RESUMO
Small molecules targeting G-quadruplex of oncogene promoter is considered as a promising anticancer therapeutics approach. Natural aloe compounds aloe emodin, and its glycoside derivative aloe emodin-8-glucoside and aloin have anticancer activity and also have potential DNA binding ability. These three compounds have promising binding ability towards quadruplex structures particularly c-KIT G-quadruplex. Here, this study demonstrates complete biophysical study of these compounds to c-KIT quadruplex structure. Aloe emodin showed highest binding stabilization with c-KIT which has been proved by absorbance, fluorescence, dye displacement, ITC and SPR studies. Moreover, comparative study of these compounds with HCT 116 cells line also agreed to their anti proliferative property which may be helpful to establish these aloe compounds as potential anticancer drugs. This study comprises a complete biophysical study along with their anti proliferative property and demonstrates aloe emodin as a potent c-KIT binding molecule.
Assuntos
Aloe , Quadruplex G , Aloe/química , Antraquinonas/farmacologiaRESUMO
G-quadruplex, a unique DNA quartet motif with a pivotal role in regulation of the gene expression, has been established as a potent therapeutic target for the treatment of cancer. Small-molecule-mediated stabilization of the G-quadruplex and thus inhibition of the expression from the oncogene promoter and telomere region may be a promising anticancer strategy. Aloe vera-derived natural compounds like aloe emodin, aloe emodin-8-glucoside, and aloin have significant anticancer activity. Comparative binding studies of these three molecules with varieties of G-quadruplex sequences were carried out using different biophysical techniques like absorption spectral titration, fluorescence spectral titration, dye displacement, ferrocyanide quenching assay, and CD and DSC thermogram studies. Overall, this study revealed aloe emodin and aloe emodin-8-glucoside as potent quadruplex-binding molecules mostly in the case of c-KIT and c-MYC sequences with a binding affinity value of 105 order that is higher than their duplex DNA binding ability. This observation may be correlated to the anticancer activity of these aloe-active compounds and also be helpful in the potential therapeutic application of natural compound-based molecules.
RESUMO
Natural anthraquinone compounds have emerged as potent anticancer chemotherapeutic agents because of their promising DNA-binding properties. Aloe vera is among one of the very well-known medicinal plants, and the anthraquinone derivatives like aloe emodin (ALM), aloins (ALN), and aloe emodin-8-glucoside (ALMG) are known to have immense biological activities. Here, we have used biophysical methods to elucidate the comparative DNA-binding abilities of these three molecules. Steady-state fluorescence study indicated complexation between calf thymus DNA (ctDNA) and both the molecules ALM and ALMG whereas ALN showed very weak interaction with DNA. Displacement assays with ctDNA-bound intercalator (ethidium bromide) and a groove binder (Hoechst 33258) indicated preferential binding of both ALM and ALMG to minor groove of DNA. Isothermal titration calorimetric (ITC) data suggested spontaneous exothermic single binding mode of both the molecules: ALM and ALMG. Entropy is the most important factor which contributed to the standard molar Gibbs energy associated with relatively small favorable enthalpic contribution. The equilibrium constants of binding to ctDNA were (6.02 ± 0.10) × 104 M-1 and (4.90 ± 0.11) × 104 M-1 at 298.15 K, for ALM and ALMG, respectively. The enthalpy vs temperature plot yielded negative standard molar heat capacity value, and a strong negative correlation between enthalpy and entropy terms was observed which indicates the enthalpy entropy compensation behavior in both systems. All these thermodynamic phenomena indicate that hydrophobic force is the key factor which is involved in the binding process. Moreover, the enhancement of thermal stability of DNA helix by ALM and ALMG fully agreed to the complexation of these molecules with DNA.
Assuntos
Aloe/química , DNA/metabolismo , Compostos Fitoquímicos/metabolismo , Animais , Calorimetria , Varredura Diferencial de Calorimetria , Bovinos , Corantes/química , DNA/química , Conformação de Ácido Nucleico , Compostos Fitoquímicos/química , Espectrometria de Fluorescência , TemperaturaRESUMO
BACKGROUND: Natural aristolochia alkaloids have attracted the attention of both chemists and biologists from the stand point of their structural and pharmacological aspects. Many of the compounds isolated in this group are potent tumor inhibitors. These are divided into nitrophenanthrinic acid, phenanthrene lactams and isoquinoline alkaloids. A number of structure-activity studies have been performed on aristolochia alkaloids. Of particular interest is the molecule with the ß-D-glucoside moiety that has similarity to the clinical anticancer agent daunomycin. OBJECTIVE: The anticancer activity of aristololactam-ß-D-glucoside has been thought to be due to its DNA and RNA binding activities among other actions. In this article we focus on the physicochemical property of this alkaloid and the structural and functional aspects of its binding to different nucleic acid and protein structures. METHODS: This review highlights a large number of biophysical studies employing various analytical techniques like absorbance, fluorescence, circular dichroism, thermal melting, viscosity, IR study, isothermal calorimetry and differential scanning calorimetry. RESULT: The detailed binding mechanism in terms of the structural and thermodynamic aspects at the molecular level has been discussed. CONCLUSION: This review enables to assess the high potential of developing aristololactam-ß-Dglucoside and related alkaloids as therapeutic agents.
Assuntos
Alcaloides/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Aristolochia/química , Ácidos Aristolóquicos/farmacologia , Produtos Biológicos/farmacologia , Glucosídeos/farmacologia , Neoplasias/tratamento farmacológico , Alcaloides/química , Alcaloides/isolamento & purificação , Animais , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Ácidos Aristolóquicos/química , Ácidos Aristolóquicos/isolamento & purificação , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Proliferação de Células/efeitos dos fármacos , Glucosídeos/química , Glucosídeos/isolamento & purificação , Humanos , Substâncias Macromoleculares/química , Substâncias Macromoleculares/isolamento & purificação , Substâncias Macromoleculares/farmacologia , Neoplasias/patologia , TermodinâmicaRESUMO
Study on anticancer agents that act via stabilization of telomeric G-quadruplex DNA has emerged as novel and exciting field for anticancer drug discovery. The interaction of carbohydrate containing anticancer alkaloid aristololactam-ß-D-glucoside (ADG) with human telomeric G-quadruplex DNA sequence was characterized by different biophysical techniques. The binding parameters were compared with daunomycin (DAN), a well-known chemotherapeutic drug. The Scatchard binding isotherms revealed noncooperative binding for both with the binding affinity values of (1.01 ± 0.05) × 106 and (1.78 ± 0.18) × 106 M-1 for ADG and DAN, respectively. Circular dichroism, ferrocyanide quenching study, anisotropy study, thiazole orange displacement, optical melting, differential scanning calorimetry study, and molecular docking study suggest significant stacking and stabilizing efficiency of ADG with comparison to DAN. The energetics of the interaction for ADG and DAN revealed that both reactions were predominantly entropy driven. Negative heat capacity values were obtained from the temperature dependence of the enthalpy change. The standard molar Gibbs energy change exhibited only marginal alterations with temperature suggesting the occurrence of enthalpy-entropy compensation. These findings indicate that ADG can act as a stabilizer of telomeric G-quadruplex DNA and thereby can be considered as a potential telomerase inhibitor.
Assuntos
Antineoplásicos/farmacologia , Ácidos Aristolóquicos/farmacologia , DNA/efeitos dos fármacos , Daunorrubicina/farmacologia , Quadruplex G/efeitos dos fármacos , Glucosídeos/farmacologia , Telômero/efeitos dos fármacos , Anisotropia , Ácidos Aristolóquicos/química , Benzotiazóis/química , Calorimetria , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Daunorrubicina/química , Glucosídeos/química , Humanos , Simulação de Acoplamento Molecular , Desnaturação de Ácido Nucleico , Quinolinas/química , Espectrometria de Fluorescência , TermodinâmicaRESUMO
The interaction of the plant alkaloid aristololactam-ß-D-glucoside (ADG) and the anticancer agent daunomycin (DAN) with human hemoglobin was studied by different spectroscopic and calorimetric methods. The binding affinity values of ADG and DAN, estimated from spectroscopic experiments, were 3.79 × 10(4) and 6.68 × 10(4) M(-1), respectively. From circular dichroism, 3D fluorescence, and FTIR studies it was observed that, DAN induced stronger conformational changes than ADG in the protein. From synchronous fluorescence spectroscopy results, a pronounced shift in the maximum emission wavelength of tyrosine residues was observed in both cases suggesting that the drugs changed the polarity around tyrosine residues with marginal change around the tryptophan residues. The thermodynamics of the binding interaction analyzed using microcalorimetry presented single binding events that were exothermic in nature in both cases. The binding was driven by large positive standard molar entropy changes with small favorable enthalpy contributions. Negative heat capacity changes in both cases are correlated to the involvement of significant hydrophobic forces in the complexation process. The affinity of DAN to Hb was higher than that of ADG.
Assuntos
Ácidos Aristolóquicos/química , Daunorrubicina/química , Glucosídeos/química , Hemoglobinas/química , Ácidos Aristolóquicos/metabolismo , Calorimetria/métodos , Daunorrubicina/metabolismo , Glucosídeos/metabolismo , Hemoglobinas/metabolismo , Humanos , Estrutura Molecular , Ligação Proteica , Análise Espectral/métodos , TermodinâmicaRESUMO
BACKGROUND: Interaction of the plant alkaloid aristololactam-ß-d-glucoside and the antitumor drug daunomycin with single stranded RNAs poly(G), poly(I), poly(C) and poly(U) has been investigated. METHODS: Biophysical techniques of absorption, fluorescence, competition dialysis, circular dichroism, and microcalorimetry have been used. RESULTS: Absorption and fluorescence studies have revealed noncooperative binding of ADG and DAN to the single stranded RNAs. The binding affinity of ADG varied as poly(G) > poly(I) > > poly(C) > poly(U). The affinity of DAN was one order higher than that of ADG and varied as poly(G) > poly(I) > poly(U) > poly(C). This binding preference was further confirmed by competition dialysis assay. The thermodynamics of the binding was characterised to be favourable entropy and enthalpic terms but their contributions were different for different systems. The major non-polyelectrolytic contribution to the binding revealed from salt dependent data appears to be arising mostly from stacking of DAN and ADG molecules with the bases leading to partial intercalation to single stranded RNA structures. Small negative heat capacity values have been observed in all the four cases. CONCLUSIONS: This study presents the comparative structural and thermodynamic profiles of the binding of aristololactam-ß-d-glucoside and daunomycin to single stranded polyribonucleotides. GENERAL SIGNIFICANCE: These results suggest strong, specific but differential binding of these drug molecules to the single stranded RNAs and highlight the role of their structural differences in the interaction profile.
Assuntos
Antibióticos Antineoplásicos/metabolismo , Ácidos Aristolóquicos/metabolismo , Daunorrubicina/metabolismo , Glucosídeos/metabolismo , Plantas/química , Polirribonucleotídeos/metabolismo , Calorimetria , Dicroísmo Circular , Concentração Osmolar , Espectrometria de FluorescênciaRESUMO
The plant alkaloid aristololactam-ß-d-glucoside and the anticancer chemotherapy drug daunomycin are two sugar bearing DNA binding antibiotics. The binding of these molecules to three double stranded ribonucleic acids, poly(A)·poly(U), poly(I)·poly(C) and poly(C)·poly(G), was studied using various biophysical techniques. Absorbance and fluorescence studies revealed that these molecules bound non-cooperatively to these ds RNAs with the binding affinities of the order 10(6) for daunomycin and 10(5) M(-1) for aristololactam-ß-d-glucoside. Fluorescence quenching and viscosity studies gave evidence for intercalative binding. The binding enhanced the melting temperature of poly(A)·poly(U) and poly(I)·poly(C) and the binding affinity values evaluated from the melting data were in agreement with that obtained from other techniques. Circular dichroism results suggested minor conformational perturbations of the RNA structures. The binding was characterized by negative enthalpy and positive entropy changes and the affinity constants derived from calorimetry were in agreement with that obtained from spectroscopic data. Daunomycin bound all the three RNAs stronger than aristololactam-ß-d-glucoside and the binding affinity varied as poly(A)·poly(U) > poly(I)·poly(C) > poly(C)·poly(G). The temperature dependence of the enthalpy changes yielded negative values of heat capacity changes for the complexation suggesting substantial hydrophobic contribution to the binding process. Furthermore, an enthalpy-entropy compensation behavior was also seen in all systems. These results provide new insights into binding of these small molecule drugs to double stranded RNA sequences.
Assuntos
Antineoplásicos/metabolismo , Ácidos Aristolóquicos/metabolismo , Daunorrubicina/metabolismo , Glucosídeos/metabolismo , RNA de Cadeia Dupla/metabolismo , Sítios de Ligação , Calorimetria , Conformação de Ácido Nucleico , Poli A-U/química , Poli A-U/metabolismo , Poli I-C/química , Poli I-C/metabolismo , Interferência de RNA , TermodinâmicaRESUMO
BACKGROUND: Interaction of aristololactam-ß-D-glucoside and daunomycin with tRNA(phe) was investigated using various biophysical techniques. METHODOLOGY/PRINCIPAL FINDINGS: Absorption and fluorescence studies revealed that both the compounds bind tRNA(phe) non-cooperatively. The binding of daunomycin was about one order of magnitude higher than that of aristololactam-ß-D-glucoside. Stronger binding of the former was also inferred from fluorescence quenching data, quantum efficiency values and circular dichroic results. Results from isothermal titration calorimetry experiments suggested that the binding of both compounds was predominantly entropy driven with a smaller but favorable enthalpy term that increased with temperature. A large favorable electrostatic contribution to the binding of daunomycin to tRNA(phe) was revealed from salt dependence data and the dissection of the free energy values. The electrostatic component to the free energy change for aristololactam-ß-D-glucoside-tRNA(phe) interaction was smaller than that of daunomycin. This was also inferred from the slope of log K versus [Na(+)] plots. Both compounds enhanced the thermal stability of tRNA(phe). The small heat capacity changes of -47 and -99 cal/mol K, respectively, observed for aristololactam-ß-D-glucoside and daunomycin, and the observed enthalpy-entropy compensation phenomenon confirmed the involvement of multiple weak noncovalent interactions. Molecular aspects of the interaction have been revealed. CONCLUSIONS/SIGNIFICANCE: This study presents the structural and energetic aspects of the binding of aristololactam-ß-D-glucoside and daunomycin to tRNA(phe).
Assuntos
Ácidos Aristolóquicos/metabolismo , Daunorrubicina/metabolismo , Glucosídeos/metabolismo , RNA de Transferência de Fenilalanina/metabolismo , RNA/metabolismo , Algoritmos , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/metabolismo , Antibióticos Antineoplásicos/farmacologia , Ácidos Aristolóquicos/química , Ácidos Aristolóquicos/farmacologia , Sítios de Ligação , Ligação Competitiva , Calorimetria , Dicroísmo Circular , Daunorrubicina/química , Daunorrubicina/farmacologia , Entropia , Glucosídeos/química , Glucosídeos/farmacologia , Cinética , Estrutura Molecular , Conformação de Ácido Nucleico/efeitos dos fármacos , RNA/química , RNA/genética , RNA de Transferência de Fenilalanina/química , RNA de Transferência de Fenilalanina/genética , Espectrometria de Fluorescência , TermodinâmicaRESUMO
BACKGROUND & AIMS: Increasing age is associated with impaired immune function and in chronic HCV infection specifically, with progressive fibrosis, liver failure, HCC and impaired responses to antiviral therapy. T-lymphocyte telomere length declines with age. We hypothesised that shorter T-lymphocyte telomere length would be associated with poor clinical outcome in HCV infection. METHODS: Circulating T-lymphocyte telomere length, an objective measure of immune senescence, was measured by flow-FISH in 135 HCV-RNA-positive, treatment-naïve patients and 41 healthy controls in relation to clinical outcome. RESULTS: Shorter CD4+CD45RO+ T-lymphocyte telomeres were associated with severe fibrosis (p=0.003), independent of male sex (p=0.04), CMV positivity (p=0.003), previous HBV infection (p=0.007), and age (p=ns) in viraemic patients compared to controls. There were inverse correlations between CD4+CD45RO+ telomere length and fibrosis stage (p<0.001), portal tract inflammatory grade (p=0.035), prothrombin time (p<0.001) and bilirubin (p=0.001). One hundred and twenty-four viraemic individuals were followed prospectively to a composite endpoint of death, hepatic decompensation or HCC. Independent of age, those with shorter CD4+CD45RO+ telomeres were less likely to be complication free after 2-years than those with longer telomeres (86% versus 96%, p=0.009) with an age-adjusted hazard ratio of 0.93 (0.90-0.96). In addition, CD4+CD45RO+ telomere length predicted successful antiviral therapy (p=0.001) independent of other factors. CONCLUSIONS: CD4+ T-lymphocyte telomere length, independent of age, was related to inflammatory grade, fibrosis stage, laboratory indices of severity, subsequent hepatic decompensation and treatment outcome in patients with chronic HCV infection.