Applying ligands profiling using multiple extended electron distribution based field templates and feature trees similarity searching in the discovery of new generation of urea-based antineoplastic kinase inhibitors

Abstract

This study provides a comprehensive computational procedure for the discovery of novel urea-based antineoplastic kinase inhibitors while focusing on diversification of both chemotype and selectivity pattern. It presents a systematic structural analysis of the different binding motifs of urea-based kinase inhibitors and the corresponding configurations of the kinase enzymes. The computational model depends on simultaneous application of two protocols. The first protocol applies multiple consecutive validated virtual screening filters including SMARTS, support vector-machine model (ROC = 0.98), Bayesian model (ROC = 0.86) and structure-based pharmacophore filters based on urea-based kinase inhibitors complexes retrieved from literature. This is followed by hits profiling against different extended electron distribution (XED) based field templates representing different kinase targets. The second protocol enables cancericidal activity verification by using the algorithm of feature trees (Ftrees) similarity searching against NCI database. Being a proof-of-concept study, this combined procedure was experimentally validated by its utilization in developing a novel series of urea-based derivatives of strong anticancer activity. This new series is based on 3-benzylbenzo[d]thiazol-2(3H)-one scaffold which has interesting chemical feasibility and wide diversification capability. Antineoplastic activity of this series was assayed in vitro against NCI 60 tumor-cell lines showing very strong inhibition of GI(50) as low as 0.9 uM. Additionally, its mechanism was unleashed using KINEX™ protein kinase microarray-based small molecule inhibitor profiling platform and cell cycle analysis showing a peculiar selectivity pattern against Zap70, c-src, Mink1, csk and MeKK2 kinases. Interestingly, it showed activity on syk kinase confirming the recent studies finding of the high activity of diphenyl urea containing compounds against this kinase. Allover, the new series, which is based on a new kinase scaffold with interesting chemical diversification capabilities, showed that it exhibits its "emergent" properties by perturbing multiple unexplored kinase pathways.

Figure 1. Classification of urea-based antineoplastic kinase inhibitors according to the general chemical structure and highlighting the general mechanism.

Figure 2. General workflow of the study which includes the computational procedure of ligand profiling using multiple field templates, the protocol of cancericidal verification using features similarity method, the in vitro cytotoxicity assays and finally the mechanistic study using high-throughput kinase profiling and cell cycle analysis.

Figure 3. Classification of urea derivatives kinases complexes deposited in literature according to their families, subfamilies and groups and listing the PDB codes of each group.

Figure 4. Human cyclin dependent kinase 2 complexed with urea-based cdk4 kinase inhibitor (1GII):

(A)The complex illustrated using the color codes that represent the different regions of the binding site: G-loop, Hyd1, alphaC, Hinge, HRD and DFG regions. The urea fragment binds to the Hinge region, (B) The corresponding field template derived from the complex. Color codes of the field template are listed in the supplementary data (Text S2).

Figure 5. P38 MAP Kinase in Complex with urea-based inhibitor (1KV1):

(A) The color codes represent the different regions of the binding site: G-loop, Hyd1, alphaC, Hinge, HRD and DFG regions. The urea fragment binds to the DFG and alphaC regions. (B) The corresponding field template derived from the complex. Color codes of the field template are listed in the supplementary data (Text S2).

Figure 6. Pipeline pilot workflow used to carry out the SVM model using R statistics package.

(A) Shows the usage of R-statistics node in pipeline pilot and its usage in learning the training set, after splitting, followed by giving the cross-validated ROC score via R plot viewer. (B) Shows the usage of the test set to validate the model using enrichment plot and R plot viewer.

Figure 7. Structure-based pharmacophore construction general outline.

The interactions are translated into pharmacophore features while the binding site amino acids are translated into excluded volumes. The shape of the binding site is also added to increase sensitivity of the pharmacophore.

Figure 8. Heat map of twelve urea-based derivatives against a panel of 90 field templates representing urea –based kinases inhibitors complexed with their corresponding kinase enzymes as retrieved previously.

The color codes used here is the red-yellow-green scale as indicative for decreasing similarity.

Figure 9. Structures of the hits mentioned in figure 8 as an example of the screening protocol results.

Figure 10. Scaffold morphing observed in the hit was used as one of the selection criteria.

The diversification is achieved by having different attachment points and thus different geometrical diversity in the virtual space of its substituents.

Figure 11. Synthesis of intermediates 7a–b.

Reagents and conditions: (a) Br₂, NH₄SCN, AcOH, below 10°C, 3 h; (b) NaNO₂, H₂SO₄, below 10°C for 15 min, then at r.t. for 1 h ; (c) H₂SO₄, NaNO₃, 0°C, 1 h; (d) KMnO₄ (10%), NaOH (25%), 80–90°C, 30 min; (e) Acetone, KOH (85%), H₂O, ArCH₂Cl, Reflux, 24 h; (f) H₂/10%Pd/C, EtOH/THF (3∶1), 35 psi, r.t., 6 h.

Figure 12. Synthesis of intermediates 11a–h.

Reagents and conditions: (a) SOCl₂, reflux, 5 h; (b) NaN₃, acetone, −10°C, 30 min; (c) Benzene, 70°C, 3 hrs.

Figure 13. Synthesis of target compounds (12a–l).

Reagents and conditions: (a) CH₂Cl₂, r.t, overnight.

Figure 14. Synthesis of target compound (13).

Reagents and conditions: (a) CH₂Cl₂, r.t, overnight.

Figure 15. Two compounds showing feature trees similarity of 0.876 to the compound 12b were also retrieved in compare analysis biological pattern similarity with correlation above 0.6.

Figure 16. Kinome map of the compound 12a inhibition % is scaled using color coding as follow: 20%–40% black circles, 40%–70% orange circles and >70% in red circles.

The radius of the circle corresponds to the inhibition % within this range.

Figure 17. Similarity of urea-based derivative 12a with a Syk kinase inhibitor using field alignment method.

The boxes highlight regions of high field similarity. Both of the inhibitors are having thiazole moiety if we considered structural similarity.