In-silico based identification of phytochemicals from Houttuynia cordata Thunb. as potential inhibitors for overexpressed HER2 and VEGFR2 cancer genes.

Human epidermal growth factor receptor2 (HER2) and Vascular endothelial growth factor receptor2 (VEGFR2) - a tyrosine kinase receptors play a key role in breast and stomach cancers. The overexpression of HER2 and VEGFR2 genes increases the number of HER2 and VEGFR2 in the cell which initiates breast and stomach cancer respectively. The phytochemicals from traditional medicinal herb Houttuynia cordata Thunb. are reported to possess anti-inflammatory and anti-cancer potential. However, isolation of phytochemicals from this herb is fraught with uncertainly and time-consuming. Here, a molecular docking approach provides probable binding affinities between the receptors and phytochemicals (ligands) which initiate the first step of anticancer drug discovery and development. In the present study, In-silico docking approaches were used to identify the top-hit phytochemicals from H. cordata as potential inhibitors for overexpressed HER2 (breast) and VEGFR2 (stomach) cancer genes. A total of 100 biologically active phytochemicals from H. cordata were screened and docked against the ligand-binding pocket of HER2 and VEGFR2 kinase domains. Docking results revealed only a few phytochemicals (molecules) which appropriately fit into the ligand-binding pocket with higher binding affinity than the natural ATP ligand. A competitive docking was used to ascertain the top-hit phytochemicals that bind perfectly to the ATP ligand-binding pocket. Among the top-hit phytochemicals docked from H. cordata, the ß-sitosterol and Quercetin showed highest binding affinity towards HER2 and VEGFR2 receptors using both hydrogen and hydrophobic interactions. This study confirmed ß-sitosterol and Quercetin as potential drug candidates against breast and stomach cancer.Communicated by Ramaswamy H. Sarma.

Integrating virtual screening and biochemical experimental approach to identify potential anti-cancer agents from drug databank.

Chemical libraries constitute a reservoir of pharmacophoric molecules to identify potent anti-cancer agents. Virtual screening of heterocyclic compound library in conjugation with the agonist-competition assay, toxicity-carcinogenicity analysis, and string-based structural searches enabled us to identify several drugs as potential anti-cancer agents targeting protein kinase C (PKC) as a target. Molecular modeling study indicates that Cinnarizine fits well within the PKC C2 domain and exhibits extensive interaction with the protein residues. Molecular dynamics simulation of PKC-Cinnarizine complex at different temperatures (300, 325, 350, 375, and 400[Formula: see text]K) confirms that Cinnarizine fits nicely into the C2 domain and forms a stable complex. The drug Cinnarizine was found to bind PKC with a dissociation constant Kd of [Formula: see text]M. The breast cancer cells stimulated with Cinnarizine causes translocation of PKC-[Formula: see text] to the plasma membrane as revealed by immunoblotting and immunofluorescence studies. Cinnarizine also dose dependently reduced the viability of MDAMB-231 and MCF-7 breast cancer cells with an IC[Formula: see text] of [Formula: see text] and [Formula: see text]g/mL, respectively. It is due to the disturbance of cell cycle of breast cancer cells with reduction of S-phase and accumulation of cells in G1-phase. It disturbs mitochondrial membrane potentials to release cytochrome C into the cytosol and activates caspase-3 to induce apoptosis in cancer cells. The cell death was due to induction of apoptosis involving mitochondrial pathway. Hence, the current study has assigned an additional role to Cinnarizine as an activator of PKC and potentials of the approach to identify new molecules for anti-cancer therapy. Thus, in silico screening along with biochemical experimentation is a robust approach to assign additional roles to the drugs present in the databank for anti-cancer therapy.

Synthesis and evaluation of oxindoles as promising inhibitors of the immunosuppressive enzyme indoleamine 2,3-dioxygenase 1.

Indoleamine 2,3-dioxygenase 1 (IDO1) is considered as an important therapeutic target for the treatment of cancer, chronic infections and other diseases that are associated with immune suppression. Recent developments in understanding the catalytic mechanism of the IDO1 enzyme revealed that conversion of l-tryptophan (l-Trp) to N-formylkynurenine proceeded through an epoxide intermediate state. Accordingly, we synthesized a series of 3-substituted oxindoles from l-Trp, tryptamine and isatin. Compounds with C3-substituted oxindole moieties showed moderate inhibitory activity against the purified human IDO1 enzyme. Their optimization led to the identification of potent compounds, 6, 22, 23 and 25 (IC50 = 0.19 to 0.62 µM), which are competitive inhibitors of IDO1 with respect to l-Trp. These potent compounds also showed IDO1 inhibition potencies in the low-micromolar range (IC50 = 0.33-0.49 µM) in MDA-MB-231 cells. The cytotoxicity of these potent compounds was trivial in different model cancer (MDA-MB-231, A549 and HeLa) cells and macrophage (J774A.1) cells. Stronger selectivity for the IDO1 enzyme (124 to 210-fold) over the tryptophan 2,3-dioxygenase (TDO) enzyme was also observed for these compounds. These results suggest that the oxindole moiety of the compounds could mimic the epoxide intermediate state of l-Trp. Therefore, the structural simplicity and low-micromolar inhibition potencies of these 3-substituted oxindoles make them quite attractive for further investigation of IDO1 function and immunotherapeutic applications.

Danazol has potential to cause PKC translocation, cell cycle dysregulation, and apoptosis in breast cancer cells.

Danazol, the established clinical drug, has given promising therapeutic results in a series of clinical trials with breast cancer patients. Danazol shares structural similarities with several known PKC agonists and fits well into the C1 domain. Danazol binds to the C1b domain of PKC with Kd of 5.64 ± 1.27 µm. MD simulation studies further support that the PKC-danazol molecular model is stable and showing minimum distortion to the structure during the simulation period. Immunofluorescence and Western blotting studies indicate that MDAMB-231 cells stimulated with danazol exhibit translocation of PKCα to the plasma membrane. Cells stimulated with danazol causes appearance of several phosphorylated proteins in lysate and plasma membrane. In addition, danazol affects carcinogenic molecule (PMA)-induced intracellular signaling in cancer cells. It halted the cancer cells in the G1 phase of the cell cycle and reduced the viability of ER+ve and triple-negative breast cancer cells with an IC50 of 31 ± 2.63 and 65 ± 4.27 µg/ml, respectively. DNA fragmentation and flow cytometry experiments revealed that the cell death follows the apoptotic pathway. It affects mitochondrial membrane potentials and releases cytochrome-C from mitochondria to induce downstream apoptosis in breast cancer cells. Hence, the current study may help clinicians to re-design their treatment strategy to optimize therapeutic potentials of the molecule.

Fused Heterocyclic Compounds as Potent Indoleamine-2,3-dioxygenase 1 Inhibitors.

Uncontrolled metabolism of l-tryptophan (l-Trp) in the immune system has been recognized as a critical cellular process in immune tolerance. Indoleamine 2,3-dioxygenase 1 (IDO1) enzyme plays an important role in the metabolism of a local l-Trp through the kynurenine pathway in the immune systems. In this regard, IDO1 has emerged as a therapeutic target for the treatment of diseases that are associated with immune suppression like chronic infections, cancer, and others. In this study, we synthesized a series of pyridopyrimidine, pyrazolopyranopyrimidine, and dipyrazolopyran derivatives. Further lead optimizations directed to the identification of potent compounds, 4j and 4l (IC50 = 260 and 151 nM, respectively). These compounds also exhibited IDO1 inhibitory activities in the low nanomolar range in MDA-MB-231 cells with very low cytotoxicity. Stronger selectivity for the IDO1 enzyme (>300-fold) over tryptophan 2,3-dioxygenase (TDO) enzyme was also observed for these compounds. Hence, these fused heterocyclic compounds are attractive candidates for the advanced study of IDO1-dependent cellular function and immunotherapeutic applications.

Nitrobenzofurazan derivatives of N'-hydroxyamidines as potent inhibitors of indoleamine-2,3-dioxygenase 1.

Tryptophan metabolism through the kynurenine pathway is considered as a crucial mechanism in immune tolerance. Indoleamine 2,3-dioxygenase 1 (IDO1) plays a key role in tryptophan catabolism in the immune system and it is also considered as an important therapeutic target for the treatment of cancer and other diseases that are linked with kynurenine pathway. In this study, a series of nitrobenzofurazan derivatives of N'-hydroxybenzimidamides (1) and N'-hydroxy-2-phenylacetimidamides (2) were synthesized and their inhibitory activities against human IDO1 enzyme were tested using in-vitro and cellular enzyme activity assay. The optimization leads to the identification of potent compounds, 1d, 2i and 2k (IC50 = 39-80 nM), which are either competitive or uncompetitive inhibitors of IDO1 enzyme. These compounds also showed IDO1 inhibition potencies in the nanomolar range (IC50 = 50-71 nM) in MDA-MB-231 cells with no/negligible amount of cytotoxicity. The stronger selectivity of the potent compounds for IDO1 enzyme over tryptophan 2,3-dioxygenase (TDO) enzyme (312-1593-fold) also makes them very attractive for further immunotherapeutic applications.

Alkyl Cinnamates Induce Protein Kinase C Translocation and Anticancer Activity against Breast Cancer Cells through Induction of the Mitochondrial Pathway of Apoptosis.

PURPOSE: The protein kinase C (PKC) family of serine-threonine kinases plays an important role in cancer cell progression. Thus, molecules that target PKC have potential as anticancer agents. The current study aims to understand the treatment of breast cancer cells with alkyl cinnamates. We have also explored the mechanistic details of their anticancer action and the underlying molecular signaling. METHODS: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to measure the viability of MDAMB-231 breast cancer cells to assess the anticancer activity of these compounds. In addition, flow cytometry was performed to study the effect of alkyl cinnamates on the cell cycle and apoptosis. Immunoblotting and immunofluorescence techniques were performed to study PKC translocation, cytochrome c release, and modulation of the mitochondrial membrane potential in breast cancer cells targeted with alkyl cinnamates. RESULTS: The PKC agonist DM-2-8 translocated 16.6%±1.7% PKCα from cytosol to the plasma membrane and showed excellent anticancer activity with an half maximal inhibitory concentration (IC50) of 4.13±0.27 µg/mL against cancer cells. The treated cells had an abnormal morphology and exhibited cell cycle defects with G2/M arrest and reduced S phase. Cancer cells treated with DM-2-3, DM-2-4, or DM-2-8 underwent apoptosis as the major pathway of cell death, further confirmed by genomic DNA fragmentation. Furthermore, the mitochondrial membrane potential was perturbed, indicating involvement of the mitochondrial pathway of apoptosis. Immunolocalization studies revealed cytochrome c release from mitochondria to cytosol. Cancer cells treated with DM-2-8 and curcumin showed activation of caspase-9 and caspase-3 as downstream molecular components of the apoptotic pathway. Alkyl cinnamates also caused oxidative stress, which regulates the apoptotic machinery (DNA fragmentation), cell death, and morphological abnormalities in cancer cells. CONCLUSION: Alkyl cinnamates specifically target cancer cells through induction of PKC translocation and the mitochondrial pathway of apoptosis, and could be promising anticancer drugs.