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1.
J Nat Prod ; 87(2): 415-423, 2024 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-38291771

RESUMO

Pyrrole-containing natural products form a large group of structurally diverse compounds that occur in both terrestrial and marine organisms. In the present study the formation of trideuteromethylated artifacts of pyrrole-containing natural products was investigated, focusing on the discorhabdins. Three deuterated discorhabdins, 1, 3, and 5, were identified to be isolation procedure artifacts caused by the presence of DMSO-d6 during NMR sample preparation and handling. Three additional semisynthetic derivatives, 7-9, were made during the investigation of the mechanism of formation, which was shown to be driven by trideuteromethyl radicals in the presence of water, methanol, TFA, and traces of iron in the deuterated solvent. Generation of trideuteromethylated artifacts was also confirmed for other classes of pyrrole-containing metabolites, namely, makaluvamines, tambjamines, and dibromotryptamines, which had also been dissolved in DMSO-d6 during the structure elucidation process. Semisynthetic discorhabdins were assessed for antiproliferative activity against a panel of human tumor cell lines, and 14-trideuteromethyldiscorhabdin L (3) averaged low micromolar potency.


Assuntos
Produtos Biológicos , Dimetil Sulfóxido , Humanos , Dimetil Sulfóxido/química , Pirróis/química , Produtos Biológicos/farmacologia , Artefatos , Solventes/química
2.
J Am Chem Soc ; 142(50): 21178-21188, 2020 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-33263997

RESUMO

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a molecular target for the sensitization of cancer cells to the FDA-approved topoisomerase inhibitors topotecan and irinotecan. High-throughput screening of natural product extract and fraction libraries for inhibitors of TDP1 activity resulted in the discovery of a new class of knotted cyclic peptides from the marine sponge Axinella sp. Bioassay-guided fractionation of the source extract resulted in the isolation of the active component which was determined to be an unprecedented 42-residue cysteine-rich peptide named recifin A. The native NMR structure revealed a novel fold comprising a four strand antiparallel ß-sheet and two helical turns stabilized by a complex disulfide bond network that creates an embedded ring around one of the strands. The resulting structure, which we have termed the Tyr-lock peptide family, is stabilized by a tyrosine residue locked into three-dimensional space. Recifin A inhibited the cleavage of phosphodiester bonds by TDP1 in a FRET assay with an IC50 of 190 nM. Enzyme kinetics studies revealed that recifin A can specifically modulate the enzymatic activity of full-length TDP1 while not affecting the activity of a truncated catalytic domain of TDP1 lacking the N-terminal regulatory domain (Δ1-147), suggesting an allosteric binding site for recifin A on the regulatory domain of TDP1. Recifin A represents both the first of a unique structural class of knotted disulfide-rich peptides and defines a previously unseen mechanism of TDP1 inhibition that could be productively exploited for potential anticancer applications.


Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Peptídeos/química , Peptídeos/farmacologia , Diester Fosfórico Hidrolases/metabolismo , Tirosina , Regulação Alostérica/efeitos dos fármacos , Sequência de Aminoácidos , Domínio Catalítico , Dissulfetos/química , Ensaios de Triagem em Larga Escala , Diester Fosfórico Hidrolases/química
3.
Small ; 16(38): e2002791, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32812339

RESUMO

Combination therapies that target multiple pathways involved in immune rejection of transplants hold promise for patients in need of restorative surgery. Herein, a noninteracting multiphase molecular assembly approach is developed to crystallize tofacitinib, a potent JAK1/3 inhibitor, within a shear-thinning self-assembled fibrillar peptide hydrogel network. The resulting microcrystalline tofacitinib hydrogel (MTH) can be syringe-injected directly to the grafting site during surgery to locally deliver the small molecule. The rate of drug delivered from MTH is largely controlled by the dissolution of the encapsulated microcrystals. A single application of MTH, in combination with systemically delivered CTLA4-Ig, a co-stimulation inhibitor, affords significant graft survival in mice receiving heterotopic heart transplants. Locoregional studies indicate that the local delivery of tofacitinib at the graft site enabled by MTH is required for the observed enhanced graft survival.


Assuntos
Transplante de Coração , Hidrogéis , Animais , Humanos , Imunomodulação , Camundongos , Peptídeos
4.
Bioorg Med Chem ; 25(12): 2971-2980, 2017 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-28392275

RESUMO

C1 domain-containing proteins, such as protein kinase C (PKC), have a central role in cellular signal transduction. Their involvement in many diseases, including cancer, cardiovascular disease, and immunological and neurological disorders has been extensively demonstrated and has prompted a search for small molecules to modulate their activity. By employing a diacylglycerol (DAG)-lactone template, we have been able to develop ultra potent analogs of diacylglycerol with nanomolar binding affinities approaching those of complex natural products such as phorbol esters and bryostatins. One current challenge is the development of selective ligands capable of discriminating between different protein family members. Recently, structure-activity relationship studies have shown that the introduction of an indole ring as a DAG-lactone substituent yielded selective Ras guanine nucleotide-releasing protein (RasGRP1) activators when compared to PKCα and PKCε. In the present work, we examine the effects of ligand selectivity relative to the orientation of the indole ring and the nature of the DAG-lactone template itself. Our results show that the indole ring must be attached to the lactone moiety through the sn-2 position in order to achieve RasGRP1 selectivity.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Indóis/química , Indóis/farmacologia , Lactonas/química , Lactonas/farmacologia , Proteína Quinase C-alfa/metabolismo , Proteína Quinase C-épsilon/metabolismo , Proteínas de Ligação a DNA/química , Fatores de Troca do Nucleotídeo Guanina/química , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Domínios Proteicos , Proteína Quinase C-alfa/química , Proteína Quinase C-épsilon/química , Relação Estrutura-Atividade
5.
J Chem Inf Model ; 56(11): 2149-2161, 2016 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-27669079

RESUMO

We investigated how many cases of the same chemical sold as different products (at possibly different prices) occurred in a prototypical large aggregated database and simultaneously tested the tautomerism definitions in the chemoinformatics toolkit CACTVS. We applied the standard CACTVS tautomeric transforms plus a set of recently developed ring-chain transforms to the Aldrich Market Select (AMS) database of 6 million screening samples and building blocks. In 30 000 cases, two or more AMS products were found to be just different tautomeric forms of the same compound. We purchased and analyzed 166 such tautomer pairs and triplets by 1H and 13C NMR to determine whether the CACTVS transforms accurately predicted what is the same "stuff in the bottle". Essentially all prototropic transforms with examples in the AMS were confirmed. Some of the ring-chain transforms were found to be too "aggressive", i.e. to equate structures with one another that were different compounds.


Assuntos
Bases de Dados Factuais , Informática/métodos , Compostos Orgânicos/química , Bases de Dados Factuais/economia , Isomerismo
6.
Biopolymers ; 104(6): 663-73, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26152807

RESUMO

Our recently discovered, selective, on-resin route to N(τ)-alkylated imidazolium-containing histidine residues affords new strategies for peptide mimetic design. In this, we demonstrate the use of this chemistry to prepare a series of macrocyclic phosphopeptides, in which imidazolium groups serve as ring-forming junctions. Interestingly, these cationic moieties subsequently serve to charge-mask the phosphoamino acid group that directed their formation. Neighbor-directed histidine N(τ)-alkylation opens the door to new families of phosphopeptidomimetics for use in a range of chemical biology contexts.


Assuntos
Histidina/química , Imidazóis/química , Compostos Macrocíclicos/química , Fosfopeptídeos/química , Alquilação , Cristalografia por Raios X
7.
Biopolymers ; 102(6): 444-55, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25283071

RESUMO

Binding of polo-like kinase 1 (Plk1) polo-box domains (PBDs) to phosphothreonine (pThr)/phosphoserine (pSer)-containing sequences is critical for the proper function of Plk1. Although high-affinity synthetic pThr-containing peptides provide starting points for developing PBD-directed inhibitors, to date the efficacy of such peptides in whole cell assays has been poor. This potentially reflects limited cell membrane permeability arising, in part, from the di-anionic nature of the phosphoryl group or its mimetics. In our current article we report the unanticipated on-resin N(τ)-alkylation of histidine residues already bearing a N(π)- alkyl group. This resulted in cationic imidazolium-containing pThr peptides, several of which exhibit single-digit nanomolar PBD-binding affinities in extracellular assays and improved antimitotic efficacies in intact cells. We enhanced the cellular efficacies of these peptides further by applying bio-reversible pivaloyloxymethyl (POM) phosphoryl protection. New structural insights presented in our current study, including the potential utility of intramolecular charge masking, may be useful for the further development of PBD-binding peptides and peptide mimetics.


Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Histidina/metabolismo , Fosfopeptídeos/síntese química , Fosfopeptídeos/farmacologia , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Alquilação , Ânions , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida , Cristalização , Estabilidade Enzimática/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Esterases/metabolismo , Polarização de Fluorescência , Células HeLa , Histidina/química , Humanos , Fosfopeptídeos/química , Pró-Fármacos/farmacologia , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Espectrometria de Massas em Tandem , Quinase 1 Polo-Like
8.
Bioorg Med Chem ; 22(12): 3123-40, 2014 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-24794745

RESUMO

The development of selective agents capable of discriminating between protein kinase C (PKC) isoforms and other diacylglycerol (DAG)-responsive C1 domain-containing proteins represents an important challenge. Recent studies have highlighted the role that Ras guanine nucleotide-releasing protein (RasGRP) isoforms play both in immune responses as well as in the development of prostate cancer and melanoma, suggesting that the discovery of selective ligands could have potential therapeutic value. Thus far, the N-methyl-substituted indololactone 1 is the agonist with the highest reported potency and selectivity for RasGRP relative to PKC. Here we present the synthesis, binding studies, cellular assays and biophysical analysis of interactions with model membranes of a family of regioisomers of 1 (compounds 2-5) that differ in the position of the linkage between the indole ring and the lactone moiety. These structural variations were studied to explore the interaction of the active complex (C1 domain-ligand) with cellular membranes, which is believed to be an important factor for selectivity in the activation of DAG-responsive C1 domain containing signaling proteins. All compounds were potent and selective activators of RasGRP when compared to PKCα with selectivities ranging from 6 to 65 fold. However, the parent compound 1 was appreciably more selective than any of the other isomers. In intact cells, modest differences in the patterns of translocation of the C1 domain targets were observed. Biophysical studies using giant vesicles as model membranes did show substantial differences in terms of molecular interactions impacting lipid organization, dynamics and membrane insertion. However, these differences did not yield correspondingly large changes in patterns of biological response, at least for the parameters examined.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Diglicerídeos/farmacologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Indóis/farmacologia , Lactonas/farmacologia , Neoplasias da Próstata/patologia , Proteína Quinase C/metabolismo , Animais , Células CHO , Membrana Celular/metabolismo , Células Cultivadas , Cricetulus , Diglicerídeos/química , Polarização de Fluorescência , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Indóis/química , Lactonas/química , Masculino , Modelos Moleculares , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Isoformas de Proteínas
9.
Chem Biodivers ; 11(5): 784-91, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24827688

RESUMO

The design and efficient synthesis of N-Fmoc-phosphothreonine protected by a mono-(pivaloyloxy)methyl (POM) moiety at its phosphoryl group (Fmoc-Thr[PO(OH)(OPOM)]-OH, 1, is reported. This reagent is suitable for solid-phase syntheses employing acid-labile resins and Fmoc-based protocols. It allows the preparation of phosphothreonine (pThr)-containing peptides bearing bis-POM-phosphoryl protection. The methodology allows the first reported synthesis of pThr-containing polypeptides having bioreversible prodrug protection, and as such it should be useful in a variety of biological applications.


Assuntos
Aminoácidos/química , Fluorenos/química , Peptídeos/química , Fosfotreonina/química , Pró-Fármacos/química , Aminoácidos/síntese química , Desenho de Fármacos , Fluorenos/síntese química , Peptídeos/síntese química , Fosfotreonina/síntese química , Pró-Fármacos/síntese química
10.
J Org Chem ; 76(6): 1513-20, 2011 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-21291260

RESUMO

L-γ-Carboxyglutamic acid (Gla) is an uncommon amino acid that binds avidly to mineral surfaces and metal ions. Herein, we report the synthesis of N-α-Fmoc-L-γ-carboxyglutamic acid γ,γ'-tert-butyl ester (Fmoc-Gla(O(t)Bu)(2)-OH), a suitably protected analogue for Fmoc-based solid-phase peptide synthesis. The residue was synthesized using a novel chiral Cu(II) complex, whose structure-based design was inspired by the blue copper protein rusticyanin. The five-coordinate complex is formed by Shiff base formation between glycine and the novel ligand (S)-2-(N-(2-methylthio)benzylprolyl)aminobenzophenone in the presence of copper. Michael addition of di-tert-butyl methylenemalonate to the α-carbon of the glycine portion of the complex occurs in a diastereoselective fashion. The resulting (S,S)-complex diastereomer can be easily purified by chromatography. Metal complex decomposition followed by Fmoc protection affords the enantiomerically pure amino acid. With the use of this novel chiral complex, the asymmetric synthesis of Fmoc-Gla(O(t)Bu)(2)-OH was completed in nine steps from thiosalicylic acid in 14.5% overall yield.


Assuntos
Cobre/química , Fluorenos/síntese química , Ácido Glutâmico/síntese química , Compostos Organometálicos/química , Fluorenos/química , Ácido Glutâmico/química , Ligantes , Modelos Moleculares , Conformação Molecular , Bases de Schiff/química , Estereoisomerismo , Especificidade por Substrato
11.
Nat Commun ; 12(1): 5856, 2021 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-34615874

RESUMO

The role of metabolite-responsive riboswitches in regulating gene expression in bacteria is well known and makes them useful systems for the study of RNA-small molecule interactions. Here, we study the PreQ1 riboswitch system, assessing sixteen diverse PreQ1-derived probes for their ability to selectively modify the class-I PreQ1 riboswitch aptamer covalently. For the most active probe (11), a diazirine-based photocrosslinking analog of PreQ1, X-ray crystallography and gel-based competition assays demonstrated the mode of binding of the ligand to the aptamer, and functional assays demonstrated that the probe retains activity against the full riboswitch. Transcriptome-wide mapping using Chem-CLIP revealed a highly selective interaction between the bacterial aptamer and the probe. In addition, a small number of RNA targets in endogenous human transcripts were found to bind specifically to 11, providing evidence for candidate PreQ1 aptamers in human RNA. This work demonstrates a stark influence of linker chemistry and structure on the ability of molecules to crosslink RNA, reveals that the PreQ1 aptamer/ligand pair are broadly useful for chemical biology applications, and provides insights into how PreQ1, which is similar in structure to guanine, interacts with human RNAs.


Assuntos
Pirimidinonas/metabolismo , Pirróis/metabolismo , Transcriptoma , Sítios de Ligação , Cristalografia por Raios X , Humanos , Ligantes , Pirimidinonas/química , Pirróis/química , RNA Bacteriano/genética , Riboswitch
12.
Bioorg Med Chem Lett ; 20(13): 3848-50, 2010 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-20627559

RESUMO

Five new naphthopyrones (1-5) along with the known compounds TMC-256A1, 5,8-dihydroxy-6-methoxy-2-propyl-4H-naphtho[2,3-b]pyran-4-one, TMC-256C1, comaparvin, 6-methoxycomaparvin, and 6-methoxycomaparvin 5-methyl ether (6-11) were isolated from crinoids of the family Comasteridae. All compounds were tested for their ability to inhibit the multidrug transporter ABCG2, which plays a role in drug resistance. Six of the seven angular naphthopyrones showed moderate activity with <60% inhibition of ABCG2-mediated transport as compared to the positive control fumitremorgin C. None of the linear naphthopyrones inhibited ABCG2-mediated efflux.


Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Equinodermos/química , Naftalenos/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Pironas/farmacologia , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Conformação Molecular , Naftalenos/química , Naftalenos/isolamento & purificação , Proteínas de Neoplasias/metabolismo , Pironas/química , Pironas/isolamento & purificação , Estereoisomerismo , Relação Estrutura-Atividade
13.
Tetrahedron ; 66(34): 6707-6717, 2010 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-21052524

RESUMO

Steric and electronic parameters such as the anomeric effect (AE) and gauche effect play significant roles in steering the North ⇆ South equilibrium of nucleosides in solution. Two isomeric oxa-bicyclo[3.1.0]hexane nucleosides that are conformationally locked in either the North or the South conformation of the pseudorotational cycle were designed to study the consequences of having the AE operational or not, independent of other parameters. The rigidity of the system allowed the orientation of the orbitals involved to be set in "fixed" relationships, either antiperiplanar where the AE is permanently "on", or gauche where the AE is impaired. The consequences of these two alternatives were subject to high-level calculations and measured experimentally by x-ray crystallography, hydrolytic stability of the glycosyl bond, and pKa values.

14.
Bioorg Med Chem ; 17(2): 548-52, 2009 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-19101152

RESUMO

Copper-64 (t(1/2)=12.7h; beta(+): 0.653 MeV, 17.4%; beta(-): 0.578 MeV, 39%) is produced in a biomedical cyclotron and has applications in both imaging and therapy. Macrocyclic chelators are widely used as bifunctional chelators to bind copper radionuclides to antibodies and peptides owing to their relatively high kinetic stability. A novel side-bridged cyclam featuring both pendant acetate and phosphonate groups was synthesized using a Kabachnik-Fields approach followed by hydrobromic acid deprotection. The Cu(II) complex of the novel ligand was synthesized, radiolabeling with (64)Cu was demonstrated, and in vitro (serum) stability was performed. In addition, in vivo distribution and clearance of the (64)Cu-labeled complex was visualized by positron emission tomography (PET) imaging. This novel chelate may be useful in (64)Cu-mediated diagnostic positron emission tomography (PET) imaging as well as targeted radiotherapeutic applications.


Assuntos
Radioisótopos de Cobre , Compostos Heterocíclicos/síntese química , Acetatos , Animais , Quelantes , Ligantes , Camundongos , Organofosfonatos , Tomografia por Emissão de Pósitrons , Relação Estrutura-Atividade
15.
Bioconjug Chem ; 19(7): 1476-84, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18597510

RESUMO

The increased use of copper radioisotopes in radiopharmaceutical applications has created a need for bifunctional chelators (BFCs) that form stable radiocopper complexes and allow covalent attachment to biological molecules. Previous studies have established that 4,11-bis-(carbo- tert-butoxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (H 2CB-TE2A), a member of the ethylene "cross-bridged" cyclam (CB-cyclam) class of bicyclic tetraaza macrocycles, forms highly kinetically stable complexes with Cu(II) and is less susceptible to in vivo transchelation than its nonbridged analogue, 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA). Herein, we report a convenient synthesis of a novel cross-bridged BFC that is structurally analogous to CB-TE2A in that it possesses two coordinating acetate arms, but in addition possesses a third orthogonally protected arm for conjugation to peptides and other targeting agents. Application of this strategy to cross-bridged chelators may also enable the development of even further improved agents for (64)Cu-mediated diagnostic positron emission tomography (PET) imaging as well as for targeted radiotherapeutic applications.


Assuntos
Quelantes/síntese química , Reagentes de Ligações Cruzadas/síntese química , Lactamas Macrocíclicas/síntese química , Peptídeos/química , Coloração e Rotulagem/métodos , Aminas/química , Quelantes/química , Radioisótopos de Cobre , Reagentes de Ligações Cruzadas/química , Lactamas Macrocíclicas/química , Ligantes , Peptídeos/uso terapêutico
16.
Chem Sci ; 9(2): 307-314, 2018 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-29619201

RESUMO

Ambiguities and errors in the structural assignment of organic molecules hinder both drug discovery and total synthesis efforts. Newly described NMR experimental approaches can provide valuable structural details and a complementary means of structure verification. The caulamidines are trihalogenated alkaloids from a marine bryozoan with an unprecedented structural scaffold. Their unique carbon and nitrogen framework was deduced by conventional NMR methods supplemented by new experiments that define 2-bond heteronuclear connectivities, reveal very long-range connectivity data, or visualize the 35,37Cl isotopic effect on chlorinated carbons. Computer-assisted structural elucidation (CASE) analysis of the spectroscopic data for caulamidine A provided only one viable structural alternative. Anisotropic NMR parameters, specifically residual dipolar coupling and residual chemical shift anisotropy data, were measured for caulamidine A and compared to DFT-calculated values for the proposed structure, the CASE-derived alternative structure, and two energetically feasible stereoisomers. Anisotropy-based NMR experiments provide a global, orthogonal means to verify complex structures free from investigator bias. The anisotropic NMR data were fully consistent with the assigned structure and configuration of caulamidine A. Caulamidine B has the same heterocyclic scaffold as A but a different composition and pattern of halogen substitution. Caulamidines A and B inhibited both wild-type and drug-resistant strains of the malaria parasite Plasmodium falciparum at low micromolar concentrations, yet were nontoxic to human cells.

17.
J Med Chem ; 50(5): 962-78, 2007 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-17284021

RESUMO

Highly rigid and geometrically well-defined rods composed of ethynylene-substituted aromatic spacers [oligo(p-phenyleneethynylene), OPE] were incorporated as acyl moieties on diacylglycerol lactones (DAG-lactones) and investigated for their ability to bind to protein kinase C (PKC) and translocate PKC alpha and delta isoforms to plasma and internal membranes. The kinetics of PKC translocation were correlated with biological responses, viz. ERK phosphorylation, induction of IL-6 secretion, inhibition of cell proliferation, and induction of cellular attachment, that display very different time courses. Because OPE rods assemble through noncovalent forces and form stable films, they may influence the microdomain environment around the DAG-lactone membrane-binding site. A comparison of two DAG-lactones (1 and 10), one with two PE units (1) and the other with an equivalent flexible acyl chain (10) of matching lipophilicity, clearly demonstrated the effect of the rigid OPE chain in substantially prolonging the translocated state of both PKC alpha and delta.


Assuntos
Membrana Celular/metabolismo , Diglicerídeos/síntese química , Lactonas/síntese química , Proteína Quinase C-alfa/metabolismo , Proteína Quinase C/metabolismo , Animais , Sítios de Ligação , Adesão Celular , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Cricetinae , Cricetulus , Diglicerídeos/química , Diglicerídeos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Interleucina-6/metabolismo , Isoenzimas/metabolismo , Cinética , Lactonas/química , Lactonas/farmacologia , Ligantes , Conformação Molecular , Fosforilação , Ligação Proteica , Proteína Quinase C beta , Transporte Proteico , Relação Estrutura-Atividade
18.
Cancer Res ; 65(11): 4673-82, 2005 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-15930285

RESUMO

A strong association exists between states of chronic inflammation and cancer, and it is believed that mediators of inflammation may be responsible for this phenomenon. Interleukin 6 (IL-6) is an inflammatory cytokine known to play a role in the growth and survival of many types of tumors, yet the mechanisms employed by this pleomorphic cytokine to accomplish this feat are still poorly understood. Another important factor in tumor development seems to be the hypermethylation of CpG islands located within the promoter regions of tumor suppressor genes. This common epigenetic alteration enables tumor cells to reduce or inactivate the expression of important tumor suppressor and cell cycle regulatory genes. Here we show that in the IL-6-responsive human multiple myeloma cell line KAS 6/1, the promoter region of p53 is epigenetically modified by methyltransferases, resulting in decreased levels of expression. Furthermore, cells treated with IL-6 exhibit an increase in the expression of the DNA maintenance methylation enzyme, DNMT-1. The DNA methyltransferase inhibitor zebularine reverses the methylation of the p53 promoter, allowing the resumption of its expression. However, when zebularine is withdrawn from the cells, the reestablishment of the original CpG island methylation within the p53 promoter does not occur in the absence of IL-6, and cells which do not receive IL-6 eventually die, as p53 expression continues unchecked by remethylation. Interestingly, this loss of viability seems to involve not the withdrawal of cytokine, but the inability of the cell to resilence the promoter. Consistent with this model, when cells that express IL-6 in an autocrine fashion are subjected to identical treatment, p53 expression is reduced shortly after withdrawal of zebularine. Therefore, it seems IL-6 is capable of maintaining promoter methylation thus representing one of the possible mechanisms used by inflammatory mediators in the growth and survival of tumors.


Assuntos
Azacitidina/análogos & derivados , Citidina/análogos & derivados , Desoxicitidina/análogos & derivados , Genes p53/fisiologia , Interleucina-6/fisiologia , Apoptose/genética , Azacitidina/farmacologia , Sequência de Bases , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Citidina/farmacologia , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/biossíntese , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/efeitos dos fármacos , Decitabina , Desoxicitidina/metabolismo , Inativação Gênica/efeitos dos fármacos , Humanos , Interleucina-6/genética , Interleucina-6/imunologia , Interleucina-6/farmacologia , Dados de Sequência Molecular , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Sulfitos/farmacologia , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/genética
19.
Cancer Res ; 64(10): 3465-73, 2004 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-15150099

RESUMO

The epigenetic control of gene transcription in cancer has been the theme of many recent studies and therapeutic approaches. Carcinogenesis is frequently associated with hypermethylation and consequent down-regulation of genes that prevent cancer, e.g., those that control cell proliferation and apoptosis. We used the demethylating drug zebularine to induce changes in DNA methylation, then examined patterns of gene expression using cDNA array analysis and Restriction Landmark Genomic Scanning followed by RNase protection assay and reverse transcription-PCR to confirm the results. Microarray studies revealed that many genes were epigenetically regulated by methylation. We concluded that methylation decreased the expression of, or silenced, several genes, contributing to the growth and survival of multiple myeloma cells. For example, a number of genes (BAD, BAK, BIK, and BAX) involved in apoptosis were found to be suppressed by methylation. Sequenced methylation-regulated DNA fragments identified by Restriction Landmark Genomic Scanning were found to contain CpG islands, and some corresponded to promoters of genes that were regulated by methylation. We also observed that after the removal of the demethylating drug, the addition of interleukin 6 restored CpG methylation and re-established previously silenced gene patterns, thus implicating a novel role of interleukin 6 in processes regulating epigenetic gene repression and carcinogenesis.


Assuntos
Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Mieloma Múltiplo/genética , Linhagem Celular Tumoral , Citidina/análogos & derivados , Metilação de DNA/efeitos dos fármacos , Genoma Humano , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Nucleosídeos de Pirimidina/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleases/metabolismo
20.
Biochem Pharmacol ; 70(1): 121-33, 2005 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-15885659

RESUMO

Zebularine (2(1H)-pyrimidinone riboside, Zeb), a synthetic analogue of cytidine that is a potent inhibitor of cytidine deaminase, has been recently identified as a general inhibitor of DNA methylation. This inhibition of DNA methyltransferase (DNMT) is hypothesized to be mechanism-based and result from formation of a covalent complex between the enzyme and zebularine-substituted DNA. Metabolic activation of Zeb thus requires that it be phosphorylated and incorporated into DNA. We have quantitatively assessed the phosphorylation and DNA incorporation of Zeb in T24 cells using 2-[(14)C]-Zeb in conjunction with gradient anion-exchange HPLC and selected enzymatic and spectroscopic analyses. The corresponding 5'-mono-, di- and triphosphates of Zeb were readily formed in a dose- and time-dependent manner. Two additional Zeb-containing metabolites were tentatively identified as diphosphocholine (Zeb-DP-Chol) and diphosphoethanolamine adducts. Intracellular concentrations of Zeb-TP and Zeb-DP-Chol were similar and greatly exceeded those of other metabolites. DNA incorporation occurred but was surpassed by that of RNA by at least seven-fold. Equivalent levels and similar intracellular metabolic patterns were also observed in the Molt-4 (human T-lymphoblasts) and MC38 (murine colon carcinoma) cell lines. For male BALB/c nu/nu mice implanted s.c. with the EJ6 variant of T24 bladder carcinoma and treated i.p. with 500mg/kg 2-[(14)C]-Zeb, the in vivo phosphorylation pattern of Zeb in tumor tissue examined 24h after drug administration was similar to that observed in vitro. The complex metabolism of Zeb and its limited DNA incorporation suggest that these are the reasons why it is less potent than either 5-azacytidine or 5-aza-2'-deoxycytidine and requires higher doses for equivalent inhibition of DNMT.


Assuntos
Citidina/análogos & derivados , Citidina/farmacocinética , Metilação de DNA/efeitos dos fármacos , Inibidores Enzimáticos/farmacocinética , Neoplasias da Bexiga Urinária/metabolismo , Animais , Biotransformação , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Citidina/farmacologia , DNA/metabolismo , Relação Dose-Resposta a Droga , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fosforilação , RNA/metabolismo , Neoplasias da Bexiga Urinária/patologia
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