An Integrated Bioinformatics Study of a Novel Niclosamide Derivative, NSC765689, a Potential GSK3β/ β-Catenin/ STAT3/ CD44 Suppressor with Anti-Glioblastoma Properties

Abstract

Despite management efforts with standard surgery, radiation, and chemotherapy, glioblastoma multiform (GBM) remains resistant to treatment, which leads to tumor recurrence due to glioma stem cells (GSCs) and therapy resistance. In this study, we used random computer-based prediction and target identification to assess activities of our newly synthesized niclosamide-derived compound, NSC765689, to target GBM oncogenic signaling. Using target prediction analyses, we identified glycogen synthase kinase 3β (GSK3β), β-Catenin, signal transducer and activator of transcription 3 (STAT3), and cluster of differentiation 44 (CD44) as potential druggable candidates of NSC765689. The above-mentioned signaling pathways were also predicted to be overexpressed in GBM tumor samples compared to adjacent normal samples. In addition, using bioinformatics tools, we also identified microRNA (miR)-135b as one of the most suppressed microRNAs in GBM samples, which was reported to be upregulated through inhibition of GSK3β, and subsequently suppresses GBM tumorigenic properties and stemness. We further performed in silico molecular docking of NSC765689 with GBM oncogenes; GSK3β, β-Catenin, and STAT3, and the stem cell marker, CD44, to predict protein-ligand interactions. The results indicated that NSC765689 exhibited stronger binding affinities compared to its predecessor, LCC09, which was recently published by our laboratory, and was proven to inhibit GBM stemness and resistance. Moreover, we used available US National Cancer Institute (NCI) 60 human tumor cell lines to screen in vitro anticancer effects, including the anti-proliferative and cytotoxic activities of NSC765689 against GBM cells, and 50% cell growth inhibition (GI₅₀) values ranged 0.23~5.13 μM. In summary, using computer-based predictions and target identification revealed that NSC765689 may be a potential pharmacological lead compound which can regulate GBM oncogene (GSK3β/β-Catenin/STAT3/CD44) signaling and upregulate the miR-135b tumor suppressor. Therefore, further in vitro and in vivo investigations will be performed to validate the efficacy of NSC765689 as a novel potential GBM therapeutic.

Keywords: drug resistance; glioblastoma multiforme (GBM); glioma stem cell (GSC); in silico molecular docking; miR-135b; stemness; tumor microenvironment (TME).

Figure 1

NSC765689 successfully met the required drug-likeness criteria. (A) Flow diagram of LCC09 and NSC765689 chemical and structural synthesis with reference to previous studies. (B) Bioavailability radar (BA), displaying the six physicochemical properties of absorption of the NSC765689 compound. The pink area represents the optimal range for each property. The accompanying table displays physicochemical properties of lipophilicity of XLOGP3 between −0.7 and 5.0; molecular weight of ≤500 g/mol; polarity of TPSA ≤ 140 Å (angstrom); flexibility of ≤9 rotatable bonds; and solubility of log S between 0 and 6, and saturation sp3 hybridization of ≤0.25.

Figure 2

Glycogen synthase kinase 3β (GSK3B)/β-Catenin/signal transduction and activator of transcription 3 (STAT3)/cluster of differentiation 44 (CD44) mRNA levels were highly expressed in glioblastoma multiforme (GBM) tumor cohorts compared to normal samples with significant p values (A). When all four genes were combined for analysis, there were positive correlations of STAT3 with GSK3β, CTNNB1 (β-Catenin) with GSK3β, STAT3 with CD44, and CTNNB1 with CD44 in GBM patients (r = 0.28~0.58) (B). The statistical significance of differentially expressed genes was evaluated using the Wilcoxon test. * p < 0.05.

Figure 3

Prediction of shorter survival times using glycogen synthase kinase 3β (GSK3β)/β-Catenin/signal transduction and activator of transcription 3 (STAT3)/cluster of differentiation 44 (CD44) signatures. Elevated mRNA levels of GSK3β/β-Catenin/STAT3/CD44 were found to be associated with shorter survival times in patients with glioblastoma multiforme (GBM) (GBM Metabase and SurvExpress). p values are indicated in each panel.

Figure 4

STRING database predicted protein-protein interacting networks (PINs) among the oncogenic markers of glycogen synthase kinase 3β (GSK3B)/β-Catenin/signal transduction and activator of transcription 3 (STAT3)/cluster of differentiation 44 (CD44). (A) The network has four initial proteins formed and was further expanded by an additional 30 nodes/protein, by using the “more” key on the STRING interface. Furthermore, a PIN enrichment p value of <1.0 × 10⁻¹⁶ was obtained from the network, with a clustering coefficient of 0.711. The accompanying table shows all other interacting proteins with GSK3β/β-Catenin/STAT3/CD44, and the confidence cutoff value representing interaction links was adjusted to 0.900 as the highest scoring link. (B) Biological processes and (C) KEGG pathways associated with of GSK3β/β-Catenin/STAT3/CD44 clustering networks.

Figure 5

Prediction of expression levels and long-term survival using miRNA-135b-5p and glycogen synthase kinase 3β (GSK3β) in glioblastoma multiforme (GBM). (A) The miRDB prediction tool revealed low expression of miR-135b in tumor cohorts compared to adjacent normal tissue samples with p < 0.05. (B) miRNA-135b-5p expression was low in GBM patients (miRondala-DB). (C) Using targetscan online software, we identified the miR-135 -5p site on the 3′-untranslated region of GSK3β mRNA, which predicted GSK3β as a target gene of miR-135b-5P.

Figure 6

NSC765689 could serve as a potential small-molecule inhibitor of multi-oncogenic proteins. (A) 3D structures of glycogen synthase kinase 3β (GSK3β)/β-Catenin/signal transduction and activator of transcription 3 (STAT3)/cluster of differentiation 44 (CD44)-NSC765689 interactions (left). (B) Surface representations of the active-site flap of the GSK3β/β-Catenin/STAT3/CD44-NSC765689 complex in the binding pocket (middle). (C) 2D structural analysis of amino acids and distances of GSK3β/β-Catenin/STAT3/CD44-NSC765689 complexes using Discovery Studio (right). Accompanying Table shows binding affinities, ligand-receptor interactions, atoms involved in H-bonding, bond distances between NSC765689 and target proteins, and interacting amino acid residues.

Figure 7

Docking results of LCC09 with glycogen synthase kinase 3β (GSK3β)/β-Catenin/signal transduction and activator of transcription 3 (STAT3)/cluster of differentiation 44 (CD44). (A) 3D structures of GSK3β/β-Catenin/STAT3/CD44-LCC09 interactions (left). (B) Surface representations of the active-site flap of the GSK3β/β-Catenin/STAT3/CD44-LCC09 complex in the binding pocket (middle). (C) 2D structural analysis of amino acids and distances of the GSK3β/β-Catenin/STAT3/CD44-LCC09 complex using Discovery Studio (right). Accompanying table shows binding affinities, ligand-receptor interactions, atoms involved in H-bond distances between LCC09 and target proteins, and the interacting amino acid residues.

Figure 8

Docking results of AZD1080 with glycogen synthase kinase 3β (GSK3β)/β-Catenin/signal transduction and activator of transcription 3 (STAT3). (A) 3D structures of GSK3β/β-Catenin/STAT3 -AZD1080 interactions (left). (B) Surface representations of the active-site flap of the GSK3β/β-Catenin/STAT3 -AZD1080 complex in the binding pocket (middle). (C) 2D structural analysis of amino acid and distances of the GSK3β/β-Catenin/STAT3 -AZD1080 complex using Discovery Studio (right). Accompanying table shows binding affinities, ligand-receptor interactions, atoms involved in H-bonding, bond distances between AZD1080 and target proteins, and interacting amino acid residues.

Figure 9

Sensitivity of the NCI-60 cancer cell lines to inhibitory activities of NSC765689. An initial single dose of 10 μM was used on each cell line to test the compound. The zero on the x-axis indicates the mean percentage of cell growth of the cell lines. The percentage growth of each cell line relative to the mean is represented by horizontal bars, which are elongated to the right side indicating greater sensitivity and to the left side indicating reduced sensitivity.

Figure 10

Dose-response curves of the cytotoxic activity of NSC765689 against central nervous system (CNS) cancer cell lines. Growth percentage value of 100 represents the growth of untreated cells, while a value of 0 represents no net growth throughout the period of the experiment, and a value of -100 indicates that all of the cells had been killed by the end of the experiment.