Targeting GSK-3β enzyme by diazepino-quinolone derivatives

Purpose: To synthesize a heterocyclic system containing quinolone and diazepine scaffolds as GSK-3β inhibitor. Methods: The diazepino-quinoline derivatives were synthesized starting from quinolone nucleus in a simple chemical reaction. The in vitro GSK-3β enzyme assay and MTT assay against cancer cell lines were carried out followed by Z´ı-LYTE GSK-3β assay. Anticancer activity was determined using U-87 glioma cell line. Results: Diazepino-quinoline derivatives were obtained in a good yield, and compound 102 exhibited significant activity against in vitro GSK-3β (IC 50 : 0.114 μM), and anticancer activity (IC 50 : 37 μM) against U-87 glioma cell line.


INTRODUCTION
One of the most important serine/threonine kinase enzymes is the GSK-3β enzyme that is active, pervasive, and vital for life [1]. The GSK-3β enzyme is a key signaling molecule that is involved in insulin and glycogen metabolism [2] and inflammatory responses. It plays a significant role in viral infections [3,4] and in cancer [5]. Its expression in cancer has been associated with survival, proliferation, and migration of cancer cells and their resistance to chemotherapy [6].
The GSK-3β enzyme inhibitors have gained a lot of interest as a potential treatment of neurodegenerative diseases such as Alzheimer´s disease [7], diabetes [2] and malignancies [8,9]. The enzyme inhibitors were previously synthesized from heterocyclic compounds having a quinoxaline nucleus that showed good inhibitory activity against GSK-3β enzyme [10].

EXPERIMENTAL Equipment and reagents
Melting point were measured by Stuart melting point apparatus, IR spectrum was recorded by Shimadzu 8400F FT-IR spectrophotometer. Bruker, Advance DPX-300 Nuclear magnetic resonance spectrometer was used to record (NMR) spectra. High-resolution mass spectrophotometer was used for mass instrument. Reagents and chemicals used are of synthetic grade and were bought from Sigma-Aldrich.

Synthon I derivatives
The synthetic procedure used in scheme I is based on reported methods [10,15]. By applying three steps of reaction, synthon I was obtained: The first step was done by reacting synthon a, and b with different 3-aminopropanoic acid derivatives under conditions shown in Figure 2, and the reaction was done under reflux at 80 o C then solution of sodium dithionite (8.0 g, 46 mmol) was added. Finally, a reflux condition was used and polyphosphoric acid was added (10 mL) to get synthon I. CO

GSK 3β in vitro assay
The Z´1-LYTE GSK-3β assay was used to make GSK-3β in vitro assay test (Z_-LYTETM Screening Protocol and Assay Conditions 2016); Z´ı-LYTE test is used to get the % Inhibition.
The compounds that have the highest percentage inhibition were tested for their IC50 values. Stock solutions of 10 mM concentration of the tested compounds (1, 4, and 102) in dimethyl sulfoxide (DMSO) was prepared. Then each was tested at 10 µM using the Select Screen Kinase Profiling Services (ThermoFisher Scientific, USA) [16].

MTT assay
The cytotoxicity of GSK-3β inhibitors was determined by MTT assay protocol based on reported procedure carried out by Al-Sha'er et al [17]. The U-87 glioma cell line was used for the MTT assay and was provided by Dr. Ahmad Sharab, of the American University of Madaba while the chemicals were purchased from Sigma Aldrich.

Molecular modeling studies
In order to support the in vitro assay results, a molecular docking procedure was carried out. The GSK-3β enzyme (PDB code: 3Q3B, resolution 2.7 Ǻ) binding pocket were determined and used in the docking procedure and the most active compound was docked using the Dock Ligands (LibDock).

Spectral characteristics
Based on the synthetic approach in Figure 2, the following compounds were obtained:

GSK-3β inhibitors activity
The synthon I derivatives were tested by using the in vitro human recombinant GSK-3β kinase (human recombinant) assay kit. The GSK-3β inhibitors was screened in this assay at 10 nM. Compounds that showed good percentage inhibition, namely, (1, 4, and 102) were screened at different concentrations to find their IC50 values. Compounds (1, 4, and 102) activities were measured against GSK 3 enzyme and the results are shown in Table 1. As presented in the Table; compound 1 showed 79 % inhibition while 4 showed 11 % inhibition and compound 102 showed 100 % inhibition the IC50 was calculated for compounds 1 and 102 and it was 4.18 and 0.114 μM, respectively. Figure 3 illustrates the concentration (nM)/percent inhibition plots of the most active compound 102. Next step was to determine if these compounds have any pharmacological activities as potential anticancer treatments. The U-87 glioma cell line was used in the MTT assay as it is known to express GSK-3β [18]. The most active (compound 102) and the least active (compound 4) compounds from the enzyme assay were chosen to assess their anti-proliferative activity. The MTT results are in accordance with the enzyme assay results with IC50 37 μM and > 100 μM for compounds 102 and 4, respectively ( Table 1). The results, confirm the importance of GSK-3β in cancer progression and open new avenues for the use of the synthon I derivatives GSK-3β inhibitors as anti-cancer drugs [18].

Docking results
The software assists in identifying the binding site in the receptor of GSK-3β that binds to the synthesized compound and determined the conformations of the active compound 102 by docking procedure that allowed the types of interactions between the binding pocket and the active compound. Figure 4 shows the most active compound (102) with different poses that might be acquired inside the binding pocket of (3Q3B).

DISCUSSION
Synthon I derivatives were obtained in a good yield and tested against GSK-3β enzyme with interesting inhibitory concentration as shown in Table 1. Compound 102 showed 100 % inhibition with IC50 (0.114 μM), while compound 1, had 79 % inhibition with IC50 (4.180 μM) while compound 4 had 11% inhibition so the IC50 was not measured and the IC50 can be explained by docking studies that revealed that there are many interactions for compound 1 GSK 3β inhibitor in the binding pocket of GSK-3β kinase (3Q3B), among these interactions, there is pi-pi stacking, pi-sigma, pi-alkyl, halogen, and Hbonding.
The results from this study, confirm the importance of GSK-3β in cancer progression and open new avenues for the use of the synthon I derivatives GSK-3β inhibitors as anti-cancer drugs [17]. The molecular docking studies also elucidated the binding modes of the compounds with the GSK-3β target CONCLUSION Synthesis of novel GSK-3β enzyme inhibitors has been carried out using simple chemical reactions. Diazepine derivatives could be a successful template for the design of inhibitors against GSK-3β enzyme with IC50 in a micromolar range. Compound 102 shows potent inhibitory activity against both GSK-3β enzyme and U-87 glioma cell line in MTT assay. With these findings, efforts are expanding for further optimization of diazepine derivatives in order to develop potent inhibitors with potential therapeutic activity against GSK-3β.