Modified thrombin-binding aptamers (TBAs) carrying uridine (U), 2’-deoxy-2’fluorouridine (FU) and North-methanocarbathymidine (NT) residues in the loop regions were synthesized and analyzed by UV thermal denaturation experiments and CD spectroscopy. The replacement of thymidines in the TGT loop by U and FU results in an increased stability of the antiparallel quadruplex structure described for the TBA while the presence of NT residues in the same positions destabilizes the antiparallel structure. The substitution of the thymidines in the TT loops for U, FU and NT induce a destabilization of the antiparallel quadruplex, indicating the crucial role of these positions. NMR studies on TBAs modified with uridines at the TGT loop also confirm the presence of the antiparallel quadruplex structure. Nevertheless, replacement of two Ts in the TT loops by uridine gives a more complex scenario in which the antiparallel quadruplex structure is present along with other partially unfolded species or aggregates.

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Lysine specific histone methylation and deacetylation and DNA hypermethylation are involved in the epigenetic silencing of tumor suppressor genes (TSG), e.g., p16 and JunB. The multi-protein complex PRC (polycomb repressive complex) 2 that contains the three core proteins EZH2, SUZ12 and EED, has intrinsic histone methyltransferase (HMTase) activity. This is mediated by the SET domain of EZH2, which induces tri-methylation (3Me) of lysine (K)-27 on histone H3, as well as promotes cell proliferation and aggressiveness of neoplastic cells. EZH2 is preferentially overexpressed in proliferating but not resting Mantle Cell Lymphoma (MCL) cells. In the present studies we demonstrate that treatment with the S-adenosylhomocysteine hydrolase inhibitor 3-Deazaneplanocin A (DZNep) dose-dependently (500 nM to 2.0 uM) depletes EZH2, SUZ12 and EED levels, as well as inhibits 3Me K27 on H3 while inducing K27 H3 acetylation. DZNep treatment also induces the levels of p21, p27, JunB and FBXO32, while depleting cyclin D1 and cyclin E levels in the cultured human MCL Jeko-1, MO2058 and Z138 cells and in primary patient-derived MCL cells. Treatment with DZNep induces PARP cleavage activity of the caspases and apoptosis in the cultured and primary MCL cells. DZNep promoted proteasomal degradation of EZH2 and SUZ12, since co-treatment with bortezpmib significantly restored EZH2 and SUZ12 levels in the MCL cells. We had previously reported that treatment with the pan-histone deacetylase (HDAC) inhibitor panobinostat (PS) (LBH589, Novartis Pharmaceutical Corp) depletes the levels of EZH2, SUZ12 and EED in cultured and primary AML cells (Mol Cancer Ther. 2006; 5:3096). Within the PRC2 complex, EZH2 bound and recruited the DNA methyltransferases DNMT1, and treatment with PS also disrupted the interaction of EZH2 with DNMT1, attenuated DNMT1 levels and its binding to the EZH2-targeted gene promoters, e,g, JunB. Here, we also demonstrate that, PS treatment depletes DNMT1 levels and induces JunB levels in cultured MCL cells. As compared to treatment with either agent alone, co-treatment with DZNep and PS caused more depletion of EZH2 and SUZ12, but not of DNMT1, more induction of JunB, p21 and p27, as well as synergistically induced apoptosis of cultured MCL cells (combination indices < 1.0). Taken together, these findings indicate that DZNep and PS mediated targeting of EZH2 and the PRC2 complex is an effective epigenetic therapy of MCL, which also results in undermining several molecular determinants of MCL cell proliferation and survival. Additionally, combined epigenetic therapy with DZNep and PS exerts synergistic in vitro activity against human MCL cells, suggesting that this combination may be a promising novel treatment for MCL.

ADVERTISEMENT RETURN TO ISSUEPREVBook ReviewNEXTCombinatorial Synthesis of Natural Product-Based Libraries. Edited by Armen M. Boldi. CRC Press, Taylor & Francis Group, Boca Raton, FL. 2006. xii + 347 pp. 18 × 26 cm. ISBN 0-8493-4000-4. $198.95.Victor E. MarquezView Author Information Laboratory of Medicinal Chemistry National Cancer Institute National Institutes of Health Frederick, Maryland 21702-1201Cite this: J. Med. Chem. 2007, 50, 5, 1083–1084Publication Date (Web):February 13, 2007Publication History Published online13 February 2007Published inissue 1 March 2007https://pubs.acs.org/doi/10.1021/jm078003ahttps://doi.org/10.1021/jm078003abook-reviewACS PublicationsCopyright © Not subject to U.S. Copyright. Published 2007 American Chemical SocietyRequest reuse permissionsArticle Views317Altmetric-Citations-LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose SUBJECTS:Carbohydrates,Genetics,Pharmaceuticals,Polymer scaffolds,Structural characteristics Get e-Alerts

Combining molecular dynamics (MD) in a hydrated phospholipid (DOPC) bilayer, a Monte Carlo search, and synthesis of locked nucleotide analogues, we discovered that the Southern conformation of the ribose is preferred for ligand recognition by the P2Y(6) receptor. 2'-Deoxy-(S)-methanocarbaUDP was found to be a full agonist of the receptor and displayed a 10-fold higher potency than that for the corresponding flexible 2'-deoxyUDP. MD results also suggested a conformational change of the second extracellular loop consequent to agonist binding.

Changes in bending of the DNA helix axis caused by the introduction of conformationally locked nucleotide analogs into the center region of the palindromic Dickerson dodecamer, d(CGCGAATTCGCG)(2), have been studied by NMR measurement of residual one-bond (13)C-(1)H dipolar couplings. Thymidine analogs, in which the deoxyribose was substituted by bicyclo[3.1.0]hexane, were incorporated in the T7, T8, and T7T8 positions. These nucleotide analogs restrict the ring pucker to the C2'-exo or "north" conformation, instead of C2'-endo or "south," which dominates in regular B-form DNA. For all three oligomers, bending toward the major groove is found relative to the native molecule. The effects are additive with bending of 5 +/- 1 degrees per locked nucleotide. Measurement of the change in bending is more accurate than measurement of the bending angle itself and requires far fewer experimental data.

BACKGROUNDGene silencing by abnormal methylation of promoter regions of regulatory genes is commonly associated with cancer. Silenced tumor suppressor genes are obvious targets for reactivation by methylation inhibitors such as 5-azacytidine (5-Aza-CR) and 5-aza-2'-deoxycytidine (5-Aza-CdR). However, both compounds are chemically unstable and toxic and neither can be given orally. We characterized a new demethylating agent, zebularine [1-(beta-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one], which is a chemically stable cytidine analog.METHODSWe tested the ability of zebularine to reactivate a silenced Neurospora crassa gene using a hygromycin gene reactivation assay. We then analyzed the ability of zebularine to inhibit DNA methylation in C3H 10T1/2 Cl8 (10T1/2) mouse embryo cells as assayed by induction of a myogenic phenotype and in T24 human bladder carcinoma cells, using the methylation-sensitive single nucleotide primer extension (Ms-SNuPE) assay. We also evaluated the effects of zebularine (administered orally or intraperitoneally) on growth of EJ6 human bladder carcinoma cells grown in BALB/c nu/nu mice (five mice per group) and the in vivo reactivation of a methylated p16 gene in these cells. All statistical tests were two-sided.RESULTSIn N. crassa, zebularine inhibited DNA methylation and reactivated a gene previously silenced by methylation. Zebularine induced the myogenic phenotype in 10T1/2 cells, which is a phenomenon unique to DNA methylation inhibitors. Zebularine reactivated a silenced p16 gene and demethylated its promoter region in T24 bladder carcinoma cells in vitro and in tumors grown in mice. Zebularine was only slightly cytotoxic to T24 cells in vitro (1 mM zebularine for 48 hours decreased plating efficiency by 17% [95% confidence interval (CI) = 12.8% to 21.2%]) and to tumor-bearing mice (average maximal weight change in mice treated with 1000 mg/kg zebularine = 11% [95% CI = 4% to 19%]). Compared with those in control mice, tumor volumes were statistically significantly reduced in mice treated with high-dose zebularine administered by intraperitoneal injection (P<.001) or by oral gavage (P<.001).CONCLUSIONSZebularine is a stable DNA demethylating agent and the first drug in its class able to reactivate an epigenetically silenced gene by oral administration.

Although protein kinase D (PKD), like protein kinase C (PKC), possesses a C1 domain that binds phorbol esters and diacylglycerol, the structural differences from PKC within this and other domains of PKD imply differential regulation by lipids and ligands. We characterized the phorbol ester and phospholipid binding properties of a glutathione S-transferase-tagged full-length PKD and compared them with those of PKC-alpha and -delta. We found that PKD is a high-affinity phorbol ester receptor for a range of structurally and functionally divergent phorbol esters and analogs and showed both similarities and differences in structure-activity relations compared with the PKCs examined. In particular, PKD had lower affinity than PKC for certain diacylglycerol analogs, which might be caused by a lysine residue at the 22 position of the PKD-C1b domain in place of the tryptophan residue at this position conserved in the PKCs. The membrane-targeting domains in PKD are largely different from those in PKC; among these differences, PKD contains a pleckstrin homology (PH) domain that is absent in PKC. However, phosphatidylinositol-4,5-bisphosphate PIP2, a lipid ligand for some PH domains, reconstitutes phorbol 12,13-dibutyrate (PDBu) binding to PKD similarly as it does to PKC-alpha and -delta, implying that the PH domain in PKD may not preferentially interact with PIP2. Overall, the requirement of anionic phospholipids for the reconstitution of [3H]PDBu binding to PKD was intermediate between those of PKC-alpha and -delta. We conclude that PKD is a high-affinity phorbol ester receptor; its lipid requirements for ligand binding are approximately comparable with those of PKC but may be differentially regulated in cells through the binding of diacylglycerol to the C1 domain.

The structures of beta-methylenethiazole-4-carboxamide adenine dinucleotide (TAD), NAD(+), and NADH as bound to ecto-ADP-ribosyltransferase 2.2 from rat and to its mutants E189I and E189A, respectively, have been established. The positions and conformations of NAD(+) and its analogues agree in general with those in other ADP-ribosyltransferases. The kinetic constants for NAD(+) hydrolysis were determined by RP-HPLC. The specific activity amounts to 26 units/mg, which is 6000-fold higher than a previously reported rate and 500-fold higher than the hydrolysis rates of other ADP-ribosyltransferases, confirming that hydrolysis is the major function of this enzyme. On the basis of structures and mutant activities, a catalytic mechanism is proposed. The known auto-ADP-ribosylation of the enzyme at the suggested position R184 is supported by one of the crystal structures where the nucleophile position is occupied by an Neta atom of this arginine which in turn is backed up by the base E159.

BACKGROUND:The cyclooxygenase-2 (COX-2) inhibitor celecoxib is thought to act as a chemopreventive agent by sensitizing cancer cells to apoptotic signals. Other COX-2 inhibitors, such as rofecoxib, are two orders of magnitude less potent than celecoxib at inducing apoptosis. The molecular structures of celecoxib and rofecoxib were used as starting points to examine the structural features that contribute to this discrepancy.METHODS:We used a systematic chemical approach to modify the structures of celecoxib and rofecoxib to produce a series of compounds that were tested for their effects on the viability of human prostate cancer PC-3 cells and their ability to induce apoptosis in these cells. Cell viability was measured by the trypan blue dye exclusion assay, and apoptosis was measured by an enzyme-linked immunosorbent assay that quantifies DNA cleavage and by western blot detection of poly(ADP-ribose) polymerase (PARP) cleavage. Western blotting was used to monitor the effects of the compounds on phosphorylation of the serine/threonine kinase Akt and extracellular signal-regulated kinase 2 (ERK2), two components of celecoxib-induced apoptosis signaling. Monte Carlo simulations were used to molecularly model the surface electrostatic potential and electron density of selected compounds. All statistical tests were two-sided.RESULTS:The structural requirements for the induction of apoptosis in PC-3 cells were different from those for COX-2 inhibition. Structure-function analysis indicated that the induction of apoptosis by compounds derived from COX-2 inhibitors required a bulky terminal phenyl ring, a heterocyclic system with negative electrostatic potential, and a benzenesulfonamide or benzenecarboxamide moiety. These derivatives mediated apoptosis by facilitating the dephosphorylation of Akt and ERK2, irrespective of their COX-2 inhibitory activities.CONCLUSION:A new class of compounds that induce apoptosis by targeting Akt and ERK2 signaling pathways in human prostate cancer cells can be synthesized by modifying existing COX-2 inhibitors.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTThe decomposition of 1-.beta.-(D-ribofuranosyl)-1,2-dihydropyrimidin-2-one (zebularine) in alkali: mechanism and products [Erratum to document cited in CA116(5):41960v]Joseph J. Barchi Jr., Steven Musser, and Victor E. MarquezCite this: J. Org. Chem. 1992, 57, 21, 5802Publication Date (Print):October 1, 1992Publication History Published online1 May 2002Published inissue 1 October 1992https://doi.org/10.1021/jo00047a052RIGHTS & PERMISSIONSArticle Views38Altmetric-Citations-LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (129 KB) Get e-Alerts Get e-Alerts

A convenient route to the anti-HIV active compound, 9-(2,3-dideoxy-2-fluoro-β-D-threo-pentofuranosyl)adenine (1, β-FddA) started with the facile introduction of fluorine at C2′ from the α-side of protected 9-(β-D-arabinofuranosyl)adenine (ara-A). Inversion of the stereochemistry at C2′ was accomplished via a stable vinyl intermediate (6), which underwent stereoselective reduction of the double bond to give the desired 2′-F-threo isomer with the opposite β-fluoro stereochemistry.