mTOR/EGFR/iNOS/MAP2K1/FGFR/TGFB1 Are Druggable Candidates for N-(2,4-Difluorophenyl)-2',4'-Difluoro-4-Hydroxybiphenyl-3-Carboxamide (NSC765598), With Consequent Anticancer Implications

Abstract

Background: The application of computational and multi-omics approaches has aided our understanding of carcinogenesis and the development of therapeutic strategies. NSC765598 is a novel small molecule derivative of salicylanilide. This study aims to investigate the ligand-protein interactions of NSC765598 with its potential targets and to evaluate its anticancer activities in vitro.

Methods: We used multi-computational tools and clinical databases, respectively, to identify the potential drug target for NSC765598 and analyze the genetic profile and prognostic relevance of the targets in multiple cancers. We evaluated the in vitro anticancer activities against the National Cancer Institute 60 (NCI60) human tumor cell lines and used molecular docking to study the ligand-protein interactions. Finally, we used the DTP-COMPARE algorithm to compare the NSC765598 anticancer fingerprints with NCI standard agents.

Results: We identified mammalian target of rapamycin (mTOR)/epidermal growth factor receptor (EGFR)/inducible nitric oxide synthase (iNOS)/mitogen-activated protein 2 kinase 1 (MAP2K1)/fibroblast growth factor receptor (FGFR)/transforming growth factor-β1 (TGFB1) as potential targets for NSC765598. The targets were enriched in cancer-associated pathways, were overexpressed and were of prognostic relevance in multiple cancers. Among the identified targets, genetic alterations occurred most frequently in EGFR (7%), particularly in glioblastoma, esophageal squamous cell cancer, head and neck squamous cell cancer, and non-small-cell lung cancer, and were associated with poor prognoses and survival of patients, while other targets were less frequently altered. NSC765598 displayed selective antiproliferative and cytotoxic preferences for NSCLC (50% growth inhibition (GI₅₀) = 1.12-3.95 µM; total growth inhibition (TGI) = 3.72-16.60 μM), leukemia (GI₅₀ = 1.20-3.10 µM; TGI = 3.90-12.70 μM), melanoma (GI₅₀ = 1.45-3.59 µM), and renal cancer (GI₅₀ = 1.38-3.40 µM; TGI = 4.84-13.70 μM) cell lines, while panels of colon, breast, ovarian, prostate, and central nervous system (CNS) cancer cell lines were less sensitive to NSC765598. Interestingly, NSC765598 docked well into the binding cavity of the targets by conventional H-bonds, van der Waal forces, and a variety of π-interactions, with higher preferences for EGFR (ΔG = -11.0 kcal/mol), NOS2 (ΔG = -11.0 kcal/mol), and mTOR (ΔG = -8.8 kcal/mol). NSC765598 shares similar anti-cancer fingerprints with NCI standard agents displayed acceptable physicochemical values and met the criteria of drug-likeness.

Conclusion: NSC765598 displayed significant anticancer and potential multi-target properties, thus serve as a novel candidate worthy of further preclinical studies.

Keywords: NSC765598; anticancer activity; molecular docking; multi-omics study; multi-target small molecule; protein-ligand interaction.

Figure 1

Synthetic protocol and potential drug target classification of N-(2,4-difluorophenyl)-2’,4’-difluoro-4-hydroxybiphenyl-3-carboxamide (NSC765598). (A) NSC765598 is a chemical derivative of diflunisal and NDMC101s (reaction conditions; SOCl₂, anhydrous THF, reflux, 8 h; and aniline, anhydrous THF, reflux 14-16) (B). The SwissTarget identified top 20 targets of NSC765598 are classified into enzymes (20%), kinases (40%) and Family A G protein-coupled receptor (40%).

Figure 2

Potential targets for NSC765598. (A) Venn diagram showing common targets of NSC765598 as revealed by the SwissTarget, PASS, and PharmMapper algorithms. Six genes including iNOS, TGFB1, vascular endothelial growth factor receptor 2, FGFR, EDFR, and MAPK2 were predicted by the three algorithms ( Figures 3A, B ), while two genes, viz., signal transducer and activator of transcription and serine/threonine-protein kinase mTOR, were predicted to be top ranked targets of NSC765598 by the two algorithms. (B) Pharmacophore-based models of the six commonly identified NSC765598 target predictions via protein-receptor interactions.

Figure 3

NSC765598 targets are enriched in cancer-associated biological processes and signaling pathways. (A) Enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway of NSC765598 targets. Enrichment was considered at p < 10-6, a gene count of ≥ 3, and a strength of ≥ 2. (B) Enriched Gene Ontology (GO) of NSC765598 targets. (C) Protein-protein interaction (PPI) network of mTOR/EGFR/iNOS/MAP2K1/FGFR/TGFB1. Second-order clustering of NSC765598 gene targets generated 26 nodes, 176 edges, 13.5 average node degrees, an 0.811 average local clustering coefficient, and a PPI enrichment p-value of < 1.0e-16. Hub genes with the highest nodes were mTOR, MAP2K1, and EGFR with 23, 14, and 13 nodes, respectively The accompanying table shows connection scores of nodes with the top three hub genes (mTOR, MAP2K1, and EGFR).

Figure 4

Expression levels and prognostic values of epidermal growth factor receptor (EGFR), mammalian target of rapamycin (mTOR), nitric oxide synthase 2 (NOS2), transforming growth factor-β1 (TGFB1), mitogen-activated protein 2 kinase 1 (MAP2K1), and fibroblast growth factor receptor 1 (FGFR1) in The Cancer Genome Atlas (TCGA) cancer cohorts. (A) Box plots showing differential gene expression levels (log2 TPM) of signal transduction and activator of transcription 3 (STAT3)/cyclin-dependent kinase 2 (CDK2)/4/6 between tumor and adjacent normal tissue samples across TCGA database. Blue labels indicate normal tissues, and red labels indicate tumor samples. (B) Kaplan-Meier curve of overall survival of cancer cohorts with low and cohorts with high RNA expression levels of EGFR, mTOR, NOS2, TGFB1, MAP2K1, and FGFR1. Higher RNA expression profiles of MAP2K1/NOS2/EGFR were correlated with low overall survival of cohorts. The statistical significance of differentially expressed genes was evaluated using the Wilcoxon test. * p<0.05; ** p<0.01; *** p<0.001.

Figure 5

Expression scatterplots of EGFR correlations with NOS2, TGFB1, MAP2K1, mTOR and FGFR1 in multiple cancer types. The strength of correlations between the genes is reflected by the purity-adjusted partial spearman’s rho value, where a value of r= 1 means a perfect positive correlation and a value of r= −1 means a perfect negative correlation.

Figure 6

Genomic alterations in the epidermal growth factor receptor (EGFR) are associated with poor prognoses of cancer cohorts. (A) Bar graph showing alteration frequencies of the EGFR in cohorts of different cancer types. The most common alteration in the EGFR gene was amplification. Kaplan-Meir survival curve of (B) overall survival, (C) disease-specific survival, and (D) disease-progression survival of cancer cohorts with altered EGFR and unaltered EGFR genes. Cohorts with altered EGFR had low overall survival, disease-specific survival, and disease-progression survival. p < 10^−10. (E). Bar showing the EGFR putative copy number alterations and messenger RNA expressions. (F) Heat map showing p values and significance levels of other gene mutations associated with cohorts with altered EGFR and cohorts with unaltered EGFR. (G) Line graph showing frequencies of altered genes in cancer cohorts with altered EGFR and cohorts with unaltered EGFR. (H) Bar graph showing gene mutations that were significantly enriched in both EGFR-altered and EGFR-unaltered cohorts.

Figure 7

Docking profiles of mTOR/MAP2K/EGFR with NSC765598 and standard inhibitors. The two dimensional (2D) representations of ligand–receptor complexes, showing the interacting amino acid residues and the type of interactions, occurring between the ligands and (A) mTOR (B) MAP2K and (C) EGFR.

Figure 8

Docking profiles of iNOS/FGFR/TGFB with NSC765598 and standard inhibitors. The two-dimensional (2D) representations of ligand–receptor complexes, showing the interacting amino acid residues and the type of interactions, occurring between the ligands and (A) iNOS (B) FGFR and (C) TGFB.

Figure 9

Inhibitory activities of NSC765598 against panels of 60 human cancer cell lines. Each cell line was treated with a single dose of 10 μM of NSC765598 for 48 h. The bar points above point zero on the y-axis indicate the mean percentage of cell growth relative to cells without drug treatment, while bar points below point zero indicate the percentage of cell death.

Figure 10

Dose-response curves of NCI60 human cancer cell lines to NSC765598 treatment. Growth percentage value of 100 on the y-axis represents the growth of untreated cells, the 0 value represents no net growth, while −100 represents complete death of cells.

Figure 11

GI₅₀ (50% inhibition of the cancer cell growth) and TGI (100% growth inhibition of the cancer cells) concentrations of NSC765598 against NCI60 panels of human cancer cell lines.