Regulatory activity of putative elements.

<p>(<b>A</b>) Mean activities of 8 replicates of Sau3AI-digested putative regulatory elements in C3A liver cells. Log₁₀ changes relative to promoter-only construct shown. Error bars, standard error of the mean. (**, P<0.01 and ***, P<0.0001 compared to promoter-only construct, both figures.) (<b>B</b>) Mean activities of 8 replicates of AluI-digested putative regulatory elements in C3A liver cells.</p

Druggability score histograms for validation set binding site structures.

<p>The top panel depicts results with the original crystal structures used rigidly, with a red line indicating the Dscore+>1.3 cutoff used in this work. The top Dscore+ value is shown for each of the 27 protein targets (17 druggable and 10 difficult, where the prodrug targets are considered difficult). The bottom panel depicts results after modeling of protein flexibility, with difficult targets in ghost outline because flexibility modeling is not usually applied to sites that score below the Dscore+>1.3 cutoff. Difficult targets are indicated by the lighter bars, while druggable targets are indicated by the darker bars. See text for further discussion.</p

Breakdown of predicted druggable pockets at known intermolecular interfaces.

<p>Pockets found where a bound ligand would disrupt a protein-protein interaction are shown in the blue circle, and pockets found where a bound ligand would disrupt a protein-ligand interaction are shown in red. The overlap region shown in purple indicates where a protein or structure contains a pocket at both a protein-ligand and protein-protein interface. The blue region indicates proteins or structures containing only protein-protein interfacial pockets, and the red region indicates proteins or structures containing only protein-ligand pockets. Ligands are defined as any molecule with molecular weight ≤1000 kDa.</p

Results for protein-protein interaction targets from 2P2PI with known druggability [10], [12].

<p>These targets have <u>protein-protein</u> co-crystal structures shown in the top half of the table, and corresponding <u>protein-ligand</u> co-crystal structures are shown in the bottom half. Values of Dscore+ and volumes falling within drug-like criteria are highlighted in bold. ‘cmpd’ indicates a small molecule compound, and volumes are in units of ų. “*” indicates ‘not applicable’ because the site's initial Dscore+ values did not meet the cut-off for flexibility modeling; however, some values were subsequently calculated for validation purposes, and are discussed in the text. We define known druggability based on the success in finding drug-like, clinical small molecules where there are efforts from multiple independent groups.</p

Histone modifications associated with fragment sequences^a.

a<p>“liver” signifies histone modifications associated with fragment in HepG2 cells; “other” signifies histone modifications in cell types other than HepG2.</p>b<p>No histone modification data for mitochondrial DNA.</p

Results of druggability analysis of mammalian crystal structures in the PDB (as of June 30, 2012) with inclusion of light protein flexibility.

<p>The “Structures” column provides the number of unique PDB entries represented, and “Proteins” represents the number of unique Swiss-Prot entries.</p>1<p>Dscore+>1.3 for original rigid structure.</p>2<p>Dscore+≥1.7, drug-like volume (160–800 ų) after flexibility modeling, protein at least 100 amino acids in size (equivalent to about 10 kDa molecular weight).</p>3<p>Intermolecular interfaces are further defined to include only protein-protein interaction dimer interfaces and protein-ligand pockets.</p>4<p>Cryptic pockets are further defined as pockets that are less than 100 ų in volume in the crystal structure, but fall into the drug-like volume range after modeling of protein flexibility. An additional criteria of enclosure <96% was applied to eliminate small buried sites.</p

Discovery and Characterization of Human Exonic Transcriptional Regulatory Elements

<div><p>We sought exonic transcriptional regulatory elements by shotgun cloning human cDNA fragments into luciferase reporter vectors and measuring the resulting expression levels in liver cells. We uncovered seven regulatory elements within coding regions and three within 3' untranslated regions (UTRs). Two of the putative regulatory elements were enhancers and eight were silencers. The regulatory elements were generally but not consistently evolutionarily conserved and also showed a trend toward decreased population diversity. Furthermore, the exonic regulatory elements were enriched in known transcription factor binding sites (TFBSs) and were associated with several histone modifications and transcriptionally relevant chromatin. Evidence was obtained for bidirectional <em>cis-</em>regulation of a coding region element within a tubulin gene, TUBA1B, by the transcription factors PPARA and RORA. We estimate that hundreds of exonic transcriptional regulatory elements exist, an unexpected finding that highlights a surprising multi-functionality of sequences in the human genome.</p> </div

Ligands used in protein modeling and docking procedure.

<p>The tetra-substituted naphthalene compound, 2, was designed to facilitate opening of pockets.</p

Flexible protein druggability modeling applied to Bcl-xL.

<p>Phe105, Leu108, and Leu130 are shown in stick in all structures. (a) Crystal structure of Bcl-xl protein bound to a BAD peptide (red and gray, respectively, PDB ID: 2bzw), (b) two naphthalene induced-fit docked models (orange), (c) one TSN induced-fit docked model (green), and (d) ABT-737-bound crystal structure (blue, PDB ID: 2yxj) with TSN induced-fit model (green).</p

Distributions and workflow.

<p>(<b>A</b>) Quantile normalized relative luciferase activity for Sau3AI-digested exonic fragments in liver C3A cells compared within, and between, plates. Relative luciferase activity is the log₁₀ ratio of firefly luciferase to <i>Renilla</i> luciferase. Batch number indicates corresponding 96-well plate. (<b>B</b>) Quantile normalized relative luciferase activity for AluI-digested exonic fragments in C3A liver cells. (<b>C</b>) Distribution of relative luciferase activities for Sau3aI-digested fragments in liver C3A cells. (<b>D</b>) Distribution of relative luciferase activities for AluI-digested fragments in liver C3A cells. (<b>E</b>) Workflow for identifying regulatory elements.</p