Structure-function relationships of wheat flavone O-methyltransferase: Homology modeling and site-directed mutagenesis

<p>Abstract</p> <p>Background</p> <p>Wheat (<it>Triticum aestivum </it>L.) <it>O</it>-methyltransferase (TaOMT2) catalyzes the sequential methylation of the flavone, tricetin, to its 3'-methyl- (selgin), 3',5'-dimethyl- (tricin) and 3',4',5'-trimethyl ether derivatives. Tricin, a potential multifunctional nutraceutical, is the major enzyme reaction product. These successive methylations raised the question as to whether they take place in one, or different active sites. We constructed a 3-D model of this protein using the crystal structure of the highly homologous <it>Medicago sativa </it>caffeic acid/5-hydroxyferulic acid <it>O</it>-methyltransferase (MsCOMT) as a template with the aim of proposing a mechanism for multiple methyl transfer reactions in wheat.</p> <p>Results</p> <p>This model revealed unique structural features of TaOMT2 which permit the stepwise methylation of tricetin. Substrate binding is mediated by an extensive network of H-bonds and van der Waals interactions. Mutational analysis of structurally guided active site residues identified those involved in binding and catalysis. The partly buried tricetin active site, as well as proximity and orientation effects ensured sequential methylation of the substrate within the same pocket. Stepwise methylation of tricetin involves deprotonation of its hydroxyl groups by a His262-Asp263 pair followed by nucleophilic attack of SAM-methyl groups. We also demonstrate that Val309, which is conserved in a number of graminaceous flavone OMTs, defines the preference of TaOMT2 for tricetin as the substrate.</p> <p>Conclusions</p> <p>We propose a mechanism for the sequential methylation of tricetin, and discuss the potential application of TaOMT2 to increase the production of tricin as a nutraceutical. The single amino acid residue in TaOMT2, Val309, determines its preference for tricetin as the substrate, and may define the evolutionary differences between the two closely related proteins, COMT and flavone OMT.</p

A Novel Synthetic Compound (E)-5-((4-oxo-4H-chromen-3-yl)methyleneamino)-1-phenyl-1H-pyrazole-4-carbonitrile Inhibits TNF alpha-Induced MMP9 Expression via EGR-1 Downregulation in MDA-MB-231 Human Breast Cancer Cells

Breast cancer is a common malignancy among women worldwide. Gelatinases such as matrix metallopeptidase 2 (MMP2) and MMP9 play crucial roles in cancer cell migration, invasion, and metastasis. To develop a novel platform compound, we synthesized a flavonoid derivative, (E)-5-((4-oxo-4H-chromen-3-yl)methyleneamino)-1-phenyl-1H-pyrazole-4-carbonitrile (named DK4023) and characterized its inhibitory effects on the motility andMMP2andMMP9expression of highly metastatic MDA-MB-231 breast cancer cells. We found that DK4023 inhibited tumor necrosis factor alpha (TNF alpha)-induced motility and F-actin formation of MDA-MB-231 cells. DK4023 also suppressed the TNF alpha-induced mRNA expression ofMMP9through the downregulation of the TNF alpha-extracellular signal-regulated kinase (ERK)/early growth response 1 (EGR-1) signaling axis. These results suggest that DK4023 could serve as a potential platform compound for the development of novel chemopreventive/chemotherapeutic agents against invasive breast cancer

Structural and functional analyses of minimal phosphopeptides targeting the polo-box domain of polo-like kinase 1

Polo-like kinase-1 (Plk1) has a pivotal role in cell proliferation and is considered a potential target for anticancer therapy. The noncatalytic polo-box domain (PBD) of Plk1 forms a phosphoepitope binding module for protein-protein interaction. Here, we report the identification of minimal phosphopeptides that specifically interact with the PBD of human PLK1, but not those of the closely related PLK2 and PLK3. Comparative binding studies and analyses of crystal structures of the PLK1 PBD in complex with the minimal phosphopeptides revealed that the C-terminal SpT dipeptide functions as a high-affinity anchor, whereas the N-terminal residues are crucial for providing specificity and affinity to the interaction. Inhibition of the PLK1 PBD by phosphothreonine mimetic peptides was sufficient to induce mitotic arrest and apoptotic cell death. The mode of interaction between the minimal peptide and PBD may provide a template for designing therapeutic agents that target PLK1.National Institutes of Health (U.S.) (Grant R01 GM60594)National Cancer Institute (U.S.)National Institutes of Health (U.S.) (Contract N01-CO-12400)National Institutes of Health (U.S.) (HHSN261200800001E

3-Methoxy-2-[5-(naphthalen-2-yl)-4,5-dihydro-1H-pyrazol-3-yl]phenol

The asymmetric unit of the title compound, C20H18N2O2, contains two independent molecules in which the dihedral angles between the naphthalene ring system [r.m.s. deviations = 0.012 (1) and 0.015 (1) Å] and the benzene ring are 71.65 (6) and 74.51 (6)°. In the crystal, pairs of N—H...O hydrogen bonds form two independent inversion dimers with graph-set notation R22(14). In addition, each molecule contains an intramolecular O—H...N hydrogen bond with an S(6) motif

3-(2-Hydroxy-4-methoxyphenyl)-N-(2-methoxyphenyl)-5-(naphthalen-1-yl)-4,5-dihydro-1H-pyrazole-1-carbothioamide

In the title molecule, C28H25N3O3S, the dihedral angles formed by the naphthalene ring system and the benzene rings are 73.03 (13) and 74.04 (11)°. The benzene rings attached to the central pyrazoline ring are almost coplanar, as indicated by the dihedral angle of 2.22 (10)° between them. The C atom of the methoxy group of the phenol ring is essentially coplanar with the ring [C—C—O—C = −0.3 (3)°], whereas the C atom of the methoxy group of the thioamide benzene ring is slightly twisted [C—C—O—C = 5.4 (3)°]. An intramolecular O—H...N hydrogen bond generates an S(6) ring motif. In the crystal, pairs of very weak C—H...S interactions form inversion dimers with an R22(18) motif

Crystal structure of 2-(3,4-dimethoxyphenyl)-3-hydroxy-4H-chromen-4-one

In the title compound, C17H14O5, the dimethoxy-substituted benzene ring is twisted relative to the 4H-chromenon skeleton (r.m.s. deviation = 0.015 Å) by 5.2 (4)°. The C atoms of the methoxy groups lie close to the plane of their attached benzene ring [deviations = 0.036 (3) and 0.290 (3)Å for the meta and para substituents, respectively]. An intramolecular O—H...O hydrogen bond closes an S(5) ring. In the cystal, inversion dimers linked by pairs of O—H...O hydrogen bonds generate R22(10) loops and C—H...O interactions connect the dimers into [010] chains

Inhibitory Effect of Alisma canaliculatum Ethanolic Extract on NF-κB-Dependent CXCR3 and CXCL10 Expression in TNFα-Exposed MDA-MB-231 Breast Cancer Cells

CXC motif chemokine ligand 10 (CXCL10) and its receptor CXC motif chemokine receptor 3 (CXCR3), play important roles in the motility of breast cancer cells. Alisma canaliculatum is a herb that has been used as a traditional medicine for thousands of years in Korea and China. Whether A. canaliculatum inhibits the motility of metastatic breast cancer cells is not clear yet. In this study, we show that A. canaliculatum ethanolic extract (ACE) prevented tumor necrosis factor-alpha (TNF&alpha;)-induced migration of MDA-MB-231 cells. ACE significantly attenuated TNF&alpha;-induced upregulation of CXCL10 and CXCR3 expression at the gene promoter level. Mechanistically, ACE inhibits TNF&alpha;-induced phosphorylation of inhibitor of &kappa;B (I&kappa;B) kinase (IKK), I&kappa;B and p65/RelA, leading to the suppression of nuclear translocation of p65/RelA nuclear factor kappa-B (NF-&kappa;B). Also, ACE inhibited NF-&kappa;B-dependent CXCR3 and CXCL10 promoter activities. These results suggest that ACE abrogates TNF&alpha;-induced migration of MDA-MB-231 breast cancer cells through down-regulation of IKK-NF-&kappa;B-dependent CXCR3 and CXCL10 expression. Our results suggest that ACE has potential as a herbal supplement for the inhibition of breast cancer metastasis

Novel anticancer agent, benzyldihydroxyoctenone, isolated from Streptomyces sp. causes G1 cell cycle arrest and induces apoptosis of HeLa cells

In the course of screening for anticancer agents, a novel active compound, F3-2-5, was isolated from culture broth of Streptomyces sp., KACC91015. Its structure was identified using nuclear magnetic resonance, mass spectrometry, and molecular modeling experiments, and confirmed by total synthesis. The growth of various human cancer cell lines was inhibited in a dose-dependent manner by 0.06-0.48 mM F3-2-5 over 24 h. Its IC(50) values were estimated at 37 microM on HeLa, 72 microM on A549, and 190 microM on HT-29 cells. However, F3-2-5 had no antiproliferative effect on normal lymphocytes and normal fibroblasts used as controls. Moreover, it affected cell cycle regulation and caused apoptosis of the HeLa cells; chromatin condensation and DNA fragmentation were observed in cells exposed to 80 microM F3-2-5. Western blot analysis revealed that F3-2-5 inhibited phosphorylation of retinoblastoma protein (pRb) and reduced expression of cyclin-dependent kinase-4 and -6, and cyclin D1 and E, while levels of p53 and p21(WAF1/CIP1) increased. Taken together, these findings show that F3-2-5 inhibits proliferation of HeLa cells by inducing G(1) phase arrest as a consequence of inhibition of pRb phosphorylation following up-regulation of p21(WAF1/CIP1) and p53. Furthermore, apoptosis in HeLa cells treated with F3-2-5 was associated with an increase in Bax and p53, leading to release of cytochrome c, activation of caspase-3, and -8, and cleavage of poly (ADP-ribose) polymerase