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. 2020 Aug 6;5(32):20170-20186.
doi: 10.1021/acsomega.0c01829. eCollection 2020 Aug 18.

Design, Synthesis, and Biological Evaluation of Novel 7 H-[1,2,4]Triazolo[3,4- b][1,3,4]thiadiazine Inhibitors as Antitumor Agents

Muhammad I Ismail  1 , Samy Mohamady  1 , Nermin Samir  2 , Khaled A M Abouzid  2   3
Affiliations

Design, Synthesis, and Biological Evaluation of Novel 7 H-[1,2,4]Triazolo[3,4- b][1,3,4]thiadiazine Inhibitors as Antitumor Agents

Muhammad I Ismail et al. ACS Omega. .

Abstract

A series of novel anticancer hydrazinotriazolothiadiazine-based derivatives were designed based on the structure-activity relationship of the previously reported anticancer triazolothiadiazines. These derivatives were synthesized and biologically screened against full NCI-60 cancer cell lines revealing compound 5l with a potential antiproliferative effect. 5l was screened over 16 kinases to study its cytotoxic mechanism which showed to inhibit glycogen synthase kinase-3 β (GSK-3β) with IC50 equal to 0.883 μM and 14-fold selectivity over CDK2. Also, 5l increased active caspase-3 levels, induced cell cycle arrest at the G2-M phase, and increased the percentage of Annexin V-fluorescein isothiocyanate-positive apoptotic cells in PC-3 prostate cancer-treated cells. Molecular docking and dynamics were performed to predict the binding mode of 5l in the GSK-3β ATP binding site. 5l can be utilized as a starting scaffold for developing potential GSK-3β inhibitors.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
(A) Representative GSK-3β inhibitors under clinical trials, (B) GSK-3 inhibitors having 5–6-fused ring system (I–III) including our discovered triazolothiadiazine (5l).
Figure 2
Figure 2
General SAR for triazolothiadiazine analogues possessing anticancer/antitumor activity.
Scheme 1
Scheme 1
Reagents and conditions: (a) HCl, EtOH, H2O, reflux 1 h, 70–80%; (b) EtOH, reflux 2 h, 85–92%.
Figure 3
Figure 3
Heat map depicting the growth inhibition percentage of the compounds 5a–t (y-axis) at concentration of 10 μM against the NCI-60 cell lines (x-axis).
Figure 4
Figure 4
Percent inhibition of compound (5l) against 22 cell lines from the NCI-60 cell lines that exhibit the percent inhibition range from 50 to 125% (red: >90%, blue: 80–90%, green: 70–80%, black: <70%).
Figure 5
Figure 5
Computational LBTF workflow.
Figure 6
Figure 6
Results of kinase screening of compound 5l on the 16 kinases represented in kinase percent inhibition at a concentration of 10 μM. The experiments were conducted in duplicates.
Figure 7
Figure 7
Caspase-3 (active) levels measured in both 5l-treated and untreated cells. Values are presented as means ± S.D. from three independent experiments performed in triplicates. *p < 0.05 significant from control untreated PC-3 cells using the Student’s t-test.
Figure 8
Figure 8
PC-3 cell cycle analysis results. (a) Histograms representing the cell cycle phases in 5l-treated cells compared to control untreated cells. (b) Annexin V-FITC and PI-double stained untreated vs 5l-treated PC-3 cells analyzed with flowcytometry. Percentages of living (bottom left), early apoptotic (bottom right), necrotic (top left), and late apoptotic (top right) cells are shown. (c) Stacked histogram representing cells percentages of each cell cycle phase in 5l-treated PC-3 cells compared to control.
Figure 9
Figure 9
Docking validation results. HYBRID-predicted docked pose of AR-A014418 (blue) is superimposed with the co-crystallized AR-A014418 (green) with rmsd = 0.599 Å, while the docked pose predicted by FRED (yellow) shows a great deviation with rmsd = 8.357 Å. Hydrogen bonds of co-crystallized AR-A014418 (green dashed lines) with Val135 and Pro136 are conserved by HYBRID docked pose (cyan dashed lines), while the FRED docked pose shows no hydrogen bonds with the protein amino acids. Valine 135 and proline 136 amino acids of the hinge region are depicted in coral, while the protein ribbon in gray colors.
Figure 10
Figure 10
Left: Reported binding mode of a pyrazolopyridazine GSK-3 inhibitor (gray) with three hydrogen bonds with Asp133 and Val135 (green dashed lines). Right: Binding mode of 5l predicted by FRED (5l_FRED, orange), showing one hydrogen bond formed with Val135 (orange dashed line), and HYBRID (5l_HYBRID, green), showing two hydrogen bonds formed with Val135 (green dashed lines).
Figure 11
Figure 11
Top: Protein rmsd of the three MD runs showing stability after 1 ns and minimal fluctuation throughout the simulation time. Middle: Per residue RMSF of the three MD runs showing RMSF < 1 Å for the binding site residues (colored rectangles) with low RMSF in the hinge region residues (red rectangle). Bottom: Protein radius of gyration (Rg) of the three MD runs showing compactness of the protein throughout the simulation time with no major conformational changes. AR-A014418, 5l_HYBRID, and 5l_FRED proteins are depicted in green, blue, and yellow colors, respectively.
Figure 12
Figure 12
rmsd of the ligand during the three MD runs: AR-A014418 (green), 5l_HYBRID docked pose (blue), and 5l_FRED docked pose (yellow). The 5l_FRED docked pose shows the highest deviation which indicates the instability of this binding pose.
Figure 13
Figure 13
Number of hydrogen bonds formed during the simulation time between the ligands and the protein (left), and the ligands and Valine 135 (right).
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