RESUMO
The interaction of daunorubicin with the self-complementary DNA fragment d(CGTACG) was studied by 31P NMR spectroscopy. The individual phosphates have been assigned for the nucleotide and the complex and signals from bound and free species in slow exchange at 19 degrees C were detected. In solution, the hexanucleotide binds two molecules of daunorubicin, which intercalate in the d(CG) sequence at both ends of the helix. Evidence for local deformations of the backbone at the sites of C5pG6, C1pG2 and G2pT3 phosphates is given. The binding constants for the stepwise equilibrium and the rate of dissociation of the intercalated duplex were also determined.
Assuntos
Adutos de DNA , DNA/metabolismo , Daunorrubicina/metabolismo , Oligodesoxirribonucleotídeos/metabolismo , Fenômenos Químicos , Química , Substâncias Intercalantes/metabolismo , Espectroscopia de Ressonância Magnética , Fosfatos/metabolismo , SoluçõesRESUMO
Among enzymes involved in the synthesis of nucleotides and DNA, some exceptions have recently been found to the universal rule that enzymes act only on one enantiomer of a chiral substrate and that only one of the enantiomeric forms of chiral molecules may bind effectively at the catalytic site, displaying biological activity. The exceptions include: herpes virus thymidine kinases, cellular deoxycytidine kinase and deoxynucloside mono- and diphosphate kinases, cellular and viral DNA polymerases, such as DNA polymerase alpha, terminal transferase and HIV-1 reverse transcriptase. The ability of these enzymes to utilize unnatural L-beta-nucleosides or -nucleotides as substrate may be exploited from chemotherapeutic point of view.
Assuntos
Antivirais/farmacologia , Enzimas/metabolismo , Herpesvirus Humano 1/efeitos dos fármacos , Nucleosídeos/farmacologia , Proteínas Virais/metabolismo , DNA/biossíntese , DNA Polimerase Dirigida por DNA , Desoxirribonucleotídeos/biossíntese , Células HeLa , Herpesvirus Humano 1/enzimologia , Humanos , Núcleosídeo-Fosfato Quinase/química , Núcleosídeo-Fosfato Quinase/metabolismo , Estereoisomerismo , Relação Estrutura-Atividade , Timidina/química , Timidina Quinase/química , Timidina Quinase/metabolismoRESUMO
The multiple drug resistance of neoplastic cells is mediated by overexpression of the human MDR1 gene, which encodes the transmembrane efflux pump P-glycoprotein. In both cell lines and human tumors the MDR phenotype closely correlates with MDR1 mRNA and P-glycoprotein levels. Reversion of the MDR phenotype was attempted in human colorectal adenocarcinoma doxorubicin (Dx)-resistant cells (Lo Vo/Dx) by long-term administration of an equimolecular mixture of three unmodified ODNs (18mer) targeted to adjacent binding sites of the MDR1 mRNA and carried by a synthetic cationic lipid (DOTAP). Three different experimental parameters were used to evaluate the antimessenger agent's effectiveness in comparison with a random sequence ODN: the level of cell resistance to Dx; the level of P-glycoprotein (determined by flow cytometry); the level of MDR1 mRNA (determined by quantitative RT-PCR). Experimental data indicate that the level of both the MDR1 mRNA and the P-glycoprotein is reduced by approximately 50% by treatment of Lo Vo/Dx cells with a 10 microM total concentration of the aODN mixture every 24 h for 15 days. In agreement with these findings, sensitivity to Dx of the antimessenger agent-treated Lo Vo/Dx cells was almost doubled in comparison with random sequence ODN-treated controls.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Doxorrubicina/toxicidade , Resistência a Múltiplos Medicamentos/genética , Expressão Gênica/efeitos dos fármacos , Oligonucleotídeos Antissenso/farmacologia , Adenocarcinoma , Sequência de Bases , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Neoplasias do Colo , Relação Dose-Resposta a Droga , Humanos , Cinética , Dados de Sequência Molecular , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Fatores de Tempo , Células Tumorais CultivadasRESUMO
The effect of the substitution of a L-nucleoside for a D-nucleoside in the duplex d(G-C-G-T-G-C-G).d(C-G-C-A-C-G-C) was studied by UV and NMR spectroscopy. These unnatural oligonucleotides have potential for antisense DNA technology [Damha, M. J., Giannaris, P. A., & Marfey, P. (1994) Biochemistry (preceding paper in this issue)]. The thermal stability of such duplexes is lower than that of the natural one and is dependent on the nucleotide type and/or sequence. Interestingly, inversion of the chirality of thymidine but not adenosine coincides with a large stabilizing enthalpy change. The structure of the heterochiral duplex d(G1-C2-G3-(L)T4-G5-C6-G7).d(C8-G9-C10-A11-C12-G13- C14), where (L)T denotes the mirror image of the natural thymidine, has been determined by NMR spectroscopy. The sugar conformation was determined using the sum of coupling constants and the distances using a model free relaxation matrix approach. The torsion angles of the backbone follow from 3JHH, 3JHP, and 4JHP coupling constants. The structure of the duplex was calculated by metric matrix distance geometry followed by simulated annealing. The structure is close to that of B-DNA. The base pair formed by (L)T and A is of the Watson-Crick type. All sugars adopt an S-type pucker. The incorporation of the L-sugar in the duplex is accomplished by changes in the backbone torsion angles around the phosphates and the glycosidic torsion angle of (L)T. The modification induces changes in the natural strand as well. The structure exhibits an unusual interaction between the aromatic rings of the (L)T4.A11 and G3.C12 base pairs, which provides a plausible explanation of the unusual thermodynamic properties of the duplex.
Assuntos
Oligodesoxirribonucleotídeos/química , Sequência de Bases , Quimera , Desoxirribose/química , Temperatura Alta , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Oligodesoxirribonucleotídeos/síntese química , Soluções , Espectrofotometria Ultravioleta , TermodinâmicaRESUMO
A simple, rapid and high-yielding method for the synthesis of oligonucleotides by the phosphotriesters approach is described. The use of polyethylene glycol (PEG) as soluble polymeric support preserves some convenient features of the solid-phase synthesis with new interesting advantages. Short oligonucleotides in hundred milligrams scale can be obtained from few grams of functionalized PEG.
Assuntos
Oligonucleotídeos/síntese química , Polietilenoglicóis , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Espectroscopia de Ressonância Magnética , Oligonucleotídeos/isolamento & purificação , SolubilidadeRESUMO
Several cyclic oligodeoxynucleotides with different base composition and size have been prepared from 5',3'-unprotected linear precursors, using a bifunctional phosphorylating reagent. The final deprotected oligomers have been characterized by 1H- and 31P-NMR. The present procedure is particularly useful for millimolar scale syntheses.
Assuntos
Nucleotídeos Cíclicos/síntese química , Oligodesoxirribonucleotídeos/síntese química , Sequência de Bases , Espectroscopia de Ressonância Magnética , Métodos , Estrutura Molecular , Nucleotídeos Cíclicos/isolamento & purificação , Oligodesoxirribonucleotídeos/isolamento & purificação , FosforilaçãoRESUMO
The effects of different DNA sequences on the photoreaction of various furocoumarin derivatives was investigated from a quantitative point of view using a number of self-complementary oligonucleotides. These contained 5'-TA and 5'-AT residues, having various flanking sequences. The furocoumarins included classical bifunctional derivatives, such as 8-methoxy- and 5-methoxypsoralen, as well as monofunctional compounds, such as angelicin and benzopsoralen. Taking into an account the thermodynamic constant for noncovalent binding of each psoralen to each DNA sequence, the rate constants for the photobinding process to each fragment were evaluated. The extent of photoreaction is greatly affected by the DNA sequence examined. While sequences of the type 5'-(GTAC)n are quite reactive towards all furocoumarins, 5'-TATA exhibited a reduced rate of photobinding using monofunctional psoralens. In addition terminal 5'-TA groups were the least reactive with 5- and 8-methoxypsoralen, but not with angelicin or benzopsoralen. Also 5'-AT-containing fragments exhibited remarkably variable responses toward monofunctional or bifunctional psoralen derivatives. As a general trend the photoreactivity rate of the former is less sequence-sensitive, the ratio between maximum and minimum being less than 2 for the examined fragments. The same ratio is about 3.4 for 8-methoxypsoralen and 6.2 for 5-methoxypsoralen. This approach, in combination with footprinting studies, appears to be quite useful for a quantitative investigation of the process of covalent binding of psoralens to specific sites in DNA.
Assuntos
DNA/química , DNA/metabolismo , Furocumarinas/metabolismo , Sequência de Bases , Dados de Sequência Molecular , Peso Molecular , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , FotoquímicaRESUMO
Conjugates obtained by linking the anthracycline intercalating chromophore to triple helix forming oligonucleotides (TFOs) have been used in a physicochemical study of the stability of triple helices with DNA sequences of pharmacological relevance. The intercalating moiety is represented by carminomycinone derivatives obtained upon O-demethylation and hydrolysis of the glycosidic linkage of daunomycin followed by the introduction of an alkylating residue at two different positions. Results of experiments with a polypurinic region present in the multidrug resistance (MDR) gene indicate that the stability of the triple helix is significantly enhanced by replacement of C's with (5-Me)C's in the TFO sequences tested. The stability is not changed when a 3'-TpT is present in place of a 3'-CpG at the presumed intercalation site of the anthraquinone chromophore. The same carminomycinone derivatives were used for the preparation of conjugates able to form triple helices with the polypurine tract (PPT) present in the human integrated genome of HIV-1 infected cells. Three different TFOs (T(4)(Me)CT(4)(Me)CC, C2; T(4)(Me)CT(4)(Me)CC(Me)CC(Me)CCT, C6; and T(4)(Me)CT(4)G(6), G6) were designed and linked to the anthraquinone moiety. These conjugates showed a significantly enhanced ability to bind the PPT region of HIV with respect to the nonconjugated TFOs.
Assuntos
Antraciclinas/síntese química , Carrubicina/análogos & derivados , Carrubicina/química , Genes MDR/genética , HIV-1/genética , Oligonucleotídeos/síntese química , Antraciclinas/metabolismo , Sequência de Bases/fisiologia , Sítios de Ligação/fisiologia , Carrubicina/metabolismo , Daunorrubicina/química , Estabilidade de Medicamentos , Genes MDR/fisiologia , HIV-1/química , HIV-1/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Substâncias Intercalantes/síntese química , Substâncias Intercalantes/metabolismo , Oligonucleotídeos/genética , Purinas/química , Purinas/metabolismoRESUMO
An oligoribonucleotide, corresponding to the Tat-interactive top half of the HIV-1 TAR RNA stem-loop, was synthesized in both the natural D- and the enantiomeric L-configurations. The affinity of Tat for the two RNAs, assessed by competition binding experiments, was found to be identical and is reduced 10-fold for both, upon replacement of the critical bulge residue U23 with cytidine. It is suggested that this interaction of the flexible Tat protein depends strongly upon the tertiary structure of a binding pocket within TAR, but not upon its handedness, and may be described by a 'hand-in-mitten' model.
Assuntos
Produtos do Gene tat/metabolismo , HIV-1/metabolismo , RNA Viral/química , Ligação Competitiva , Humanos , Conformação de Ácido Nucleico , Oligorribonucleotídeos/síntese química , Oligorribonucleotídeos/química , Ligação Proteica , Estrutura Terciária de Proteína , Estereoisomerismo , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
L-beta-Deoxythymidine (L-dT), the optical enantiomer of D-beta-deoxythymidine (D-dT), and L-enantiomers of nucleoside analogs, such as 5-iodo-2'-deoxy-L-uridine (L-IdU) and E-5-(2-bromovinyl)-2'-deoxy-L-uridine (L-BVdU), are not recognized in vitro by human cytosolic thymidine kinase (TK), but are phosphorylated by herpes simplex virus type 1 (HSV-1) TK and inhibit HSV-1 proliferation in infected cells. Here we report that: (i) L-dT is selectively phosphorylated in vivo to L-dTMP by HSV-1 TK and L-dTMP is further phosphorylated to the di- and triphosphate forms by non-stereospecific cellular kinases; (ii) L-dTTP not only inhibits HSV-1 DNA polymerase in vitro, but also human DNA polymerase alpha, gamma, delta and epsilon, human immunodeficiency virus reverse transcriptase (HIV-1 RT), Escherichia coli DNA polymerase 1 and calf thymus terminal transferase, although DNA polymerase beta was resistant; (iii) whereas DNA polymerase beta, gamma, delta and epsilon are unable to utilize L-dTTP as a substrate, the other DNA polymerases clearly incorporate at least one L-dTMP residue, with DNA polymerase alpha and HIV-1 RT able to further elongate the DNA chain by catalyzing the formation of the phosphodiester bond between the incorporated L-dTMP and an incoming L-dTTP; (iv) incorporated L-nucleotides at the 3'-OH terminus make DNA more resistant to 3'-->5' exonucleases. In conclusion, our results suggest a possible mechanism for the inhibition of viral proliferation by L-nucleosides.
Assuntos
DNA Nucleotidilexotransferase/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , HIV-1/enzimologia , DNA Polimerase Dirigida por RNA/metabolismo , Nucleotídeos de Timina/metabolismo , Animais , Bovinos , DNA Nucleotidilexotransferase/antagonistas & inibidores , Escherichia coli/enzimologia , Exodesoxirribonuclease V , Exodesoxirribonucleases/metabolismo , Transcriptase Reversa do HIV , Células HeLa/enzimologia , Herpesvirus Humano 1/enzimologia , Humanos , Inibidores da Síntese de Ácido Nucleico , Poli T/metabolismo , Inibidores da Transcriptase Reversa , Estereoisomerismo , Especificidade por Substrato , Timidina/química , Timidina/metabolismo , Timidina Quinase/genética , Timidina Quinase/metabolismo , Timidina Monofosfato/química , Timidina Monofosfato/metabolismo , Nucleotídeos de Timina/química , Nucleotídeos de Timina/farmacologiaRESUMO
An oligodeoxynucleotide (ODN), capable of reducing the growth of human B lymphocytes carrying the t(14;18) chromosome translocation, was prepared in different 'chemical versions': unmodified phosphodiester, phosphorothioate and phosphodiester capped with L-2'-deoxycytidine. Their binding affinity to the complementary synthetic target was studied by the melting point assay. The ODNs, administered to DOHH2 cells, were compared for stability in the culture medium, cellular uptake, time course of the intact sequence concentration within the cell and ability to inhibit cell growth. The 5', 3'-L-capped derivative and the phosphorothioate had comparable potency, superior to that of the unmodified ODN, in agreement with the concentration of undegraded ODNs within the cell.
Assuntos
Antineoplásicos/farmacologia , Oligonucleotídeos/química , Oligonucleotídeos/farmacologia , Antineoplásicos/química , Divisão Celular/efeitos dos fármacos , Densitometria , Estabilidade de Medicamentos , Humanos , Linfoma Folicular/tratamento farmacológico , Linfoma Folicular/patologia , Oligonucleotídeos/metabolismo , Temperatura , Tionucleotídeos/química , Tionucleotídeos/farmacologia , Fatores de Tempo , Células Tumorais Cultivadas , Raios UltravioletaRESUMO
Conjugation of an anthracycline to a triplex-forming oligonucleotide (TFO) allows delivery of this drug to a specific DNA site, preserving the intercalation geometry of this class of anticancer agents. Conjugate 11, in which the TFO is linked via a hexamethylene bridge to the O-4 on the D ring of the anthraquinone moiety, affords the most stable triple helix, through intercalation of the planar chromophore between DNA bases and binding of both the TFO and the amino sugar to the major and the minor groove respectively.
Assuntos
Antibióticos Antineoplásicos/metabolismo , Daunorrubicina/análogos & derivados , Oligonucleotídeos/metabolismo , DNA/metabolismo , Daunorrubicina/metabolismo , Conformação de Ácido Nucleico , Espectrometria de Fluorescência , Espectrofotometria UltravioletaRESUMO
It is known that the Herpes simplex virus type 1 (HSV-1)-encoded thymidine kinase (TK) co-purifies with an associated thymidylate kinase (TMPK) activity and that thymidylate (TMP) inhibits the phosphorylation of thymidine by the HSV-1 TK. Here we demonstrate that: (i) TMP phosphorylation catalysed by the viral TMPK is competitively inhibited by thymidine (TdR) with a Ki equal to its Km as substrate for the viral TK; (ii) L-thymidine (L-TdR), the enantiomer of the naturally occurring D-TdR and a substrate for the HSV-1 TK [Spadari, Maga, Focher, Ciarrocchi, Manservigi, Arcamone, Capobianco, Caruso, Colonna, Iotti and Garbesi (1992) J. Med. Chem. 35, 4214-4220], is a powerful inhibitor of the HSV-1 TMPK activity with a Ki value identical with its Km as a substrate for the viral TK; (iii) both viral TK and TMPK activities are inhibited, in a competitive way and with identical Ki values, by novel, non-substrate inhibitors of HSV-1 TK, N2-phenylguanines; (iv) L-TdR is phosphorylated to L-TMP by the viral TK, but L-TMP is not phosphorylated to L-TDP by the viral TMPK activity; and (v) L-TMP inhibits competitively and with identical potencies the phosphorylation of TdR and TMP catalysed respectively by the HSV-1 TK and TMPK activities. In conclusion, our data demonstrate that both TK and TMPK activities encoded by HSV-1 share a common active site which is very tolerant in accepting modified nucleosides, but cannot readily accommodate modified nucleoside monophosphates.
Assuntos
Guanina/análogos & derivados , Herpesvirus Humano 1/enzimologia , Núcleosídeo-Fosfato Quinase/metabolismo , Timidina Quinase/metabolismo , Timidina/metabolismo , Sítios de Ligação , Ligação Competitiva , Catálise , Guanina/metabolismo , Cinética , Fosforilação , Estereoisomerismo , Especificidade por SubstratoRESUMO
The binding between Distamycin 3 and the palindromic duplexes d(CGTTTAAACG)2 and d(CGTACGTACG)2 was investigated by two independent techniques: UV-Vis absorption in the Job's plot approach and Induced Circular Dichroism. Both decamers bind two molecules of peptide per duplex, with close overall affinities. This result indicates that a row of six A:T base pairs can accommodate two molecules of drug and that the minimal binding site of Distamycin 3 can consist of just two A:T base pairs.
Assuntos
Distamicinas/metabolismo , Oligodesoxirribonucleotídeos/metabolismo , Sequência de Bases , Dicroísmo Circular , Dados de Sequência Molecular , Ácidos Nucleicos Heteroduplexes , Oligodesoxirribonucleotídeos/química , Espectrofotometria UltravioletaRESUMO
We have partially purified suid pseudorabies virus (PRV) thymidine kinase from infected thymidine kinase- mouse cells, and cytosolic swine thymidine kinase from lymphatic glands, and we have found that PRV thymidine kinase, unlike the host enzyme, shows no stereospecificity for D- and L-beta-nucleosides. In vitro, unnatural L-enantiomers, except L-deoxycytidine, function as specific inhibitors for the viral enzyme in the order: L-thymidine >> L-deoxyguanosine > L-deoxyuridine > L-deoxyadenosine. Contrary to human and swine thymidine kinases and like herpes simplex virus-1 and -2 thymidine kinases, PRV thymidine kinase phosphorylates both the natural (D-) and the unnatural (L-) thymidine enantiomers to their corresponding monophosphates with comparable efficiency. The kinetic parameters Vmax/Km for D- and L-thymidine are 3.7 and 2.3 respectively. Our results demonstrate that the lack of stereospecificity might be a common feature of the thymidine kinases that are encoded by human and animal herpes viruses. These observations could lead to the development of a novel class of antiviral drugs.
Assuntos
Herpesvirus Suídeo 1/enzimologia , Nucleosídeos/química , Nucleosídeos/metabolismo , Timidina Quinase/metabolismo , Ligação Competitiva , Desoxiadenosinas/farmacologia , Desoxiguanosina/farmacologia , Desoxiuridina/farmacologia , Fosforilação , Estereoisomerismo , Especificidade por Substrato , Timidina/metabolismo , Timidina/farmacologia , Timidina Quinase/antagonistas & inibidores , Timidina Quinase/isolamento & purificaçãoRESUMO
We demonstrate that human 2'-deoxycytidine kinase (dCK) is a nonenantioselective enzyme because it phosphorylates beta-D-2'-deoxycytidine (D-dCyd), the natural substrate, and beta-L-2'-deoxycytidine (L-dCyd), its enantiomer, with the same efficiency. Kinetic studies showed that L-dCyd is a competitive inhibitor of the phosphorylation of D-dCyd with a Kl value of 0.12 microM, which is lower than the K(m) value for D-dCyd (1,2 microM). Chemical modifications of either the base or the pentose ring strongly decrease the inhibitory potency of L-dCyd, L-dCyd is resistant to cytidine deaminase and competes in cell cultures with the natural D-dCyd as substrate for dCK, thus reducing the incorporation of exogenous [3H]dCyd into DNA. L-dCyd had no effect on the pool of dTTP deriving from the salvage or from the de novo synthesis, does not inhibit short term RNA and protein syntheses, and shows little or no cytotoxicity. Our results indicate a catalytic similarity between human dCK and herpetic thymidine kinases, enzymes that also lack stereospecificity. This functional analogy underlines the potential role of dCK as activator of L-deoxycytidine analogs as antiviral and antineoplastic agents and lends support to the hypothesis that herpesvirus thymidine kinase might have evolved from a captured cellular dCK gene, developing the ability to phosphorylate thymidine and retaining that to phosphorylate deoxycytidine.
Assuntos
Antineoplásicos/metabolismo , Antivirais/metabolismo , Desoxicitidina Quinase/metabolismo , Desoxicitidina/metabolismo , Biotransformação , Citidina Desaminase/metabolismo , Desoxicitidina/farmacologia , Células HeLa , Humanos , Cinética , Fosforilação , EstereoisomerismoRESUMO
The equilibrium and kinetic aspects of the interaction between four anthracyclines and two synthetic self-complementary hexanucleotides was investigated by fluorescence detection. Two of the studied anthracyclines are widely used antitumor drugs: doxorubicin (1, formerly adriamycin) and daunorubicin (2, formerly daunomycin). The other two, 9-deoxydoxorubicin (3) and 3'-deamino-3'-hydroxy-4'-epidoxorubicin (4), are doxorubicin analogues with modifications of the chemical groups that have been proposed as responsible for sequence specificity (Chen, K.-X., Gresh, N. and Pullman, B. (1985). J. Biomol. Struct. Dyn. 3, 445-466). One of the oligonucleotides, d(CGTACG), is identical to that used in the high resolution x-ray structure determination of the daunorubicin intercalative complex (Wang, A. H.-J., Ughetto, G., Quigley, G. J. & Rich, A. (1987). Biochemistry 26, 1152-1163). Binding to this hexanucleotide is compared with intercalation into the d(CGCGCG) duplex, revealing sequence preferences of the four anthracyclines. Taking into account the anthracycline aggregation and the dissociation of the hexanucleotide double standard form, results can be interpreted with a model that assumes complete fluorescence quenching at intercalative sites containing the CG base pair, and a large residual fluorescence after intercalation within the TpA fragment. All four anthracyclines show preferential intercalation at sites near the ends of both hexanucleotide duplexes, partly as a result of positive cooperativity in the formation of di-intercalated species at these sites. Within the limits of experimental error, complete site specificity for the CpG fragment is found in the intercalation of 1 and 2 into d(CGTACG) duplex, whereas analogues 3 and 4 give increasing evidence of intercalation at other sites including the fluorescence-preserving TpA fragment. Site specificity is less pronounced in the association with d(CGCGCG), when cooperativity is taken into account. Kinetic data corroborate the results of equilibrium studies and are interpreted with a mechanism that includes formation of an intermediate bound species followed by drug redistribution to preferential sites. Finally, from a comparison of pertinent site binding constants, approximate free energy contributions to sequence specific DNA interaction, due to C9-OH on the aglycone and -NH3+ on daunosamine, are estimated not to exceed 2 kcal/mol.
Assuntos
Antibióticos Antineoplásicos/farmacocinética , DNA/efeitos dos fármacos , Oligonucleotídeos/metabolismo , Sequência de Bases , Sítios de Ligação , Fenômenos Químicos , Química , Daunorrubicina/farmacocinética , Doxorrubicina/farmacocinética , Espectrometria de FluorescênciaRESUMO
Unnatural L-2'-deoxyribonucleosides L-T, L-dC, L-dA and L-dG were prepared from L-arabinose and assembled, by solution or solid phase synthesis, to give L-oligonucleotides (L-DNAs), which contain all four natural bases. The affinity of these modified oligomers for complementary D-ribo- and D-deoxyribo-oligomers was studied with NMR, UV and CD spectroscopies and mobility shift assay on native PAGE. All experimental results indicate that L-DNAs do not, in general, recognize single-stranded, natural DNA and RNA. Hence, contrary to previous suggestions, it is not possible to envisage their use as wide scope antimessenger agents in the selective control of gene expression.
Assuntos
DNA Antissenso/química , DNA/química , Conformação de Ácido Nucleico , Sequência de Bases , Dicroísmo Circular , DNA/metabolismo , DNA Antissenso/metabolismo , Eletroforese em Gel de Poliacrilamida , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , RNA Complementar/metabolismo , Espectrofotometria UltravioletaRESUMO
5-Iodo-2'-deoxy-L-uridine (L-IdU) and (E)-5-(2-bromovinyl)-2'-deoxy-L-uridine (L-BVdU) have been prepared and found to inhibit herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) with activities comparable to those of their analogs with the natural D-sugar configuration. The mechanism of inhibition is purely competitive for L-IdU (Ki = 0.24 microM) and mixed-type for L-BVdU (Ki = 0.13 microM). High performance liquid chromatographic analysis of the reaction products demonstrated that the viral enzyme phosphorylates both L-enantiomers to their corresponding monophosphates with efficiency comparable to that for D-enantiomers. Neither L-enantiomer inhibits the human cytosolic TK. In contrast to their D-enantiomers, L-IdU and L-BVdU have no effect on human thymidylate synthase, either in HeLa cells or in TK-deficient HeLa cells transformed with the HSV-1 TK gene. Both L-enantiomers (i) have no effect on HeLa cell growth, (ii) are 1000-fold less cytotoxic toward TK-deficient HeLa cells transformed with the HSV-1 TK gene than are their D-enantiomers, (iii) in contrast to their D-enantiomers, are fully resistant to hydrolysis by nucleoside phosphorylase, and, (iv) in spite of their much lower cytotoxicity, most probably due to the very low affinity of L-BVdU monophosphate and L-IdU monophosphate for thymidylate synthase, are only 1 or 2 orders of magnitude less potent than their D-enantiomers in inhibiting viral growth, with potency comparable to that of acyclovir.
Assuntos
Antineoplásicos/farmacologia , Antivirais/farmacologia , Bromodesoxiuridina/análogos & derivados , Herpesvirus Humano 1/enzimologia , Idoxuridina/metabolismo , Timidina Quinase/metabolismo , Plaquetas/efeitos dos fármacos , Plaquetas/enzimologia , Plaquetas/metabolismo , Bromodesoxiuridina/farmacologia , Divisão Celular/efeitos dos fármacos , Células HeLa , Herpesvirus Humano 1/efeitos dos fármacos , Humanos , Fosforilação , EstereoisomerismoRESUMO
Among a series of unmodified phosphodiester (PO)-oligodeoxynucleotides (PO-ODNs) complementary to some of the human immunodeficiency virus type 1 (HIV-1) regulatory genes, several PO-ODN sequences complementary to the vpr gene (PO-ODNs-a-vpr, where a-vpr is the antisense vpr sequence) emerged as potent inhibitors (at concentrations of 0.8 to 3.3 microM) of HIV-1 multiplication in de novo infected MT-4 cells, while they showed no cytotoxicity for uninfected cells at concentrations up to 100 microM. Unlike phosphorothioate counterparts, PO-ODN-a-vpr sequences were not inhibitory to HIV-2 multiplication in de novo infected C8166 cells and neither prevented the fusion between chronically infected and bystander CD4+ cells nor inhibited the activity of the HIV-1 reverse transcriptase in enzyme assays. Moreover, they were not inhibitory to HIV-1 multiplication in chronically infected cells. Delayed addition experiments showed that PO-ODNs-a-vpr inhibit an event in the HIV-1 replication cycle following adsorption to the host cell, but preceding reverse transcription. Structure-activity relationship studies indicated that the antiviral activity of the test PO-ODN-a-vpr sequences is not related to an antisense mechanism but to the presence, within the active sequences, of contiguous guanine residues. Physical characterization of the test PO-ODNs suggested that the active structure is a tetramer stabilized by G quartets (i.e., four G residues connected by eight hydrogen bonds).