Methyl-Thiol-Bridged Oxadiazole and Triazole Heterocycles as Inhibitors of NF-κB in Chronic Myelogenous Leukemia Cells

Nuclear factor kappa beta (NF-κB) is a transcriptional factor that plays a crucial role in regulating cancer cell proliferation. Therefore, the inhibition of NF-κB activity by small molecules may be beneficial in cancer therapy. In this report, methyl-thiol-bridged oxadiazole and triazole heterocycles were synthesized via click chemistry and it was observed that the lead structure, 2-(((1-(3,4-dichlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)thio)-5-(4-methoxybenzyl)-1,3,4-oxadiazole (4c), reduced the viability of MCF-7 cells with an IC50 value of 7.4 µM. Compound 4c also caused concentration-dependent loss of cell viability in chronic myelogenous leukemia (CML) cells. Furthermore, compound 4c inhibited the activation of NF-κB in human CML cells as observed by nuclear translocation and DNA binding assays. Functionally, compound 4c produced PARP cleavage and also suppressed expression of Bcl-2/xl, MMP-9, COX-2, survivin, as well as VEGF, resulting in apoptosis of CML cells. Moreover, ChIP assay showed that compound 4c decreased the binding of COX-2 to the p65 gene promoter. Detailed in silico analysis also indicated that compound 4c targeted NF-κB in CML cells. In conclusion, a novel structure bearing both triazole and oxadiazole moieties has been identified that can target NF-κB in CML cells and may constitute a potential novel drug candidate.


Introduction
Chronic myelogenous leukemia (CML) primarily affects the blood and bone marrow [1]. According to the American Cancer Society, CML accounts for approximately 15% of all cases of leukemia in adults, and it is estimated that in the US, there is over 9000 new cases and more than 1000 deaths from the disease per year [2]. The most common drugs used to target CML cells are tyrosine kinase inhibitors such as imatinib, dasatinib, nilotinib, bosutinib, and ponatinib [3][4][5][6]. These drugs inhibit the BCR-ABL protein, which can also activate nuclear factor-kappa B (NF-κB) and promote cell proliferation, survival, and resistance to chemotherapy [7]. Inhibition of NF-κB has been reported to be a potential strategy

Materials and Methods
The various chemicals and solvents were obtained from Sigma-Aldrich, Bangalore, India. The completion of the reaction was monitored. 1 H and 13 C NMR were recorded on an Agilent (Santa Clara, CA, USA) NMR spectrophotometer (500 MHz), and CDCl3 was used as solvent.

Materials and Methods
The various chemicals and solvents were obtained from Sigma-Aldrich, Bangalore, India. The completion of the reaction was monitored. 1 H and 13 C NMR were recorded on an Agilent (Santa Clara, CA, USA) NMR spectrophotometer (500 MHz), and CDCl 3 was used as solvent.

Cell Lines and Culture Conditions
MCF-7 cells were purchased from Procell Life Science and Technology Co., Ltd., Wuhan, China, and cultured in MEM or Leibovitz's L-15 medium containing 2% FBS. MCF-7 cells were maintained in a 5% CO 2 -humidified atmosphere at 37 • C. KCL22, LAMA84, and K562 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). The KBM5 cell line was previously provided by Dr. Bharat Aggarwal (University of Texas M.D. Anderson Cancer Center, Houston, TX, USA). KBM5 cells were cultured in IMDM medium with 10% inactivated FBS and 1% penicillin-streptomycin. KCL22, LAMA84, and K562 cells were cultured in RPMI with 10% FBS and 1% penicillin-streptomycin antibiotics and maintained at 37 • C in a 5% CO 2 chamber.

Immunocytochemistry
CML cells (1 × 10 6 cells/well) were treated with 4c (0 or 30 µM) for 2 h, followed by TNFα (0.5 nM) addition for 10 min. Further, the CML cells were fixed, permeabilized, and blocked with 5% BSA-PBS. The cells were visualized by using primary anti-p65 antibodies and secondary antibodies called Alexa Fluor ® 594 donkey anti-rabbit IgG (H + L). Nuclei were stained with DAPI, and the immobilized cells were mounted with Fluorescent Mounting Medium (Golden Bridge International Labs, Mukilteo, WA, USA) and finally quantified using an Olympus FluoView FV1000 confocal microscope (Tokyo, Japan).

Western Blot Analysis
CML cells were treated with compound 4c, harvested, and the cell lysates were obtained. The required proteins of our interest were separated on SDS-PAGE and transferred into the nitrocellulose membrane. The membrane was incubated with primary antibody and HRP-conjugated secondary antibody in a sequential manner. The ECL kit (EZ-Western Lumi Femto, DOZEN) was used to detect the respective proteins.

Annexin V Assay
CML cells (1 × 10 6 cells/well) were incubated with compound 4c (0 or 30 µM) for 24 h and then treated with TNFα (0.5 nM) for 10 min. The washed cells were tagged with FITCbound annexin V antibodies and stained with PI for 15 min at room temperature. Finally, CML cells were analyzed by the BD Accuri™ C6 Plus flow cytometer (BD Biosciences, Becton-Dickinson, Franklin Lakes, NJ, USA) and analyzed with BD Accuri C6 Plus software.

Cell Cycle Analysis
CML cells (1 × 10 6 cells/well) were incubated with compound 4c (0 or 30 µM) for 24 h, then treated with TNFα (0.5 nM) for 10 min, washed, fixed, and finally incubated with 1 mg/mL RNase A for 1 h at 37 • C. After staining with PI, the CML cells were analyzed by the BD Accuri™ C6 Plus flow cytometer (BD Biosciences, Becton-Dickinson, Franklin Lakes, NJ, USA) and quantified using BD Accuri C6 Plus software.

In Silico DFT Calculations
Computational DFT studies for the molecules were performed using Gaussian09 [35]. The molecule was built and visualized by the Gaussview 5 program package. The molecular geometry of the molecules was optimized at the B3LYP level with a 6-311++G (d,p) basis set [36].

Molecular Docking and Dynamics Analysis
The molecular docking simulation was performed with Autodock 4.2 [37] to analyze the best binding pose and the binding affinity of 4c in the binding pocket of the P65 subunit of NF-κB. The crystal structure of the IκBalpha/NF-κB complex was obtained from the RCSB Protein Data Bank, having PDB ID: 1IKN (https://www.rcsb.org/) (accessed on 23 January 2023). Initially, to begin with, the receptor was prepared to dock by removing the heteroatoms and water molecules present in it. Followed by the addition of polar hydrogens and Kollman charges. The docking model was built with a grid box size of cell dimensions of 126 × 126 × 126 Å with 0.219 Å spacing. The empirical free-energy function and the Lamarckian genetic algorithm were used to perform molecular docking with the macromolecule. The initial population consisted of 150 randomly assigned individuals, a maximum of 2,500,000 energy evaluations, a mutation rate of 0.02, a crossover rate of 0.80, and 30 docking runs. The binding energy value of the ligand and the contact bonds with NF-κB were investigated. The docking interactions were visualized and studied using Maestro and PyMOL (v2.5.2) [38][39][40].
Desmond, the Schrödinger program [41], was used for MD calculations. The chosen ligand-protein combinations were first submerged in a TIP3P water box 10 Å beyond any of the complex's atoms. Counter ions (Na + and Cl − ions) were introduced to balance charges. The salt content was adjusted to 0.15 M sodium and chloride ions to simulate physiological conditions. The MD was carried out in the NPT ensemble over 50 ns at 300 K and 1.63 bar pressure, with recording periods of 1.2 ps for energy and 100 ps for trajectory. The OPLS-3e force field was used in the simulations.

Statistical Analysis
All the numerical values have been represented as the mean ± SD. Statistical significance of the data compared with the untreated control was determined using the Student's unpaired t-test. Significance was set at * p < 0.05, ** p < 0.01, and *** p < 0.001.
K and 1.63 bar pressure, with recording periods of 1.2 ps for energy and 100 ps for traje tory. The OPLS-3e force field was used in the simulations.

Statistical Analysis
All the numerical values have been represented as the mean ± SD. Statistical sign cance of the data compared with the untreated control was determined using the St dent's unpaired t-test. Significance was set at * p < 0.05, ** p < 0.01, and *** p < 0.001.

Efficacy of Title Compounds in Breast Cancer Cells
A number of studies have shown that the NF-κB pathway is crucial for the growth and progression of breast cancer, and thereby it serves as a potential target for breast cancer prevention and treatment [44]. We therefore screened all title compounds for loss of viability of MCF-7 cells. The results summarized in Table 1 show that the title compounds significantly inhibited the viability of MCF-7 cells. Compounds bearing 4-methoxyphenyl derivatives such as 4a, 4b, 4c, and 4e produced significant loss of viability in MCF-7 cells with IC 50 values of 9.58, 8.44, 7.40, and 16.86 µM, respectively. Furthermore, the most active compounds, such as 4a, 4b, 4c, 4e, and 4q, exhibited IC 50 values in MCF-10A cells greater than 100 µM, thus indicating a minimal effect on normal mammary epithelial cells (please refer to Supplementary Data S1). Compound 4c was observed to be the most effective among the tested title compounds. Among the newly synthesized oxadiazole and triazole compounds, compounds bearing a 4-methoxyphenyl group were observed to be more active than those bearing a 3,4-dimethoxyphenyl group. Furthermore, triazole compounds bearing halogen (4a, 4b, 4c/AG, and 4e) showed the lowest IC 50 values of 9 to 16 µM. Hence, it was assumed that 4-methoxy and halides substituted for oxadiazole and triazole, respectively, are the most active among methyl-thiol-bridged oxadiazole and triazole molecules.

4c Suppressed the Viability of CML Cells
Preclinical studies of IT-901, a novel and selective NF-κB inhibitor, revealed that it significantly reduced the tumor burden in a xenograft model that was implanted with chronic lymphocytic leukemia patient-derived cells [45]. Therefore, we tested the most active compound 4c against leukemia cell viability (KMB5, KCL22, LAMA84, and K562 cell lines) using a cell viability assay. Compound 4c was applied to cells in a dose-dependent manner (0, 5, 10, 15, 30, and 50 µM) for 24 h. Compound 4c was observed to produce loss of cell viability in CML cells (30 µM). However, cell viability in the normal PBMC cells was maintained at greater than 80% even at drug concentrations of 30 and 50 µM (Figure 2A). These results encouraged us to understand the mode of action of compound 4c.

Compound 4c Inhibited the TNFα Induced NF-κB Activation in CML Cells
TNFα, acting as a proinflammatory cytokine, can activate NF-κB, which leads to the transcriptional regulation of genes that are involved in cancer cell proliferation. An EMSA assay was used to detect the binding of NF-κB to its respective DNA binding sites. The inhibitory effects of 4c on TNFα-induced NF-κB activation in CML cells were therefore investigated. For this purpose, CML cells were incubated with 4c (0, 5, 15, 30 µM) for 2 h, followed by TNFα (0.5 nM) incubation for 10 min. After the reaction, nuclear extracts of CML cells were used to equally quantify NF-κB oligonucleotides that bound to DNA. It was observed that in all the tested CML cells, the binding of NF-κB to DNA was induced by TNFα, whereas the binding pattern was substantially inhibited by compound 4c ( Figure 2B).
Furthermore, immunocytochemistry (ICC) was used to determine the effect of 4c on the translocation of NF-κB-p65 in CML cells. CML cells were grown on coverslips and treated with compound 4c (0 or 30 µM) for 2 h, followed by TNFα (0.5 nM) for 10 min. Treatment with compound 4c inhibited translocation of NF-κB-p65 in CML cells compared with non-treated (NT) cells; NF-B-p65 antibodies stained the nucleus more strongly in cells treated with TNFα alone. Despite TNFα stimulation, 4c suppressed the translocation of NF-κB-p65 to nuclei ( Figure 2C). The results of this study revealed that compound 4c inhibited NF-κB activation in human CML cells.

Compound 4c Inhibited NF-κB Complex Formation in CML Cells
CML cells were stimulated by TNFα, which binds to the TNF receptor via several intermediate steps to engage the IκB kinase (IKK) complex, which then phosphorylates IκB, and ultimately causes IκB degradation. The heterodimer (consisting of p65 and p50 subunits) then translocate into the nucleus and bind to the target DNA sequences. Since NF-κB targeting drugs inhibit the activation of the NF-κB complex, a Western blot analysis was performed. Therefore, CML cells were pre-treated compound 4c (0 or 30 µM), followed by the incubation with TNFα (0.5 nM) at various time points. The cytosol and nuclear extracts of CML cells were prepared and the expression of protein levels was analyzed by Western blot. It was confirmed that compound 4c inhibited NF-κB complex-related subunits such as p-IKKα/β and p-IκBα, which were induced by TNFα in the cytoplasmic region of CML cells, when compared to 4c-untreated cells ( Figure 3A,B). Furthermore, p65 activation by TNFα was determined, wherein 4c suppressed phospho-p65 in nuclei ( Figure 3C). In summary, compound 4c targets NF-κB activation in CML cells by diverse mechanisms.

Compound 4c Inhibits Binding of COX-2 to p65 Promoter in CML Cells
It is important to note that the EMSA method only detects the binding of NF-κB to DNA and does not directly measure the downstream effects of NF-κB activation. Therefore, ChIP assays were performed to confirm the inhibition of the binding of COX-2 to the promoter region of p65, a subunit of the NF-κB transcription factor, in the presence and absence of 4c. For this study, CML cells were treated with compound 4c (0, 5, 15, 30 µM), and the respective chromatin was fragmented, followed by immunoprecipitation using an antibody specific to COX-2. It was demonstrated that compound 4c treatment markedly inhibited COX-2 binding to the p65 promoter compared to the untreated control sample. It is therefore concluded that compound 4c disrupted the interaction between COX-2 and the p65 promoter and exerted inhibitory effects on the expression of genes regulated by NF-κB ( Figure 4).

Compound 4c Inhibits Binding of COX-2 to p65 Promoter in CML Cells
It is important to note that the EMSA method only detects the binding of NF-κB to DNA and does not directly measure the downstream effects of NF-κB activation. Therefore, ChIP assays were performed to confirm the inhibition of the binding of COX-2 to the promoter region of p65, a subunit of the NF-κB transcription factor, in the presence and absence of 4c. For this study, CML cells were treated with compound 4c (0, 5, 15, 30 µM), and the respective chromatin was fragmented, followed by immunoprecipitation using an antibody specific to COX-2. It was demonstrated that compound 4c treatment markedly inhibited COX-2 binding to the p65 promoter compared to the untreated control sample. It is therefore concluded that compound 4c disrupted the interaction between COX-2 and the p65 promoter and exerted inhibitory effects on the expression of genes regulated by NF-κB ( Figure 4).

Frontier Molecular Orbitals (FMO) Calculations for the Title Compounds That Targets NF-κB
FMO analysis of compound 4c was performed using the B3LYP method with a 6-311 (2d, p) basis set. The HOMO of a molecule represents the site where the electron density is highest and is involved in electron donation, whereas the LUMO represents the site where the electron density is lowest and is involved in electron acceptance. The ELUMO-EHOMO energy gap is directly related to the molecule's chemical reactivity and kinetic stability. The molecule has a smaller energy gap, which is attributed to less stability and more reactivity. In contrast, the molecule has a large energy gap, attributed to being less chemically reactive and having high chemical stability with greater hardness. The computed HOMO-LUMO energy gaps of 4c and 4i were found to be 4.37 eV and 4.62 eV, respectively. Thus, the title compounds had high stability, were less chemically reactive, and exhibited poor conductivity. The HOMO-LUMO energy gap of alpha and beta electrons of the 4l was found to be 3.13 eV and 3.33 eV, respectively. It is observed that the HOMO-LUMO energy gap of 4l decreased by 1.24 eV and 1.04 eV in alpha and beta orbitals, respectively, when compared with the 4c molecule. The HOMO-LUMO plots of 4c, 4i, and also the alpha and beta molecular orbitals with the unpaired electrons of 4l are depicted in Figure 5, indicating that HOMO orbitals were localized on the methoxybenzyl group and the oxadiazole ring except for the oxygen atom and sulphur atom. In contrast, LUMO orbitals were localized on dichlorophenyl, triazole groups, and sulphur atoms of the lead molecule 4c. The FMO's energy and chemical reactivity descriptor values were calculated and are summarized in Table 2. The chemical potential values for 4c and 4i were estimated to be −4.23 eV and −3.91 eV, respectively, and reflects their significant electron-accepting

Frontier Molecular Orbitals (FMO) Calculations for the Title Compounds That Targets NF-κB
FMO analysis of compound 4c was performed using the B3LYP method with a 6-311 (2d, p) basis set. The HOMO of a molecule represents the site where the electron density is highest and is involved in electron donation, whereas the LUMO represents the site where the electron density is lowest and is involved in electron acceptance. The E LUMO -E HOMO energy gap is directly related to the molecule's chemical reactivity and kinetic stability. The molecule has a smaller energy gap, which is attributed to less stability and more reactivity. In contrast, the molecule has a large energy gap, attributed to being less chemically reactive and having high chemical stability with greater hardness. The computed HOMO-LUMO energy gaps of 4c and 4i were found to be 4.37 eV and 4.62 eV, respectively. Thus, the title compounds had high stability, were less chemically reactive, and exhibited poor conductivity. The HOMO-LUMO energy gap of alpha and beta electrons of the 4l was found to be 3.13 eV and 3.33 eV, respectively. It is observed that the HOMO-LUMO energy gap of 4l decreased by 1.24 eV and 1.04 eV in alpha and beta orbitals, respectively, when compared with the 4c molecule. The HOMO-LUMO plots of 4c, 4i, and also the alpha and beta molecular orbitals with the unpaired electrons of 4l are depicted in Figure 5, indicating that HOMO orbitals were localized on the methoxybenzyl group and the oxadiazole ring except for the oxygen atom and sulphur atom. In contrast, LUMO orbitals were localized on dichlorophenyl, triazole groups, and sulphur atoms of the lead molecule 4c. The FMO's energy and chemical reactivity descriptor values were calculated and are summarized in Table 2. The chemical potential values for 4c and 4i were estimated to be −4.23 eV and −3.91 eV, respectively, and reflects their significant electron-accepting capacity. A high chemical potential indicates a strong electron-accepting ability, whereas a low chemical potential indicates a strong donating ability [46]. 4c showed a high chemical hardness value (i.e., 2.18 eV −1 ) and a low value of chemical softness (0.22 eV −1 ). According to these findings, 4c exhibited a lower tendency to exchange its electron cloud with the surrounding environment. A similar result was observed with 4i, possessing chemical hardness and softness values of 2.312 eV and 0.216 eV −1 . The electrophilicity index quantifies the energy required to stabilize a molecule, and it was found to be 4.100 eV, and 3.307 eV for 4c and 4i, respectively.

Molecular Electrostatic Potential (MEP) Analysis of Title Compounds
Molecular electrostatic potential (MEP) analysis is used to determine the chemical reactivity of the molecule and predict the electrophilic and nucleophilic sites on the molecule and their intermolecular interactions. The MEP surfaces of 4c, 4i, and 4l were generated from the optimized structure using the B3LYP method with a 6-311++G (d, p) basis set ( Figure 6). The potential surface's colors ranged from −5.455 × 10 −2 to 5.455 × 10 −2 a.u, −6.258 × 10 −2 to 6.258 × 10 −2 a.u., and −5.380 × 10 −2 to 5.380 × 10 −2 a.u. for 4c, 4i, and 4l molecules, respectively. The electrostatic potential regions have been represented by different color codes: red represents the nucleophilic (i.e., region of negative electrostatic potential or electron rich), blue represents the electrophilic (i.e., region of positive electrostatic potential or electron deficient), and green denotes the region of zero potential. From Figure 6, the negative potential spots (−5.455 × 10 −2 a.u.) in the red color regions are located on the nitrogen atom of the oxadiazole group, representing the most reactive site for the electrophilic attack. In contrast, the positive potential (5.455 × 10 −2 a.u.) in the blue color regions localized at the hydrogen atom of the triazole group is the most reactive site for nucleophilic attack of the 4c molecule. Similar electrophilic and nucleophilic attacks were observed in 4i and 4l molecules ( Figure 6).

Molecular Electrostatic Potential (MEP) Analysis of Title Compounds
Molecular electrostatic potential (MEP) analysis is used to determine the chemical reactivity of the molecule and predict the electrophilic and nucleophilic sites on the molecule and their intermolecular interactions. The MEP surfaces of 4c, 4i, and 4l were generated from the optimized structure using the B3LYP method with a 6-311++G (d, p) basis set ( Figure 6). The potential surface's colors ranged from −5.455 × 10 −2 to 5.455 × 10 −2 a.u, -6.258 × 10 −2 to 6.258 × 10 −2 a.u., and −5.380 × 10 −2 to 5.380 × 10 −2 a.u. for 4c, 4i, and 4l molecules, respectively. The electrostatic potential regions have been represented by different color codes: red represents the nucleophilic (i.e., region of negative electrostatic potential or electron rich), blue represents the electrophilic (i.e., region of positive electrostatic potential or electron deficient), and green denotes the region of zero potential. From Figure  6, the negative potential spots (−5.455 × 10 −2 a.u.) in the red color regions are located on the nitrogen atom of the oxadiazole group, representing the most reactive site for the electrophilic attack. In contrast, the positive potential (5.455 × 10 −2 a.u.) in the blue color regions localized at the hydrogen atom of the triazole group is the most reactive site for nucleophilic attack of the 4c molecule. Similar electrophilic and nucleophilic attacks were observed in 4i and 4l molecules ( Figure 6).

Natural Bond Orbital (NBO) Analysis
Natural bond orbital analysis is an efficient method for evaluating intra-and intermolecular bonding and anti-bonding interactions in molecular systems, and it also provides a useful platform for researching charge transfer or conjugative interactions. The second-order Fock matrix was employed in the NBO analysis to examine the donor-acceptor interactions [47]. For each donor (i) and acceptor (j), the stabilization energy E(2) is related to the delocalization i→j . The bigger the E(2) value, the more intense the interaction between electron donors and electron acceptors, i.e., the greater the giving tendency of electron donors to electron acceptors and the stronger the overall system's conjugation. The possible intensive interactions are listed in Table 3. Electron density delocalization between occupied Lewis-type (bond or lone pair) orbitals and formally unoccupied non-Lewis orbitals corresponds to a stabilizing donor-acceptor interaction.

Natural Bond Orbital (NBO) Analysis
Natural bond orbital analysis is an efficient method for evaluating intra-and intermolecular bonding and anti-bonding interactions in molecular systems, and it also provides a useful platform for researching charge transfer or conjugative interactions. The secondorder Fock matrix was employed in the NBO analysis to examine the donor-acceptor interactions [47]. For each donor (i) and acceptor (j), the stabilization energy E(2) is related to the delocalization i→j. The bigger the E(2) value, the more intense the interaction between electron donors and electron acceptors, i.e., the greater the giving tendency of electron donors to electron acceptors and the stronger the overall system's conjugation. The possible intensive interactions are listed in Table 3. Electron density delocalization between occupied Lewis-type (bond or lone pair) orbitals and formally unoccupied non-Lewis orbitals corresponds to a stabilizing donor-acceptor interaction. Table 3. Second-order perturbation theory analysis of Fock matrix (F (i,j) b a.u) at 6-311++G (d, p) basis level corresponding to the intra-molecular interactions of the 4c molecule. a  F (i,j) (2) is the stabilization energy in KJ/mol. a Energy difference between donor orbital i and acceptor j orbital ∆E = E(i) − E(j) a.u. b F(i,j) is the Fock matrix element between i and j NBO orbitals in a.u. unit. * represents anti-bonding molecular orbital.

Molecular Docking and Dynamics Analysis of Compound 4c That Targets NF-κB
Molecular docking studies were performed to identify the specific regions of the p65 subunit of NF-κB that interacted with 4c by using reported methods [48]. Figure 7A illustrates the binding pose of the ligand molecule in the active site of the p65 subunit of NF-κB. Figure 7B displays a 2D interaction plot of the ligand in the binding pocket of p65. The docking resulted in a binding score of −9.13 kcal/mol. The oxygen atoms of the 1-methoxy-4-methylbenzene group and 1,3,4-oxadiazole groups in 4c have been linked to GLN29 and LYS28 amino acids via hydrogen bonding with a length of 2.03 Å and 2.06 Å, respectively. Further, the chlorine atom of 1,2-dichlorobenzene has formed halogen interactions with LYS56. The complex's stability is further enhanced by the hydrophobic interaction in the active site of the protein. Because of these interactions with amino acids, precise interactions with DNA are prohibited. This results in the synthesized 4c acting as a potent inhibitor for the transcription factor NF-κB.  Table 3, which shows the intra-molecular charge transfer of the 4c molecule corresponding to the stabilization of the molecule.

Molecular Docking and Dynamics Analysis of Compound 4c That Targets NF-κB
Molecular docking studies were performed to identify the specific regions of the p65 subunit of NF-κB that interacted with 4c by using reported methods [48]. Figure 7A illustrates the binding pose of the ligand molecule in the active site of the p65 subunit of NF-κB. Figure 7B displays a 2D interaction plot of the ligand in the binding pocket of p65. The docking resulted in a binding score of −9.13 kcal/mol. The oxygen atoms of the 1-methoxy-4-methylbenzene group and 1,3,4-oxadiazole groups in 4c have been linked to GLN29 and LYS28 amino acids via hydrogen bonding with a length of 2.03 Å and 2.06 Å, respectively. Further, the chlorine atom of 1,2-dichlorobenzene has formed halogen interactions with LYS56. The complex's stability is further enhanced by the hydrophobic interaction in the active site of the protein. Because of these interactions with amino acids, precise interactions with DNA are prohibited. This results in the synthesized 4c acting as a potent inhibitor for the transcription factor NF-κB. Molecular dynamics simulations were performed to determine the stability of the compound in the p65 subunit for 50 ns, along with other coordinates associated with the trajectory file. Figure 8A depicts the root-mean-square deviations (RMSDs) of 4c concerning its target and binding pocket (red colored) with respect to protein (blue color). It was observed that the values of ligand RMSD were lower than protein RMSD throughout the simulation time, especially after 10 ns, when the ligand in the active site is bound firmly and there are no fluctuations. Root mean square fluctuation (RMSF) observations (Figure Molecular dynamics simulations were performed to determine the stability of the compound in the p65 subunit for 50 ns, along with other coordinates associated with the trajectory file. Figure 8A depicts the root-mean-square deviations (RMSDs) of 4c concerning its target and binding pocket (red colored) with respect to protein (blue color). It was observed that the values of ligand RMSD were lower than protein RMSD throughout the simulation time, especially after 10 ns, when the ligand in the active site is bound firmly and there are no fluctuations. Root mean square fluctuation (RMSF) observations ( Figure 8B) depict the changes along protein chains. It was observed that the fluctuations of residues interacting with the ligand atoms were considerably reduced. Figure 8C explains the interaction contacts of proteins concerning the ligand. The hydrogen bond was formed with the residues LYS28, GLN29, ARG30, and SER276. Out of which, the formation of hydrogen bonds was present with LYS28 for about 20% of the simulation time, and later it was mediated by hydrophobic and water bridges. This resulted in a strong H-bond and the stability of the molecule within the pocket site. 8B) depict the changes along protein chains. It was observed that the fluctuations of residues interacting with the ligand atoms were considerably reduced. Figure 8C explains the interaction contacts of proteins concerning the ligand. The hydrogen bond was formed with the residues LYS28, GLN29, ARG30, and SER276. Out of which, the formation of hydrogen bonds was present with LYS28 for about 20% of the simulation time, and later it was mediated by hydrophobic and water bridges. This resulted in a strong H-bond and the stability of the molecule within the pocket site.

Compound 4c Induces Apoptosis in CML Cells
A number of drugs that target NF-κB activation decrease the expression of anti-apoptotic and oncogenic genes in CML cells. The decrease in the expression levels of survivin, MMP-9, Bcl-2/xl, VEGF, and COX-2 suggests that the NF-κB targeting drugs could modulate the survival and growth of CML cells by promoting apoptosis. Therefore, we investigated the effect of compound 4c on apoptosis by measuring the protein expression of various NF-κB regulated genes. For this purpose, CML cells were pretreated with 4c (0 or 30 µM), and incubated with TNFα (0.5 nM) for 0, 6, 12, and 24 h.. The results of the study revealed that TNFα induced the expression of various anti-apoptotic proteins in CML cells whereas compound 4c suppressed the expression of these proteins ( Figure 9A).

Compound 4c Induces Apoptosis in CML Cells
A number of drugs that target NF-κB activation decrease the expression of antiapoptotic and oncogenic genes in CML cells. The decrease in the expression levels of survivin, MMP-9, Bcl-2/xl, VEGF, and COX-2 suggests that the NF-κB targeting drugs could modulate the survival and growth of CML cells by promoting apoptosis. Therefore, we investigated the effect of compound 4c on apoptosis by measuring the protein expression of various NF-κB regulated genes. For this purpose, CML cells were pretreated with 4c (0 or 30 µM), and incubated with TNFα (0.5 nM) for 0, 6, 12, and 24 h.. The results of the study revealed that TNFα induced the expression of various anti-apoptotic proteins in CML cells whereas compound 4c suppressed the expression of these proteins ( Figure 9A). The cleavage of PARP (poly ADP-ribose polymerase) ultimately leads to apoptosis. Therefore, 4c-induced PARP cleavage was determined using Western blot analysis. For this purpose, CML cells were pretreated with compound 4c (0 or 30 µM) and incubated with TNFα (0.5 nM) for 0, 6, 12, and 24 h. Compound 4c enhanced the cleavage of PARP in CML cells which indicated that it increased apoptosis of these cells ( Figure 9B).

Compound 4c Induced Apoptotic-Cell Death in CML Cells
Next, live and dead assays were performed following treatment with compound 4c. CML cells were treated with compound 4c (0 or 30 µM) alone or with TNFα (0.5 nM) in The cleavage of PARP (poly ADP-ribose polymerase) ultimately leads to apoptosis. Therefore, 4c-induced PARP cleavage was determined using Western blot analysis. For this purpose, CML cells were pretreated with compound 4c (0 or 30 µM) and incubated with TNFα (0.5 nM) for 0, 6, 12, and 24 h. Compound 4c enhanced the cleavage of PARP in CML cells which indicated that it increased apoptosis of these cells ( Figure 9B).

Compound 4c Induced Apoptotic-Cell Death in CML Cells
Next, live and dead assays were performed following treatment with compound 4c. CML cells were treated with compound 4c (0 or 30 µM) alone or with TNFα (0.5 nM) in combination, and the fluorescent signal was observed, which showed an increased percentage of red signal in CML cells. Additionally, 4c and TNFα combined treatment displayed an enhanced effect on cell death, resulting in a much higher percentage of dead cells than 4c alone ( Figure 10A). combination, and the fluorescent signal was observed, which showed an increased percentage of red signal in CML cells. Additionally, 4c and TNFα combined treatment displayed an enhanced effect on cell death, resulting in a much higher percentage of dead cells than 4c alone ( Figure 10A). In another assay, CML cells were treated with 4c and TNFα and the apoptotic cells were determined by using Annexin V and FITC/PI in FACS. In the scatter plot, the upper right panel showed late apoptotic cells, and the lower right panel showed early apoptotic cells. In CML cells, treatment with 4c mainly increased late apoptotic cells more than early apoptotic cells, which was further augmented in the presence of TNFα ( Figure 10B).
Finally, an effect of 4c on cell cycle arrest was also observed. Cell cycle analysis showed that compound 4c arrested the cell cycle, and cells were accumulated in the sub G1 phase before the G0/G1 phase ( Figure 10C). In another assay, CML cells were treated with 4c and TNFα and the apoptotic cells were determined by using Annexin V and FITC/PI in FACS. In the scatter plot, the upper right panel showed late apoptotic cells, and the lower right panel showed early apoptotic cells. In CML cells, treatment with 4c mainly increased late apoptotic cells more than early apoptotic cells, which was further augmented in the presence of TNFα ( Figure 10B).
Finally, an effect of 4c on cell cycle arrest was also observed. Cell cycle analysis showed that compound 4c arrested the cell cycle, and cells were accumulated in the sub G1 phase before the G0/G1 phase ( Figure 10C).

Discussion
The representative characteristic of CML cells is the presence of the Philadelphia chromosome (Ph), which arises due to the reciprocal translocation of the (9;22) chromosome and produces BCR-ABL [49]. When CML enters the myeloid blast crisis phase, BCR-ABL promotes disease progression. A number of small molecule kinase inhibitors, which inhibit BCR-ABL, have been successful to control CML progression [50,51]. However, resistance to these inhibitors may develop in some patients [52]. The reports of the development of drug resistance indicate the necessity of developing further therapeutic strategies for CML patients. Therefore, the goal of this study was to investigate the possible effects of 4c on NF-κB in human CML cells.
First, we confirmed cell viability at selected cancer cell-specific toxic concentrations and evaluated the inhibitory effect of NF-κB activity in CML cells at selected concentrations. NF-κB is a complex of p50, p65, and IκBα, and remains under unstimulated conditions in the cytoplasm [53]. When upstream kinases such as IκB kinase (IKK) are activated and IκBα is ubiquitinated as well as degraded, NF-κB is released into the nucleus to initiate the transcription of various genes involved in tumorigenesis. Therefore, we investigated whether 4c could inhibit the oncogenic activity of CML cells by targeting NF-κB. 4c reduced the activity of NF-κB by inhibiting its binding to DNA and translocation into the nucleus. Moreover, it was confirmed that 4c modulated the activation of upstream kinases rather than simply reducing the activity of NF-κB. First, 4c inhibited expression of p-IKKα/β, and then also inhibited p-IκBα, which activates NF-κB by being degraded through phosphorylation. Thereafter, the protein expression levels of p65 and p-p65 were also found to be decreased by 4c.
Cancer cells often undergo apoptosis upon exposure to therapeutic agents by modulation of survival protein function [54]. Hence, herein the inhibitory effect of 4c on the expression of survivin, MMP-9, VEGF, Bcl-2/xl, and COX-2 was observed, with 4c reducing the expression of these proteins even in presence of TNFα. It was also confirmed that PARP cleavage was induced upon treatment with 4c. Also, 4c enhanced apoptosis of CML cells as evidenced in Annexin V assays. Compound 4c therefore promotes loss of cell viability by multiple downstream mechanisms ( Figure 11) and could potentially serve as a hit to develop a molecule abrogating the development of resistance in CML.

Discussion
The representative characteristic of CML cells is the presence of the Philadelphia chromosome (Ph), which arises due to the reciprocal translocation of the (9;22) chromosome and produces BCR-ABL [49]. When CML enters the myeloid blast crisis phase, BCR-ABL promotes disease progression. A number of small molecule kinase inhibitors, which inhibit BCR-ABL, have been successful to control CML progression [50,51]. However, resistance to these inhibitors may develop in some patients [52]. The reports of the development of drug resistance indicate the necessity of developing further therapeutic strategies for CML patients. Therefore, the goal of this study was to investigate the possible effects of 4c on NF-κB in human CML cells.
First, we confirmed cell viability at selected cancer cell-specific toxic concentrations and evaluated the inhibitory effect of NF-κB activity in CML cells at selected concentrations. NF-κB is a complex of p50, p65, and IκBα, and remains under unstimulated conditions in the cytoplasm [53]. When upstream kinases such as IκB kinase (IKK) are activated and IκBα is ubiquitinated as well as degraded, NF-κB is released into the nucleus to initiate the transcription of various genes involved in tumorigenesis. Therefore, we investigated whether 4c could inhibit the oncogenic activity of CML cells by targeting NF-κB. 4c reduced the activity of NF-κB by inhibiting its binding to DNA and translocation into the nucleus. Moreover, it was confirmed that 4c modulated the activation of upstream kinases rather than simply reducing the activity of NF-κB. First, 4c inhibited expression of p-IKKα/β, and then also inhibited p-IκBα, which activates NF-κB by being degraded through phosphorylation. Thereafter, the protein expression levels of p65 and p-p65 were also found to be decreased by 4c.
Cancer cells often undergo apoptosis upon exposure to therapeutic agents by modulation of survival protein function [54]. Hence, herein the inhibitory effect of 4c on the expression of survivin, MMP-9, VEGF, Bcl-2/xl, and COX-2 was observed, with 4c reducing the expression of these proteins even in presence of TNFα. It was also confirmed that PARP cleavage was induced upon treatment with 4c. Also, 4c enhanced apoptosis of CML cells as evidenced in Annexin V assays. Compound 4c therefore promotes loss of cell viability by multiple downstream mechanisms ( Figure 11) and could potentially serve as a hit to develop a molecule abrogating the development of resistance in CML. Figure 11. A schematic diagram depicting the possible mechanism through which compound 4c targets TNFα-induced NF-κB inactivation in CML cells. Figure 11. A schematic diagram depicting the possible mechanism through which compound 4c targets TNFα-induced NF-κB inactivation in CML cells.

Conclusions
In conclusion, this study demonstrated that compound 4c can effectively inhibit NF-κB activation while inducing apoptosis in CML cells. Compound 4c not only regulates NF-κB but also upstream kinases, and regulation of these molecular mechanisms by 4c may contribute to the development of novel therapies for CML.
Author Contributions: Y.Y.J., A.R., Z.X., A.S. and B.B. conceptualization, methodology, formal analysis, and writing; M.M., V.P., P.E.L. and G.S. methodology and data curation; K.S.A. and B.B. conceptualization, methodology, software, data curation, original draft, validation, writing, and editing. All authors have read and agreed to the published version of the manuscript.