The list of active DR candidates.

<p>The list of active DR candidates.</p

Performance evaluations using the public anti-cancer HTS dataset as a benchmark.

<p>The seven classifiers (S, T, E, ST, SE, TE, and STE) were evaluated based on the AUCs of the ROC curve for glioblastoma, lung cancer, and breast cancer. Only compounds in the core set were evaluated. The AUC values were calculated by averaging 100 rounds of 3-fold cross validation. (A) Typical examples of performance evaluation using the HTS data set for glioblastoma (AID45), lung cancer (AID5), and breast cancer (AID97). The AUCs were independently calculated using two distinct sets of hit compounds as a benchmark (or positives)—i) the hit compounds of known anti-cancer activity (red lines) and ii) the novel hits (green lines). The distribution of AUCs using (B) the compounds of known anti-cancer activity as a benchmark, and (C) the novel hits as a benchmark.</p

Pathway enrichment pattern of the eight active DR candidates for glioblastoma.

<p>The p-values and their adjusted q-values were calculated by hypergeometric test and the Benjamini-Hochberg method, respectively.</p

Cluster analysis of DR candidates with other cancer drugs using their pathway enrichment patterns.

<p>The eight DR candidates and 69 cancer drugs in the LINCS dataset were clustered using the 32 up- and the 17 down-regulated pathway enrichment patterns. The eight DR candidates (red) belong to five clusters (I ~ V) with 15 cancer drugs. The bar plot on the right side shows the number of significantly enriched cancer drugs (q-value<0.05) for the corresponding pathway. The significance of up- and down-regulation is presented in red and green, respectively.</p

The high-scoring DR candidates for glioblastoma among the FDA-approved drugs that were predicted based only on the expression signatures.

<p>(A) DR scores, (B) the fraction of significantly inhibited cells summarizing the results of (C), (C) the anti-proliferative activities (% growth inhibition) for the four glioblastoma cell lines (four cell lines of TG98, A172, U251MG, and U87MG) and the eight patient-derived primary cells (the GBLs) at 10 μM, (D) in silico prediction scores for BBB transport based on <a href="http://www.cbligand.org/BBB" target="_blank">http://www.cbligand.org/BBB</a>. The red asterisk indicates experimental support for passing the BBB according to the literature. Overall, anti-proliferative activities across glioblastoma cells strongly correlated with the rankings by the DR score. Most DR candidates were shown to be able to pass the BBB.</p

Overview of the <i>in silico</i> DR procedure.

<p>(A) The structural (S), target (T), and expression (E) signatures for each compound (circles on the left) and disease (squares on the right) were compared. The associations are indicated by dashed lines in three categories (S: yellow, T: green, E: red) depending on the type of compound signature. (B) In total, seven different classifiers were constructed based on the similarity between the compound and the target signature or their combinations (S, T, E, ST, SE, TE, and STE). The DR scores were calculated using a series of classifiers based on a logistic regression with the known drug set (KD set) used as a benchmark. (C) The performance was evaluated using three independent datasets: I) the mean AUC of 100 rounds of 3-fold cross validation, II) comparison with the 29 sets of NCI-60 DTP human tumor cell line HTS data, and III) experimental validation of anti-proliferative activities using cancer cell lines and primary cells. A pathway-level interpretation of the drug mode of action was performed for active DR candidates for glioblastoma (IV).</p

Variable domain sequence redundancies of the constructed library.

<p>The variable domain sequences of the unselected library were obtained by 300 bp paired-end sequencing on Illumina MiSeq platform, and the number of replicates (n) for the variable domain sequences was analyzed. Approximately 98% of V_H and V_λ, and 88.5% of V_κ sequences were found only once (n = 1).</p

Dot blot analysis of random pre-selection scFv clones from the library.

<p>Ninety-two clones were randomly chosen from the unpanned library, grown and induced with IPTG, and periplasmic extracts were obtained and blotted on a nitrocellulose membrane. The clones that express soluble, full-length scFv were detected by the presence of a HA-tag at the C-terminus. Out of 92 clones, 58 were detected by HRP-conjugated anti-HA antibody (63%).</p

Binding kinetics of selected target-specific scFv clones determined by SPR.

<p>Binding kinetics of selected target-specific scFv clones determined by SPR.</p

Number of total and unique CDR sequences designed for the scFv library.

<p>Number of total and unique CDR sequences designed for the scFv library.</p