RESUMO
In Chinese hamster ovary cells, stable mutants that exhibit 250- to 350-fold resistance to camptothecin (CptR mutants) have been isolated from mutagen-treated cultures. The CptR mutants exhibited no cross-resistance towards drugs such as colchicine, vinblastine, taxol, or puromycin but showed slightly (2- to 3-fold) enhanced sensitivity towards various drugs that inhibit DNA topoisomerase II (namely teniposide, etoposide, doxorubicin, mitoxantrone, amsacrine, ellipticine), suggesting that the genetic lesion in these mutants was highly specific. In contrast to the wild-type cells, the CptR line was resistant to camptothecin-induced DNA strand breaks as measured by alkaline elution. Biochemical studies revealed that in CptR mutants the cellular activity as well as protein content of DNA topoisomerase I were reduced to about 40-50% of the level in wild-type cells. Normal levels of activity and content were observed for the related enzyme DNA topoisomerase II. Studies with DNA topoisomerase I purified from the wild-type and the mutant cells showed that the enzyme from the CptR cells was markedly resistant to camptothecin as assayed by the drug's effects either on relaxation of supercoiled DNA or on stabilization of the covalent enzyme-DNA intermediate. The presence of a camptothecin-resistant form of DNA topoisomerase I in the mutant cells provides further evidence that this enzyme is the cellular target of camptothecin. Cell hybridization studies between the CptR and CptS cells showed that the hybrids formed between these two cell lines were sensitive to camptothecin. The recessive behavior of the CptR mutation provides a plausible explanation for the reduced topoisomerase I content (about one-half of wild-type cells) of the mutant cells and also for their enhanced sensitivity towards inhibitors of topoisomerase II.
Assuntos
Camptotecina/farmacologia , DNA Topoisomerases Tipo I/análise , Animais , Cricetinae , Cricetulus , Dano ao DNA , DNA Topoisomerases Tipo I/isolamento & purificação , DNA Topoisomerases Tipo II/análise , Resistência a Medicamentos , Células Híbridas , MutaçãoRESUMO
Merbarone has previously been shown to have antitumor activity of unknown mechanism in P388 and L1210 tumor models (A. D. Brewer et al., Biochem. Pharmacol., 34:2047-2050, 1985) and is currently undergoing Phase I clinical trials. Here we report that merbarone is an inhibitor of topoisomerase II. Merbarone inhibited purified mammalian topoisomerase II with a 50% inhibitory concentration of 20 microM, as assessed by ATP-dependent unknotting of P4 phage DNA or relaxation of supercoiled pBR322 plasmid. In contrast to the type II enzyme, inhibition of catalytic activity of topoisomerase I required about 10-fold higher concentrations of merbarone, with a 50% inhibitory concentration of approximately 200 microM. Unlike epipodophyllotoxin analogues and certain DNA intercalative agents which stabilize the topoisomerase II-DNA "cleavable complex," merbarone did not cause detectable topoisomerase II-induced DNA cleavage. Furthermore, merbarone inhibited the production by amsacrine or teniposide of topoisomerase II-associated DNA strand breaks; under identical conditions novobiocin did not decrease these breaks, setting merbarone apart from a novobiocin-like class of topoisomerase II inhibitor. In L1210 cells, merbarone produced only small numbers of protein-associated DNA strand breaks, and only at very high concentrations. Merbarone reduced in a concentration-dependent manner the number of amsacrine- or teniposide-stimulated protein-associated DNA strand breaks in L1210 cells or their isolated nuclei. The data suggest that merbarone represents a novel type of topoisomerase II inhibitor.
Assuntos
Antineoplásicos/farmacologia , Tiobarbitúricos/farmacologia , Inibidores da Topoisomerase II , Amsacrina/farmacologia , Animais , Camptotecina/farmacologia , DNA/metabolismo , Dano ao DNA , Leucemia Experimental/enzimologia , Camundongos , Teniposídeo/farmacologia , Inibidores da Topoisomerase IRESUMO
New technologies in high-throughput screening have significantly increased throughput and reduced assay volumes. Key advances over the past few years include new fluorescence methods, detection platforms and liquid-handling technologies. Screening 100,000 samples per day in miniaturized assay volumes will soon become routine. Furthermore, new technologies are now being applied to information-rich cell-based assays, and this is beginning to remove one of the key bottlenecks downstream from primary screening.
Assuntos
Avaliação Pré-Clínica de Medicamentos/tendências , Preparações Farmacêuticas/análise , Avaliação Pré-Clínica de Medicamentos/métodos , HumanosRESUMO
This past year has seen a number of new developments in recombinant cell based assays. Lead compounds are beginning to be identified using mechanism-based whole cell assays to discover inhibitors of cellular processes, such as transactivation and signaling. These assays offer advantages for many targets over subcellular assay systems with purified macromolecules.
Assuntos
Bioensaio , Animais , Antineoplásicos/análise , Humanos , Transcrição Gênica/efeitos dos fármacosRESUMO
Several camptothecin derivatives containing a modified hydroxy lactone ring have been synthesized and evaluated for inhibition of topoisomerase I and cytotoxicity to mammalian cells. Each of the groups of the hydroxy lactone moiety, the carbonyl oxygen, the ring lactone oxygen, and the 20-hydroxy group, were shown to be critical for enzyme inhibition. For example the lactol, lactam, thiolactone, and 20-deoxy derivatives did not stabilize the covalent DNA-topoisomerase I complex. With a few exceptions, those compounds that did not inhibit topoisomerase I were not cytotoxic to mammalian cells. Two cytotoxic derivatives that did not inhibit topoisomerase I were shown to produce non-protein-associated DNA single-strand breaks and are likely to have a different mechanism of action. One of these compounds was tested for antitumor activity and was found to be inactive. The present findings, as well as other reports that the hydroxy lactone ring of camptothecin is critical for antitumor activity in vivo, correlate with the structure-activity relationships at the level of topoisomerase I and support the hypothesis that antitumor activity is related to inhibition of this target enzyme.
Assuntos
Antineoplásicos Fitogênicos/síntese química , Camptotecina/análogos & derivados , Inibidores da Topoisomerase I , Animais , Antineoplásicos Fitogênicos/farmacologia , Camptotecina/síntese química , Camptotecina/farmacologia , Fenômenos Químicos , Química , Ensaios de Seleção de Medicamentos Antitumorais , Leucemia L1210/tratamento farmacológico , Camundongos , Relação Estrutura-AtividadeRESUMO
The transition from manual to robotic high throughput screening (HTS) in the last few years has made it feasible to screen hundreds of thousands of chemical entities against a biological target in less than a month. This rate of HTS has increased the visibility of bottlenecks, one of which is assay optimization. In many organizations, experimental methods are generated by therapeutic teams associated with specific targets and passed on to the HTS group. The resulting assays frequently need to be further optimized to withstand the rigors and time frames inherent in robotic handling. Issues such as protein aggregation, ligand instability, and cellular viability are common variables in the optimization process. The availability of robotics capable of performing rapid random access tasks has made it possible to design optimization experiments that would be either very difficult or impossible for a person to carry out. Our approach to reducing the assay optimization bottleneck has been to unify the highly specific fields of statistics, biochemistry, and robotics. The product of these endeavors is a process we have named automated assay optimization (AAO). This has enabled us to determine final optimized assay conditions, which are often a composite of variables that we would not have arrived at by examining each variable independently. We have applied this approach to both radioligand binding and enzymatic assays and have realized benefits in both time and performance that we would not have predicted a priori. The fully developed AAO process encompasses the ability to download information to a robot and have liquid handling methods automatically created. This evolution in smart robotics has proven to be an invaluable tool for maintaining high-quality data in the context of increasing HTS demands.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Robótica , Automação , Avaliação Pré-Clínica de Medicamentos/normas , Avaliação Pré-Clínica de Medicamentos/estatística & dados numéricosRESUMO
A subline of P388 leukemia made 10-fold resistant to camptothecin (CPT) by serial passage in drug-treated mice was adapted to growth in tissue culture and made hyper-resistant to CPT by passage in the presence of increasing concentrations of the drug. Cells were obtained that were 1,000-fold resistant to CPT, compared to wild-type P388 cells. Neither topoisomerase I mRNA nor 100 kDa topoisomerase I enzyme was detectable in these cells, and topoisomerase I activity extracted from nuclei was less than 4% of that extracted from nuclei of wild-type cells. An immunoreactive 130 kDa protein that could be an altered, inactive form of topoisomerase I was evident in the hyper-resistant cells. In addition, the cells deficient in topoisomerase I contained enhanced topoisomerase II activity. Maintenance of the hyper-resistant phenotype required continued exposure to CPT; growth in its absence led to loss of hyper-resistance, increased topoisomerase I content and activity, and decreased topoisomerase II activity. The sensitivity of the cells to killing by a number of inhibitors of topoisomerases I and II was consistent with these observations. Thus, P388 cells have the potential to become highly resistant to CPT by severely curtailing topoisomerase I expression; in these circumstances, topoisomerase I and II activities are regulated coordinately.
Assuntos
Camptotecina/farmacologia , DNA Topoisomerases Tipo I/metabolismo , Macrófagos/enzimologia , Animais , Núcleo Celular/enzimologia , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo II/metabolismo , Resistência a Medicamentos , Expressão Gênica , Genes , Técnicas In Vitro , Metilação , Camundongos , RNA Mensageiro/genética , Células Tumorais CultivadasRESUMO
The synthesis of methidiumpropyl-EDTA (MPE) is described. The binding affinities of MPE, MPE.Ni(II), and MPE.Mg(II) to calf thymus DNA are 2.4 X 10(4) M-1, 1.5 X 10(5) M-1, and 1.2 X 10(5) M-1, respectively, in 50 mM NaCl, pH 7.4. The binding site size is two base pairs. MPE.Mg(II) unwinds PM2 DNA 11 +/- 3 degrees per bound molecule. MPE.Fe(II) in the presence of O2 efficiently cleaves DNA and with low sequence specificity. Reducing agents significantly enhance the efficiency of the cleavage reaction in the order sodium ascorbate greater than dithiothreitol greater than NADPH. At concentrations of 0.1-0.01 microM in MPE.Fe(II) and 10 microM in DNA base pairs, optimum ascorbate and dithiothreitol concentrations for DNA cleavage are 1-5 mM. Efficient cleavage of DNA (10 microM in base pairs) with MPE.Fe(II) (0.1-0.01 microM) occurs over a pH range of 7-10 with the optimum at 7.4 (Tris-HCl buffer). The optimum cleavage time is 3.5 h (22 degrees C). DNA cleavage is efficient in a Na+ ion concentration range of 5 mM to 1 M, with the optimum at 5 mM NaCl. The number of single-strand scissions on supercoiled DNA per MPE.Fe(II) under optimum conditions is 1.4. Metals such as Co(II), Mg(II), Ni(II), and Zn(II) inhibit strand scission by MPE. The released products from DNA cleavage by MPE.Fe(II) are the four nucleotide bases. The DNA termini at the cleavage site are 5'-phosphate and roughly equal proportions of 3'-phosphate and 3'-(phosphoglycolic acid). The products are consistent with the oxidative degradation of the deoxyribose ring of the DNA backbone, most likely by hydroxy radical.
Assuntos
DNA/metabolismo , Ácido Edético/análogos & derivados , Animais , Ácido Ascórbico/farmacologia , Catalase/farmacologia , Bovinos , DNA Super-Helicoidal/metabolismo , Desferroxamina/farmacologia , Ditiotreitol/farmacologia , Ácido Edético/metabolismo , Ácido Edético/farmacologia , Metais/farmacologia , Plasmídeos , Espectrofotometria , Superóxido Dismutase/farmacologiaRESUMO
Bleomycin-mediated DNA strand scission has been shown to be diminished at certain sequences in proximity to 5-methylcytidines. We have investigated the molecular basis of this observed diminution using selective bleomycin (BLM) modifications at the C-terminus. Of the four different bleomycin congeners investigated, only bleomycin A2 and bleomycin BAPP were substantially affected by cytidine methylation. We have also examined the effect of other DNA modifications on bleomycin-mediated strand scission. Methylation at the N6 position of adenosine resulted in diminution of DNA cleavage by all four bleomycin congeners. The presence of bulky 5-(glucosyloxy)methyl groups in the major groove of T4 DNA had little effect on the efficiency of DNA strand scission mediated by bleomycin A2 or B2, suggesting the absence of important steric interactions between Fe(II).BLM and DNA in the major groove. In contrast, DNA cleavage mediated by bleomycin congeners was very sensitive to a major DNA conformational change, the B----Z transition. Salt and MgCl2 titrations of the DNA copolymers poly(dG-dC).poly(dG-dC) and poly(dG-MedC).poly(dG-MedC) demonstrated that bleomycin A2 and B2 did not cleave Z-DNA efficiently. In addition, circular dichroism titrations of these copolymers revealed that both bleomycin congeners increased the cation concentration necessary to induce the B----Z transition, implying that bleomycin preferentially binds to and stabilizes B-form DNA. These results are consistent with a model in which cytidine methylation at appropriate sequences of DNA is sufficient to induce subtle conformational changes that render the helix unreceptive to cleavage by some bleomycin congeners.
Assuntos
Bleomicina , Dano ao DNA , DNA , Enzimas de Restrição do DNA , Metilação , Concentração Osmolar , Plasmídeos , Polidesoxirribonucleotídeos , Cloreto de SódioRESUMO
Camptothecin, a cytotoxic antitumor compound, has been shown to produce protein-linked DNA breaks mediated by mammalian topoisomerase I. We have investigated the mechanism by which camptothecin disrupts DNA processing by topoisomerase I and have examined the effect of certain structurally related compounds on the formation of a DNA-topoisomerase I covalent complex. Enzyme-mediated cleavage of supercoiled plasmid DNA in the presence of camptothecin was completely reversed upon the addition of exogenous linear DNA or upon dilution of the reaction mixture. Camptothecin and topoisomerase I produced the same amount of cleavage from supercoiled DNA or relaxed DNA. In addition, the alkaloid decreased the initial velocity of supercoiled DNA relaxation mediated by catalytic quantities of topoisomerase I. Inhibition occurred under conditions favoring processive catalysis as well as under conditions favoring distributive catalysis. By use of [3H]camptothecin and an equilibrium dialysis assay, the alkaloid was shown to bind reversibly to a DNA-topoisomerase I complex, but not to isolated enzyme or isolated DNA. These results are consistent with a model in which camptothecin reversibly traps an intermediate involved in DNA unwinding by topoisomerase I and thereby perturbs a set of equilibria, resulting in increased DNA cleavage. By examining certain compounds that are structurally related to camptothecin, it was found that the 20-hydroxy group, which has been shown to be essential for antitumor activity, was also necessary for stabilization of the covalent complex between DNA and topoisomerase I. In contrast, no such correlation existed for UV-light-induced cleavage of DNA by Cu(II)-camptothecin derivatives.
Assuntos
Camptotecina/farmacologia , DNA/metabolismo , Inibidores da Topoisomerase I , Camptotecina/análogos & derivados , Camptotecina/metabolismo , Catálise , Colódio , Cobre/farmacologia , DNA/efeitos da radiação , DNA Topoisomerases Tipo I/análise , DNA Topoisomerases Tipo I/metabolismo , DNA Super-Helicoidal/análise , Proteínas de Ligação a DNA/análise , Hidrólise , Hidroxilação , Compostos Organometálicos/farmacologia , Plasmídeos , Raios UltravioletaRESUMO
Three DNA duplexes differing substantially in sequence were derived from pBR322 plasmid DNA and supercoiled SV40 DNA by digestion with appropriate restriction endonucleases. Following treatment with the restriction methylase HhaI (recognition sequence: GCGC) or HhaI and HpaII (CCGG), the unmethylated and methylated DNAs were compared as substrates for the antitumor agent bleomycin. Bleomycin-mediated strand scission was shown to diminish substantially at a number of sites in proximity to the methylated cytidine moieties, especially where multiple sites had been methylated within a DNA segment of limited size. Detailed analysis of the DNA substrates revealed that both strands of DNA within a methylated region became more refractory to cleavage by bleomycin and that the protective effect could extend as many as 14 base pairs in proximity to the 5-methylcytidine moieties. Among the methylated DNA segments that became more resistant to bleomycin cleavage was a HpaII site of SV40 DNA, methylation of which has previously been shown to diminish the synthesis of the major late viral capsid protein following microinjection into Xenopus laevis oocytes. Study of the cleavage reaction at varying salt levels suggested that diminished bleomycin strand scission may be due, at least in part, to local conformational changes of the DNA to Z form (or other non-B-form structures). The results are generally consistent with the hypothesis that one mechanism for the expression of selective therapeutic action by certain DNA damaging agents could involve the recognition of specific methylation patterns.
Assuntos
Bleomicina , DNA , Sequência de Bases , Enzimas de Restrição do DNA , DNA Super-Helicoidal , DNA Viral , Escherichia coli/genética , Metilação , Plasmídeos , Vírus 40 dos Símios/genética , Relação Estrutura-AtividadeRESUMO
Methidiumpropyl-EDTA . iron(II) [MPE . Fe (II)] cleaves double-helical DNA with considerably lower sequence specificity than micrococcal nuclease. Moreover, digestions with MPE . Fe(II) can be performed in the presence of certain metal chelators, which will minimize the action of many endogenous nucleases. Because of these properties MPE . Fe(II) would appear to be a superior tool for probing chromatin structure. We have compared the patterns generated from the 1.688 g/cm3 complex satellite, 5S ribosomal RNA, and histone gene sequences of Drosophila melanogaster chromatin and protein-free DNA by MPE . Fe(II) and micrococcal nuclease cleavage. MPE . Fe(II) at low concentrations recognizes the nucleosome array, efficiently introducing a regular series of single-stranded (and some double-stranded) cleavages in chromatin DNA. Subsequent S1 nuclease digestion of the purified DNA produces a typical extended oligonucleosome pattern, with a repeating unit of ca. 190 base pairs. Under suitable conditions, relatively little other nicking is observed. Unlike micrococcal nuclease, which has a noticeable sequence preference in introducing cleavages, MPE . Fe(II) cleaves protein-free tandemly repetitive satellite and 5S DNA sequences in a near-random fashion. The spacing of cleavage sites in chromatin, however, bears a direct relationship to the length of the respective sequence repeats. In the case of the histone gene sequences a faint, but detectable, MPE . Fe(II) cleavage pattern is observed on DNA, in some regions similar to and in some regions different from the strong chromatin-specified pattern. The results indicate that MPE . Fe(II) will be very useful in the analysis of chromatin structure.
Assuntos
Cromatina/efeitos dos fármacos , DNA/metabolismo , Ácido Edético/análogos & derivados , Animais , Núcleo Celular/efeitos dos fármacos , Cromatina/fisiologia , DNA/isolamento & purificação , Drosophila melanogaster/metabolismo , Ácido Edético/farmacologia , Histonas/isolamento & purificação , Peso MolecularRESUMO
We report a direct technique for determining the binding sites of small molecules on naturally occurring heterogeneous DNA. Methidiumpropyl-EDTA.Fe(II) [MPE.Fe(II) cleaves double helical DNA with low sequence specificity. Using a combination of MPE.Fe(II) cleavage of drug-protected DNA fragments and Maxam-Gilbert gel methods of sequence analysis, we have determined the preferred binding sites on a Rsa I-EcoRI restriction fragment from pBR322 for the intercalator actinomycin D and the minor groove binders netropsin and distamycin A. Netropsin and distamycin A gave identical DNA cleavage-inhibition patterns and bound preferentially to A+T-rich regions with a minimal protected site of four base pairs. We were able to observe the effect of increasing concentration on site selection by netropsin and distamycin A. Actinomycin D afforded a completely different cleavage-inhibition pattern, with 4- to 16-base-pair-long protected regions centered around one or more G.C base pairs.
Assuntos
DNA Bacteriano , Dactinomicina , Distamicinas , Guanidinas , Netropsina , Plasmídeos , Pirróis , Fenômenos Químicos , Química , Daunorrubicina , Escherichia coli , Relação Estrutura-AtividadeRESUMO
Anthramycin, tomaymycin, and sibiromycin are members of the pyrrolo[1,4]benzodiazepine [P(1,4)B] antitumor antibiotic group. These drugs bind covalently through N2 of guanine and lie within the minor groove of DNA [Petrusek, R. L., Anderson, G. L., Garner, T. F., Fannin, Q. L., Kaplan, D. J., Zimmer, S. G., & Hurley, L. H. (1981) Biochemistry 20, 1111-1119]. The DNA sequence specificity of the P(1,4)B antibiotics has been determined by a footprinting method using methidiumpropyl-EDTA-iron(II) [MPE.Fe(II)], and the results show that each of the drugs has a two to three base pair sequence specificity that includes the covalently modified guanine residue. While 5'PuGPu is the most preferred binding sequence for the P(1,4)Bs, 5'PyGPy is the least preferred sequence. Footprinting analysis by MPE.Fe(II) reveals a minimum of a three to four base pair footprint size for each of the drugs on DNA with a larger than expected offset (two to three base pairs) on opposite strands to that observed in previous analyses of noncovalently bound small molecules. There is an extremely large enhancement of MPE.Fe(II) cleavage between drug binding sites in AT rich regions, probably indicating a drug-induced change in the conformational features of DNA which encourages interaction with MPE.Fe(II). In the presence of sibiromycin or tomaymycin the normally guanine-specific methylene blue reaction used in Maxam and Gilbert sequencing cleaves at other bases in defined positions relative to the drug binding sites. Finally, modeling studies are used to rationalize the differences and similarities in sequence specificities between the various drugs in the P(1,4)B group and their reactions with DNA.
Assuntos
Aminoglicosídeos , Antramicina/metabolismo , Antibióticos Antineoplásicos/metabolismo , Benzodiazepinonas/metabolismo , DNA/metabolismo , Ácido Edético/análogos & derivados , Quelantes de Ferro , Sequência de Bases , DNA Viral/metabolismo , Plasmídeos , Vírus 40 dos Símios/metabolismo , Espectrofotometria Ultravioleta , Relação Estrutura-AtividadeRESUMO
A human Burkitt's lymphoma cell line (Raji-HN2) made resistant to nitrogen mustard, a bifunctional alkylating agent, was used to study the mechanism of resistance to nitrogen mustard. A comparative study of Raji-HN2 and the parental sensitive Raji cell lines revealed the following: (1) The DNA of Raji-HN2 cells was crosslinked by nitrogen mustard to a lower extent than Raji DNA; (2) once interstrand crosslinks were formed, they were repaired at the same rate in both cell lines; (3) DNA crosslink formation in Raji-HN2, but not in Raji cells, was enhanced by novobiocin, a topoisomerase II inhibitor; (4) Raji-HN2 cells had elevated topoisomerase II activity and were hypersensitive to topoisomerase inhibitors (amsacrine, novobiocin, teniposide); (5) similar amounts of topoisomerase I were found in both cell lines; and (6) the chromatin of Raji-HN2 but not of Raji cells, was hypersensitive to DNase I digestion. The relationship between DNA repair, topoisomerase II activity, chromatin structure and drug resistance is discussed.
Assuntos
Amsacrina/farmacologia , Cromatina/efeitos dos fármacos , DNA Topoisomerases Tipo II/metabolismo , Mecloretamina/farmacologia , Novobiocina/farmacologia , Podofilotoxina/análogos & derivados , Teniposídeo/farmacologia , Linfoma de Burkitt , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cromatina/ultraestrutura , Resistência a Medicamentos , Humanos , Cinética , Ensaio Tumoral de Célula-TroncoRESUMO
Camptothecin (CPT) binds reversibly to, and thereby stabilizes, the cleavable complex formed between DNA and topoisomerase I. The nature of the interaction of CPT with the DNA-topoisomerase I binary complex was studied by the use of two affinity labeling reagents structurally related to camptothecin: 10-bromoacetamidomethylcamptothecin (BrCPT) and 7-methyl-10-bromoacetamidomethylcamptothecin (BrCPTMe). These compounds have been shown to trap the DNA-topoisomerase I complex irreversibly. Although cleavage of DNA plasmid mediated by topoisomerase I and camptothecin was reduced significantly by treatment with high salt or excess competitor DNA, enzyme-mediated DNA cleavage stabilized by BrCTPMe persisted for at least 4 h after similar treatment. The production of irreversible topoisomerase I-DNA cleavage was time-dependent, suggesting that BrCPTMe first bound noncovalently to the enzyme-DNA complex and, in a second slower step, alkylated the enzyme or DNA in a manner that prevented DNA ligation. The formation of a covalent linkage was supported by experiments that employed [3H]BrCPT, which was shown to label topoisomerase I within the enzyme-DNA complex. [3H]BrCPT labeling of topoisomerase I was enhanced greatly by the presence of DNA; very little labeling of isolated topoisomerase I or isolated DNA occurred. Even in the presence of DNA, [3H]BrCPT labeling of topoisomerase I was inhibited by camptothecin, suggesting that both CPT and BrCPT bound to the same site on the DNA-topoisomerase I binary complex. These studies provide further evidence that a binding site for camptothecin is created as the DNA-topoisomerase I complex is formed and suggest that the A-ring of camptothecin is proximate to an enzyme residue.
Assuntos
Marcadores de Afinidade/metabolismo , Camptotecina/análogos & derivados , Camptotecina/metabolismo , DNA Topoisomerases Tipo I/metabolismo , DNA/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Bovinos , Cinética , Dados de Sequência Molecular , Estrutura Molecular , Plasmídeos , Especificidade por Substrato , Timo/enzimologiaRESUMO
A camptothecin-resistant subline of P388 leukemia (P388/CPT) was developed by repeated transplantation of P388 cells in mice treated with therapeutic doses of camptothecin. In mice bearing the resistant tumor, a maximally tolerated dose of camptothecin produced no net reduction in tumor cell burden, in contrast to a 5-log cell kill in the parental P388 (P388/S). The IC50 of camptothecin, as determined by colony formation assays of cultured cells, was 8 times greater for the cloned P388/CPT cell line than for P388/S. P388/CPT cells were not cross-resistant to other antineoplastic agents, including topoisomerase II inhibitors. The type I topoisomerases purified from P388/CPT and P388/S cells were identical with respect to molecular weight, specific activity, in vitro camptothecin sensitivity, and DNA cleavage specificity. Camptothecin induced fewer protein-associated DNA single-strand breaks in the resistant cells than in the wild-type P388 cells. Topoisomerase I mRNA, immunoreactivity, and extractable enzymatic activity were 2-4 times lower for P388/CPT cells than for P388/S cells. As resistance to camptothecin developed, topoisomerase I extractable activity decreased, concomitant with an increase in topoisomerase II extractable activity. Furthermore, the appearance of camptothecin resistance was associated with specific rearrangements of the topoisomerase I gene. These results suggest that development of resistance to inhibitors of topoisomerase I can occur by down-regulation of the target enzyme, thus reducing the production of lethal enzyme-mediated DNA damage. The enhanced topoisomerase II activity in these cells suggests that resistance to camptothecin may be overcome by co-treatment with topoisomerase II inhibitors.
Assuntos
Camptotecina/farmacologia , DNA Topoisomerases Tipo I/análise , Leucemia P388/enzimologia , Animais , DNA/efeitos dos fármacos , DNA/metabolismo , Dano ao DNA , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/imunologia , DNA Topoisomerases Tipo II/análise , Resistência a Medicamentos , Rearranjo Gênico , Camundongos , Camundongos Endogâmicos DBA , RNA Mensageiro/análise , Células Tumorais CultivadasRESUMO
Inophyllums are novel non-nucleoside inhibitors of human immunodeficiency virus (HIV) type 1 reverse transcriptase identified through an enzyme screening program and isolated from the plant Calophyllum inophyllum. The kinetics of reverse transcriptase inhibition by inophyllum B were characterized using recombinant purified enzyme, a heteropolymeric RNA template, and a scintillation proximity assay. Preincubation of inhibitor with the enzyme-template-primer complex for 11 min was required for maximal inhibition of reverse transcriptase to occur, suggesting that inophyllum B had a slow on-rate and that template-primer must bind to reverse transcriptase prior to inhibitor binding. Inhibition of reverse transcriptase by inophyllums was shown to be reversible. When thymidine triphosphate was the variable substrate, inophyllum B inhibited reverse transcriptase noncompetitively with a Ki of 42 nM. Enzyme inhibition with respect to template-primer was uncompetitive with a Ki of 26 nM. Reverse transcriptase enzymes containing point mutations in which tyrosine 181 was changed to either cysteine or isoleucine exhibited marginal resistance to inophyllums but were resistant to (+)-(5S)-4,5,6,7-tetrahydro-9-chloro-5-methyl-6- (3-methyl-2-butenyl)-imidazo[4,5,1-j,k][1,4]benzodiazepin-2-(1H)-t hione (TIBO R82913). A mutant enzyme in which tyrosine 188 was changed to leucine was cross-resistant to both inophyllum B and TIBO R82913, as was HIV type 2 reverse transcriptase. These studies suggest that inophyllum B and TIBO R82913 bind to distinct but overlapping sites. Inhibition of avian myeloblastosis virus reverse transcriptase and Moloney murine leukemia virus reverse transcriptase by inophyllum B was detectible, suggesting that these inhibitors may be more promiscuous than other previously described non-nucleoside inhibitors. Inophyllums were active against HIV type 1 in cell culture with IC50 values of approximately 1.5 microM. These studies imply that the inophyllums have a novel mechanism of interaction with reverse transcriptase and as such could conceivably play a role in combination therapy.
Assuntos
Antivirais/farmacologia , Benzodiazepinas/farmacologia , Cumarínicos/farmacologia , HIV-1/enzimologia , Imidazóis/farmacologia , Mutagênese Sítio-Dirigida , Inibidores da Transcriptase Reversa , Sequência de Bases , Cumarínicos/química , Cumarínicos/isolamento & purificação , Transcriptase Reversa do HIV , Cinética , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , DNA Polimerase Dirigida por RNA/biossíntese , DNA Polimerase Dirigida por RNA/isolamento & purificação , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
The DNA alkylation and sequence specificity of a group of natural and synthetic pyrrolo-[1,4]benzodiazepines [P(1,4)Bs] were evaluated by using an exonuclease III stop assay, and the results were compared with in vitro and in vivo biological potency and antitumor activity. The P(1,4)B antibiotics are potent antitumor agents produced by various Actinomycetes, which are believed to mediate their cytotoxic effects by covalent bonding through N-2 of guanine in the minor groove of DNA. In this article we describe the results of a sensitive DNA alkylation assay using exonuclease III which permits both estimation of the extent of DNA modification as well as location of the precise guanines to which the drugs are covalently bound. Using this assay, we have evaluated a series of natural and synthetic compounds of the P(1,4)B class for their ability to bind to DNA and also determined their DNA sequence preference. The compounds included in this study are P(1,4)Bs carrying different substituents in the aromatic ring, having varying degrees of saturation in the five-membered ring, or differing in the stereochemistry at C-11a. These same compounds were evaluated for in vitro cytotoxic activity against B16 melanoma cells, for potency in vivo in B6D2F1 mice (LD50), and for antitumor activity (ILSmax) against P388 leukemia cells. A good correlation was found between extent of DNA alkylation and in vitro and in vivo potency. Furthermore, on the basis of electronic and steric considerations, it was possible to rationalize why those compounds that showed negligible biological activity were unable to bond covalently to DNA. Last, we have determined that the degree of saturation in the five-membered ring of the P(1,4)Bs has a significant effect on the DNA bonding reactivity and biological activity of this class of compounds.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Benzodiazepinas/farmacologia , DNA/metabolismo , Alquilação , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/metabolismo , Sequência de Bases , Benzodiazepinas/química , Benzodiazepinas/metabolismo , Sítios de Ligação , DNA/química , Dados de Sequência Molecular , Pirróis/química , Pirróis/metabolismo , Pirróis/farmacologia , Relação Estrutura-AtividadeRESUMO
In a human neutrophil cDNA library, an orphan G-protein-coupled receptor, HNFAG09, with 37% nucleotide identity to the C5a receptor (C5a-R, CD88) was identified. A novel feature of this gene, unlike C5a-R and other G-protein-coupled receptors, is the presence of an extraordinarily large predicted extracellular loop comprised of in excess of 160 amino acid residues between transmembrane domains 4 and 5. Northern blot analysis revealed that expression of mRNA for this receptor in human tissues, while similar, was distinct from C5a-R expression. Although there were differences in expression, transcripts for both receptors were detected in tissues throughout the body and the central nervous system. Mammalian cells stably expressing HNFAG09 specifically bound 125I-C3a and responded to a C3a carboxyl-terminal analogue synthetic peptide and to human C3a but not to rC5a with a robust calcium mobilization response. HNFAG09 encodes the human anaphylatoxin C3a receptor.