be detected. Finally, CT enrichments are used to probe global patterns of activity in combined ToxCast and Tox21 activity data sets relative to test-set size and chemical versus biological end point diversity, illustrating the power of CT approaches to discern patterns in chemical− activity data sets. These results support a central premise of the Tox21 program: A collaborative merging of programmatically distinct compound libraries would yield greater rewards than could be achieved separately.

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Sodium absorption in epithelial cells is rate-limited by the epithelial sodium channel (ENaC) activity in lung, kidney, and the distal colon. Pathophysiological conditions, such as cystic fibrosis and Liddle syndrome, result from water-electrolyte imbalance partly due to malfunction of ENaC regulation. Because the quaternary structure of ENaC is yet undetermined, the bases of pathologically linked mutations in ENaC subunits ␣, ␤, and ␥ are largely unknown. Here, we present a structural model of heterotetrameric ENaC ␣ 1 ␤␣ 2 ␥ that is consistent with previous cross-linking results and site-directed mutagenesis experiments. By using this model, we show that the diseasecausing mutation ␣W493R rewires structural dynamics of the intersubunit interfaces ␣ 1 ␤ and ␣ 2 ␥. Changes in dynamics can allosterically propagate to the channel gate. We demonstrate that cleavage of the ␥-subunit, which is critical for full channel activation, does not mediate activation of ENaC by ␣W493R. Our molecular dynamics simulations led us to identify a channel-activating electrostatic interaction between ␣ 2 Arg-493 and ␥Glu-348 at the ␣ 2 ␥ interface. By neutralizing a sodium-binding acidic patch at the ␣ 1 ␤ interface, we reduced ENaC activation of ␣W493R by more than 2-fold. By combining homology modeling, molecular dynamics, cysteine cross-linking, and voltage clamp experiments, we propose a dynamics-driven model for the gain-of-function in ENaC by ␣W493R. Our integrated computational and experimental approach advances our understanding of structure, dynamics, and function of ENaC in its disease-causing state.The epithelial sodium channel (ENaC) 3 regulates Na ϩ absorption by epithelia, thereby maintaining essential waterelectrolyte balance (1, 2). Therefore, altered ENaC activity contributes to pathological conditions that are partly mediated by sodium transport dysregulation, such as cystic fibrosis and Liddle syndrome (1). ENaC is composed of structurally homologous subunits ␣, ␤, and ␥. Mutations in the ␣-subunit, such as the gain-of-function mutation ␣W493R in exon 10, decrease lung functionality (3-7). Carrying the ␣W493R polymorphism in ␣ENaC can result in chronic bronchitis (4). Heterologous expression of ␣W493R with ␤ and ␥ subunits in Xenopus oocytes produced an ϳ4-fold increase in ENaC-mediated current and an increase in the channel open probability (8). However, the structural determinants of this activation remain unknown. Wild type ENaC is normally proteolytically activated by intracellular furin-like convertases and extracellular trypsinlike serine proteases (9 -12). Cleavage of an inhibitory fragment in the finger domain of the ␣-subunit is thought to reorganize the extracellular domains to favor active conformations of the channel (13). Kashlan et al. (13) have elucidated the mechanism of inhibition by the cleaved fragment using synthetic peptides derived from the inhibitory tract of the ␣-subunit. Releasing the inhibitory tract by proteolysis was found to activate ENaC by eliminating interactions at the thumb and f...