Molecular docking, synthesis, and biological evaluation of 7-azaindole-derivative (7AID) as novel anti-cancer agent and potent DDX3 inhibitor:-an in silico and in vitro approach.

The DEAD-box helicase family member DDX3 is involved in many diseases, such as viral infection, inflammation, and cancer. Many studies in the last decade have revealed the role of DDX3 in tumorigenesis and metastasis. DDX3 has both tumour suppressor and oncogenic effect, in the present study we have evaluated the expression levels of DDX3 in cervical squamous cell carcinoma at mRNA level via real-time PCR and protein level via Immunohistochemistry. DDX3 has become a molecule of interest in cancer biology that promotes drug resistance by adaptive response inevitably leading to treatment failure. One approach to avoid the development of resistant to disease is to create novel drugs that target the overexpressed proteins, we designed and synthesized a novel 7-azaindole derivative (7-AID) compound, {5-[1H-pyrrolo (2, 3-b) pyridin-5-yl] pyridin-2-ol]} that could lodge within the adenosine-binding pocket of the DDX3 (PDB ID: 2I4I). The binding efficacy of 7-AID compound with DDX3 was analysed by molecular docking studies. 7-AID was found to interact with the key residues Tyr200 and Arg202 from the Q-motif rendered by π-interactions and hydrogen bonds within the binding pocket with good docking score - 7.99 kcal/mol. The cytotoxicity effect of 7-AID compound was evaluated using MTT assay on human cervical carcinoma cells (HeLa) and breast cancer cells (MCF-7 and MDA MB-231) and the compound shown effective inhibitory concentration (IC50) on Hela cells 16.96 µM/ml and 14.12 and 12.69 µM/ml on MCF-7 and MDA MB-231, respectively. Further, the in-vitro, in-vivo anti-cancer and anti-angiogenic assessment of 7-AID compound was evaluated on Hela cells using scratch wound-healing assay, DAPI staining, cell cycle analysis, immunoblotting, and chorioallontoic membrane assay. Furthermore, the inhibitory effect of derivative compound on DDX3 was investigated in HeLa, MCF-7, and MDA MB-231 cells at the mRNA and protein levels. The results showed that the 7-AID compound effectively inhibited DDX3 in a dose-dependent manner, and the findings suggest that the compound could be used as a potential DDX3 inhibitor.

Overexpression of Secreted Phosphoprotein 1 (SPP1) predicts poor survival in HPV positive cervical cancer.

Cervical cancer (CC) is the most prevalent malignant gynecological tumor with limited treatments. The present study describes the role of SPP1 in cancer progression, SPP1 emerged as one of the most overexpressed genes identified through clariom D transcriptome microarray. This investigation aims towards identifying a potential gene with significant prognostic value for detection and early diagnosis of cervical cancer. The elevated expression of SPP1 in cervical squamous cell carcinoma tissue was validated across GEO (Gene Expression Omnibus) microarray data sets, TCGA (The Cancer Genome Atlas), and Oncomine databases. SPP1 expression was found to be prognostically significant, showing association with poor survival rate of the patients. Our study intended to assess the expression of secreted phosphoprotein (SPP1) gene at mRNA and protein levels, and to explore the association of single nucleotide polymorphisms of SPP1 with risk of CC. Further, receiver operating characteristics (ROC) curve was plotted to determine the levels of SPP1 to differentiate CC against control. Results revealed significant (p < 0.01) stage-wise upregulation of SPP1 in CC compared to the normal cervical tissue and this was further confirmed using Immunohistochemistry and real-time PCR. The ROC for SPP1 demonstrated good selective power to differentiate malignant CC and non-malignant cervical tissues. The SPP1 gene -443 T > C promoter polymorphisms are found to be significantly predominant in the disease group and Insilico analysis by the TRANSFAC software confirms its association with loss of STAT6 transcription factor binding site leading to overexpression of the SPP1.

Screening and Identification of Potential iNOS Inhibitors to Curtail Cervical Cancer Progression: an In Silico Drug Repurposing Approach.

Cervical cancer is the second most common cause of cancer deaths in women worldwide and remains the main reason of mortality among women of reproductive age in developing countries. Nitric oxide is involved in several physiological functions inclusive of inflammatory and immune responses. However, the function of NO in tumor biology is debatable. The inducible NOS (iNOS/NOS2) isoform is the one responsible to maintain the levels of NO, and it exhibits pleotropic effects in various cancers with concentration-dependent pro- and anti-tumor effects. iNOS triggers angiogenesis and endothelial cell migration in tumors by regulating the levels of vascular endothelial growth factor (VEGF). In drug discovery, drug repurposing involves investigations of approved drug candidates to treat various other diseases. In this study, we used anti-cancer drugs and small molecules to target iNOS and identify a potential selective iNOS inhibitor. The structures of ligands were geometrically optimized and energy minimized using Hyperchem software. Molecular docking was performed using Molegro virtual docker, and ligands were selected based on MolDock score, Rerank score, and H-bonding energy. In the study shown, venetoclax compound demonstrated excellent binding affinity to iNOS protein. This compound exhibited the lowest MolDock score and Rerank score with better H-bonding energy to iNOS. The binding efficacy of venetoclax was analyzed by performing molecular docking and molecular dynamic simulations. Multiple parameters were used to analyze the simulation trajectory, like root mean square deviation (RMSD), radius of gyration (Rg), and hydrogen bond interactions. Based on the results, venetoclax emerges to be a promising potential iNOS inhibitor to curtail cervical cancer progression.

Inhibition of Inducible Nitric Oxide Synthase (iNOS) by Andrographolide and In Vitro Evaluation of Its Antiproliferative and Proapoptotic Effects on Cervical Cancer.

This work is aimed at investigating the expression levels of inducible nitric oxide synthase (iNOS) in cervical cancer and identifying a potential iNOS inhibitor. The data mining studies performed advocated iNOS to be a promising biomarker for cancer prognosis, as it is highly overexpressed in several malignant cancers. The elevated iNOS was found to be associated with poor survival and increased tumor aggressiveness in cervical cancer. Immunohistochemical and RT-PCR investigations of iNOS showed significant upregulation of endogenous iNOS expression in the cervical tumor samples, thus making iNOS a potent target for decreasing tumor inflammation and aggressiveness. Andrographolide, a plant-derived diterpenoid lactone, is widely reported to be effective against infections and inflammation, causing no adverse side effects on humans. In the current study, we investigated the effect of andrographolide on the prognostic value of iNOS expression in cervical cancer, which has not been reported previously. The binding efficacy of andrographolide was analyzed by performing molecular docking and molecular dynamic simulations. Multiple parameters were used to analyze the simulation trajectory, like root mean square deviation (RMSD), torsional degree of freedom, protein-root mean square fluctuations (P-RMSF), ligand RMSF, total number of intramolecular hydrogen bonds, secondary structure elements (SSE) of the protein, and protein complex with the time-dependent functions of MDS. Ligand-protein interactions revealed binding efficacy of andrographolide with tryptophan amino acid of iNOS protein. Cancer cell proliferation, cell migration, cell cycle analysis, and apoptosis-mediated cell death were assessed in vitro, post iNOS inhibition induced by andrographolide treatment (demonstrated by Western blot). Results. Andrographolide exhibited cytotoxicity by inhibiting the in vitro proliferation of cervical cancer cells and also abrogated the cancer cell migration. A significant increase in apoptosis was observed with increasing andrographolide concentration, and it also induced cell cycle arrest at G1-S phase transition. Our results substantiate that andrographolide significantly inhibits iNOS expression and exhibits antiproliferative and proapoptotic effects on cervical cancer cells.

Identification of Differentially Expressed Genes in Cervical Cancer Patients by Comparative Transcriptome Analysis.

Cervical cancer is one of the most malignant reproductive diseases seen in women worldwide. The identification of dysregulated genes in clinical samples of cervical cancer may pave the way for development of better prognostic markers and therapeutic targets. To identify the dysregulated genes (DEGs), we have retrospectively collected 10 biopsies, seven from cervical cancer patients and three from normal subjects who underwent a hysterectomy. Total RNA isolated from biopsies was subjected to microarray analysis using the human Clariom D Affymetrix platform. Based on the results of principal component analysis (PCA), only eight samples are qualified for further studies; GO and KEGG were used to identify the key genes and were compared with TCGA and GEO datasets. Identified genes were further validated by quantitative real-time PCR and receiver operating characteristic (ROC) curves, and the highest Youden index was calculated in order to evaluate cutoff points (COPs) that allowed distinguishing of tissue samples of cervical squamous carcinoma patients from those of healthy individuals. By comparative microarray analysis, a total of 108 genes common across the six patients' samples were chosen; among these, 78 genes were upregulated and 26 genes were downregulated. The key genes identified were SPP1, LYN, ARRB2, COL6A3, FOXM1, CCL21, TTK, and MELK. Based on their relative expression, the genes were ordered as follows: TTK > ARRB2 > SPP1 > FOXM1 > LYN > MELK > CCL21 > COL6A3; this generated data is in sync with the TCGA datasets, except for ARRB2. Protein-protein interaction network analysis revealed that TTK and MELK are closely associated with SMC4, AURKA, PLK4, and KIF18A. The candidate genes SPP1, FOXM1, LYN, COL6A3, CCL21, TTK and MELK at mRNA level, emerge as promising candidate markers for cervical cancer prognosis and also emerge as potential therapeutic drug targets.

Short communication for targeting natural compounds against HER2 kinase domain as potential anticancer drugs applying pharmacophore based molecular modelling approaches- part 2.

Breast cancer is one of the common causes of death noticed in women globally. In order to find effective therapeutics, the current investigation has focussed on identifying candidate compounds for EGFR and HER2. Accordingly, the pharmacophore modelling approaches were adapted to identify two prospective compounds and were docked against the target 3RCD that is complexed with TAK-285 a known dual inhibitor. Focussing on the target 3RCD, our results have showed that the compounds have demonstrated a good binding affinity towards the target occupying the binding pocket. They have established key residue interactions with stable molecular dynamics simulation results. The Hit compounds have demonstrated a potential to penetrate the blood brain barrier thereby enriching their therapeutics towards breast cancer brain metastasis. Taken together, our findings propose two candidate compounds as EGFR/HER2 inhibitors that might serve as novel chemical spaces for designing and developing new inhibitors.

Celecoxib sensitizes imatinib-resistant K562 cells to imatinib by inhibiting MRP1-5, ABCA2 and ABCG2 transporters via Wnt and Ras signaling pathways.

Imatinib mesylate, a tyrosine kinase inhibitor, is very effective in the treatment of chronic myeloid leukemia (CML). However, development of resistance to imatinib therapy is also a very common mechanism observed with long-term administration of the drug. Our previous studies have highlighted the role of cyclooxygenase-2 (COX-2) in regulating the expression of multidrug resistant protein-1 (MDR1), P-gp, in imatinib-resistant K562 cells (IR-K562) via PGE2-cAMP-PKC-NF-κB pathway and inhibition of COX-2 by celecoxib, a COX-2 specific inhibitor, inhibits this pathway and reverses the drug resistance. Studies have identified that not only MDR1 but other ATP-binding cassette transport proteins (ABC transporters) are involved in the development of imatinib resistance. Here, we tried to study the role of COX-2 in the regulation of other ABC transporters such as MRP1, MRP2, MRP3, ABCA2 and ABCG2 that have been already implicated in imatinib resistance development. The results of the study clearly indicated that overexpression of COX-2 lead to upregulation of MRP family proteins in IR-K562 cells and celecoxib down-regulated the ABC transporters through Wnt and MEK signaling pathways. The study signifies that celecoxib in combination with the imatinib can be a good alternate treatment strategy for the reversal of imatinib resistance.