Multiomics Study of a Novel Naturally Derived Small Molecule, NSC772864, as a Potential Inhibitor of Proto-Oncogenes Regulating Cell Cycle Progression in Colorectal Cancer

Abstract

Colorectal cancer (CRC) is one of the most prevalent malignant tumors, and it contributes to high numbers of deaths globally. Although advances in understanding CRC molecular mechanisms have shed significant light on its pathogenicity, current treatment options, including combined chemotherapy and molecular-targeted agents, are still limited due to resistance, with almost 25% of patients developing distant metastasis. Therefore, identifying novel biomarkers for early diagnosis is crucial, as they will also influence strategies for new targeted therapies. The proto-oncogene, c-Met, a tyrosine kinase that promotes cell proliferation, motility, and invasion; c-MYC, a transcription factor associated with the modulation of the cell cycle, proliferation, apoptosis; and cyclin D1 (CCND1), an essential regulatory protein in the cell cycle, all play crucial roles in cancer progression. In the present study, we explored computational simulations through bioinformatics analysis and identified the overexpression of c-Met/GSK3β/MYC/CCND1 oncogenic signatures that were associated with cancer progression, drug resistance, metastasis, and poor clinical outcomes in CRC. We further demonstrated the anticancer activities of our newly synthesized quinoline-derived compound, NSC772864, against panels of the National Cancer Institute's human CRC cell lines. The compound exhibited cytotoxic activities against various CRC cell lines. Using target prediction tools, we found that c-Met/GSK3β/MYC/CCND1 were target genes for the NSC772864 compound. Subsequently, we performed in silico molecular docking to investigate protein-ligand interactions and discovered that NSC772864 exhibited higher binding affinities with these oncogenes compared to FDA-approved drugs. These findings strongly suggest that NSC772864 is a novel and potential antiCRC agent.

Keywords: colorectal cancer; drug resistance; molecular docking simulation; protein–ligand interaction; small molecule.

Figure 1

Schematic diagram representing the study design.

Figure 2

Overexpression of c-Met/GSK3β/MYC/CCND1 promotes colon cancer progression. (A,B) Volcano plots showing top overexpressed DEGs in COAD tissues compared to normal tissues were retrieved from two NCBI GEO datasets, GSE4107 and GSE41328. (C) Venn diagram showing the overlapping upregulated genes from the two datasets. (D) Heatmaps showing the top differentially expressed genes in colon adenocarcinoma (COAD). (E–H) c-Met, GSK3β, MYC, and CCND1 were highly expressed in colon cancer tissues compared to adjacent normal tissues. (I–L) c-Met/GSK3β/MYC/CCND1 were overexpressed in colon cancer from stages 1~4 compared to normal tissues, with p < 0.05. considered statistically significant.

Figure 3

Protein–protein interaction (PPI) network revealed interactions among the c-Met/GSK3β/MYC/CCND1 oncogenes in colorectal cancer (CRC). (A) The average local clustering coefficient was 0.920 with interaction enrichment of p = 0.0135. Interactions were based on coexpressions, gene fusion, and co-occurrences. (B) The top 10 biological processes (BPs), (C) KEGG pathways, with p < 0.05 were considered significant.

Figure 4

Network topology profiling of miRNAs that targets c-Met, GSK3β, MYC, and CCND1 oncogenes specifically, with * p < 0.05 considered statistically significant.

Figure 5

Crosstalk between overexpression of Met/GSK3β/MYC/CCND1 oncogenes and upregulated miR-26a is associated with CRC cancer progression. (A) expressed miRNA interacting with Met/GSK3β/MYC/CCND1 within the same cluster. (B) miRNA-26a is significantly downregulated in CRC samples compared to normal samples. (C–E) miR-26a-1-3p site in the 3′ untranslated region (UTR) of GSK3β, MYC, and CCND1 mRNA, thus predicting a link between miR-26a and Met/GSK3β/MYC/CCND1 in cancers. (F–G) Enriched GO, including biological processes (BPs), and affected KEGG pathways, with p < 0.05 considered statistically significant.

Figure 6

Genomic alterations in c-Met/MYC/cyclin D1 (CCND1) signatures are associated with poor prognoses of colorectal cancer (CRC) cohorts. (A) Mutation of the c-Met gene was associated with high expression levels of MSANTD3. (B–D) Changes in expression levels of the c-Met/GSK3β/MYC/CCND1 oncogenes were linked to mutations of the BRAF, NOTCH2, and ODF2 genes at the target level, compared to the wild-type (WT) group. (E) Bar graph showing higher alteration frequencies of c-Met/GSK3β/MYC/CCND1 gene signatures in CRC, compared to colorectal adenoma (COAD) and rectal adenocarcinoma (READ). (F) Bar graph showing alterations of the c-Met/GSK3β/MYC/CCND1 genes according to sample subtypes. (G) Volcano plot showing unaltered and altered genes associated with c-Met/GSK3β/MYC/CCND1 signatures with −log10(p values) set as a standard. (H–J) A lollipop diagram showing the distribution of c-Met/GSK3β/MYC/CCND1 mutations in CRC across protein domains. Mutations are color-coded as missense, truncating, and in-frame mutations.

Figure 7

Rational scaffold-hopping protocol for the design of NSC772684.

Figure 8

Identified potential targets for the NSC772864 small molecule. (A–C) Pharmacophore-based models of c-Met/cMYC/CCND1 identified as NSC772864 target genes through protein–receptor interactions.

Figure 9

Drug sensitivity of the c-Met/GSK3β/MYC/CCND1 oncogenes from the GSCA. Correlations among genomics of drug sensitivity in cancer (GDSC) of FDA-approved drugs. Positive Spearman correlation coefficients (orange circles) indicate that increased gene expression levels were resistant to the drug, compared to negative correlations shown in blue, which indicate sensitivity to the drug.

Figure 10

NSC772864 passed the required physicochemical properties, medicinal chemistry, pharmacokinetics (PKs), and drug-likeness criteria. (A) Structure of the NSC772864 small molecule, bioavailability (BA) radar, displaying the six physicochemical properties of absorption including lipophilicity (XLOGP3 = 5.04), molecular weight (379.88 g/mol), polarity (PSA = 45.23 Ų), solubility (Log S (ESOL) = −5.41), flexibility (rotation = 6), saturation (fraction Csp3 = 0.27), and pKa of the most basic or acidic group (=0.6) of the NSC772864 compound. In addition, the NSC772864 compound demonstrated a highly probable gastrointestinal absorption (GIA), BA score (55%), and good synthetic accessibility (3.26). (B) The compound passed the blood–brain barrier (BBB) assessment with a score of 5.98 and further displayed a drug-like model score of −0.48. A structural characterization of the compound was performed with the help of spectroscopic studies including IR, proton NMR, 13C NMR, MS, and an elemental analysis (C). Pie chart showing targetable protein candidates for NSC772864 (Table 2). Results are shown of the physiochemical properties, pharmacokinetics, drug-likeness, and medical chemistry.

Figure 11

NSC772864 demonstrated anticancer effects in NC-I60 human colon cancer cell lines. (A–I) Single-dose treatment administered at 10 µM revealed growth percentage antiproliferative effects against COLO205, HCC-2998, HCT-116, HCT-15, KM12, and SW620 cells and cytotoxic effects against the HT29 cell line. (J) Dose-dependent responses of colon cancer cell lines evaluated using the 50% growth inhibition (GI50) and tumor growth inhibition (TGI). (K) In vitro IG50 results ranged from 0.16 to 2.85 μM in colon cancer cell lines, with HCT-15 being the most responsive at 0.16 μM, followed by HCT116 at 0.38 μM, SW-620 at 0.39 μM, HT-29 at 0.57 μM, KW12 at 0.94 μM, and COLO 205 at 2.15 μM, with HCC-2998 at 2.85 μM showing the least responsiveness compared to the aforementioned cell lines.

Figure 12

Docking profiles of c-Met with NSC772864 and standard inhibitors. (A,B) NSC772864 bound with c-Met with Gibbs’ free energy (ΔG) of −8.0 kcal/mol. (C,D) Binding of crizotinib with c-Met showed a slightly higher affinity of ΔG = −8.1 kcal/mol compared to NSC772864. Table 3 gives binding energies of ligand–receptor interactions, including different types of interactions and the amino acid residues involved.

Figure 13

Docking profiles of GSK3β with NSC772864 and standard inhibitors. (A,B) NSC772864 bound with GSK3β with Gibbs’ free energy (ΔG) of −8.6 kcal/mol. (C,D) Binding of AZD1080 with GSK3β showed a slightly higher affinity of ΔG = −8.4 kcal/mol compared to NSC772864. Table 4 gives binding energies of ligand–receptor interactions, including different types of interactions and the amino acid residues involved.

Figure 14

Docking profiles of c-MYC with NSC772864 and standard inhibitors. (A,B) NSC772864 bound with c-MYC with Gibbs’ free energy (ΔG) of −9.1 kcal/mol. (C,D) Binding of crizotinib with c-Met showed a slightly higher affinity of ΔG = −7.6 kcal/mol compared to NSC772864. Table 5 gives binding energies of ligand–receptor interactions, including different types of interactions and the amino acid residues involved.

Figure 15

Docking profiles of cyclin D1 (CCND1) with NSC772864 and standard inhibitors. (A,B) NSC772864 bound to CCND1 with Gibbs’ free energy (ΔG) of −8.0 kcal/mol. (C,D) Binding of trilaciclib with c-Met showed a slightly higher affinity at ΔG = −7.4 kcal/mol compared to NSC772864. Table 6 gives the binding energies of ligand–receptor interactions, including different types of interactions and the amino acid residues involved.

Figure 16

Schematic diagram shows NSC772864 may directly target Met receptor-mediated cell cycle progression through WNT/β-catenin/GSK3β pathway and MYC transcription factor. c-Met expression enhances the Wnt/β-catenin signaling and impedes GSK3β from phosphorylating β-catenin in CRC; this subsequently promotes β-catenin translocation into the nucleus leading to cancer initiation; moreover, proto-oncogene, c-MYC, is associated with the regulation of CCND1, a key regulatory protein through the G1 to S phases of the cell cycle via binding to cyclin-dependent kinase 4 (CDK4) and CDK6 cell cycle, and play crucial roles in cell cycle progression. Therefore, blockade of c-Met/cMYC/CCND1 inhibits CRC cell progression, stemness, and metastasis.