RESUMEN
BACKGROUND: Treatment failure and resistance to the commonly used drugs remains a major obstacle for successful chemotherapy against visceral leishmaniasis (VL). Since the development of novel therapeutics involves exorbitant costs, the effectiveness of the currently available antitrypanosomatid drug suramin has been investigated as an antileishmanial, specifically for VL,in vitro and in animal model experiments. METHODOLOGY/PRINCIPAL: Leishmania donovani promastigotes were treated with suramin and studies were performed to determine the extent and mode of cell mortality, cell cycle arrest and other in vitro parameters. In addition, L. donovani infected BALB/c mice were administered suramin and a host of immunological parameters determined to estimate the antileishmanial potency of the drug. Finally, isothermal titration calorimetry (ITC) and enzymatic assays were used to probe the interaction of the drug with one of its putative targets namely parasitic phosphoglycerate kinase (LmPGK). FINDINGS: The in vitro studies revealed the potential efficacy of suramin against the Leishmania parasite. This observation was further substantiated in the in vivo murine model, which demonstrated that upon suramin administration, the Leishmania infected BALB/c mice were able to reduce the parasitic burden and also generate the host protective immunological responses. ITC and enzyme assays confirmed the binding and consequent inhibition of LmPGK due to the drug. CONCLUSIONS/SIGNIFICANCE: All experiments affirmed the efficacy of suramin against L. donovani infection, which could possibly lead to its inclusion in the repertoire of drugs against VL.
Asunto(s)
Antiprotozoarios/farmacología , Antiprotozoarios/uso terapéutico , Leishmaniasis Visceral/tratamiento farmacológico , Suramina/farmacología , Suramina/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Citocinas/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Concentración 50 Inhibidora , Leishmania donovani/efectos de los fármacos , Leishmaniasis Visceral/parasitología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Óxido Nítrico/metabolismo , Fosfoglicerato Quinasa/efectos de los fármacos , Células RAW 264.7/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Accumulating evidences indicate the involvement of epigenetic deregulations in cancer. While some epigenetic regulators with aberrant functions in cancer are targeted for improving therapeutic outcome in patients, reinstating the functions of tumor-suppressor-like epigenetic regulators might further potentiate anti-cancer therapies. Epigenetic reader zinc-finger MYND-type-containing 8 (ZMYND8) has been found to be endowed with multiple anti-cancer functions like inhibition of tumor cell migration and proliferation. Here, we report another novel tumor suppressor role of ZMYND8 as an inducer of differentiation in breast cancer cells, by upregulating differentiation genes. Interestingly, we also demonstrated that ZMYND8 mediates all its antitumor roles through a common dual-histone mark binding to H4K16Ac and H3K36Me2. We validated these findings by both biochemical and biophysical analyses. Furthermore, we also confirmed the differentiationinducing potential of ZMYND8 in vivo, using 4T1 murine breast cancer model in Balb/c mice. Differentiation therapy holds great promise in cancer therapy, since it is non-toxic and makes the cancer cells therapysensitive. In this scenario, we propose epigenetic reader ZMYND8 as a potential therapeutic candidate for differentiation therapy in breast cancer.
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Neoplasias de la Mama/genética , Epigénesis Genética , Histonas/genética , Proteínas Supresoras de Tumor/genética , Animales , Neoplasias de la Mama/patología , Diferenciación Celular/genética , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Xenoinjertos , Humanos , Ratones , Unión Proteica/genética , Activación Transcripcional/genéticaRESUMEN
BACKGROUND: p300 (KAT3B) lysine acetyltransferase activity is modulated under different physiological and pathological contexts through the induction of trans-autoacetylation. This phenomenon is mediated by several factors, mechanisms of which are not fully understood. METHODS: Through acetyltransferase assays using full-length, baculovirus-expressed KATs, the specificity of NPM1-mediated enhancement of p300 autoacetylation was tested. Chaperone assays and tryptophan fluorescence studies were performed to evaluate the NPM1-induced protein folding. The NPM1 oligomer-defective mutant characterization was done by glutaraldehyde-crosslinking. The small-molecule inhibitor of NPM1 oligomerization was used to confirm the absolute requirement of multimeric NPM1 in vivo. Immunohistochemistry analysis of oral cancer patient samples was done to uncover the pathophysiological significance of NPM1-induced p300 autoacetylation. RESULTS: We find that the histone chaperone NPM1 is a specific inducer of p300 autoacetylation. Distinct from its histone chaperone activity, NPM1 is a molecular chaperone of p300. The biophysical experiments suggest that there is a reversible binding between NPM1 and p300 which can modulate p300 acetyltransferase activity. Disruption of NPM1 oligomerization suggests that oligomeric NPM1 is essential for the induction of p300 autoacetylation. Significantly, we observe a concomitant hyper-autoacetylation of p300 with overexpression of NPM1 in oral cancer samples. CONCLUSION: NPM1 can specifically modulate p300 acetyltransferase activity through the enhancement of autoacetylation. The molecular chaperone activity and oligomerization of NPM1 play a pivotal role in this phenomenon. GENERAL SIGNIFICANCE: NPM1 is overexpressed in several solid cancers, the significance of which is unknown. Induction of p300 autoacetylation could be the cause of NPM1-mediated tumorigenicity.
Asunto(s)
Proteína p300 Asociada a E1A/química , Proteína p300 Asociada a E1A/metabolismo , Histonas/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Pliegue de Proteína , Multimerización de Proteína , Neoplasias de la Lengua/metabolismo , Acetilación , Humanos , Nucleofosmina , Unión Proteica , Conformación Proteica , Neoplasias de la Lengua/patología , Células Tumorales CultivadasRESUMEN
Transcription factor 19 (TCF19) has been reported as a type 1 diabetes-associated locus involved in maintenance of pancreatic ß cells through a fine-tuned regulation of cell proliferation and apoptosis. TCF19 also exhibits genomic association with type 2 diabetes, although the precise molecular mechanism remains unknown. It harbors both a plant homeodomain and a forkhead-associated domain implicated in epigenetic recognition and gene regulation, a phenomenon that has remained unexplored. Here, we show that TCF19 selectively interacts with histone 3 lysine 4 trimethylation through its plant homeodomain finger. Knocking down TCF19 under high-glucose conditions affected many metabolic processes, including gluconeogenesis. We found that TCF19 overexpression represses de novo glucose production in HepG2 cells. The transcriptional repression of key genes, induced by TCF19, coincided with NuRD (nucleosome-remodeling-deacetylase) complex recruitment to the promoters of these genes. TCF19 interacted with CHD4 (chromodomain helicase DNA-binding protein 4), which is a part of the NuRD complex, in a glucose concentration-independent manner. In summary, our results show that TCF19 interacts with an active transcription mark and recruits a co-repressor complex to regulate gluconeogenic gene expression in HepG2 cells. Our study offers critical insights into the molecular mechanisms of transcriptional regulation of gluconeogenesis and into the roles of chromatin readers in metabolic homeostasis.
Asunto(s)
Gluconeogénesis , Hepatocitos/metabolismo , Histonas/metabolismo , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Modelos Moleculares , Procesamiento Proteico-Postraduccional , Factores de Transcripción/metabolismo , Línea Celular , Epigénesis Genética , Regulación Enzimológica de la Expresión Génica , Hepatocitos/citología , Hepatocitos/enzimología , Histonas/química , Histonas/genética , Humanos , Lisina , Metilación , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/química , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/genética , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , Mutación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Regiones Promotoras Genéticas , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: A and B-type lamins are integral scaffolding components of the nuclear lamina which impart rigidity and shape to all metazoan nuclei. Over 450 mutations in A-type lamins are associated with 16 human diseases including dilated cardiomyopathy (DCM). Here, we show that DCM mutants perturb the self-association of lamin A (LA) and it's binding with lamin B1 (LB1). METHODS: We used confocal and superresolution microscopy (NSIM) to study the effect of LA mutants on the nuclear lamina. We further used circular dichroism, fluorescence spectroscopy and isothermal titration calorimetry (ITC) to probe the structural modulations, self-association and heteropolymeric association of mutant LA. RESULTS: Transfection of mutants in cultured cell lines result in the formation of nuclear aggregates of varied size and distribution. Endogenous LB1 is sequestered into these aggregates. This is consistent with the ten-fold increase in association constant of the mutant proteins compared to the wild type. These mutants exhibit differential heterotypic interaction with LB1, along with significant secondary and tertiary structural alterations of the interacting proteins. Thermodynamic studies demonstrate that the mutants bind to LB1 with different stoichiometry, affinity and energetics. CONCLUSIONS: In this report we show that increased self-association propensity of mutant LA modulates the LA-LB1 interaction and precludes the formation of an otherwise uniform laminar network. GENERAL SIGNIFICANCE: Our results might highlight the role of homotypic and heterotypic interactions of LA in the pathogenesis of DCM and hence laminopathies in the broader sense.
Asunto(s)
Cardiomiopatía Dilatada/genética , Lamina Tipo A/química , Lamina Tipo B/química , Mapas de Interacción de Proteínas/genética , Cardiomiopatía Dilatada/patología , Núcleo Celular/química , Núcleo Celular/genética , Humanos , Lamina Tipo A/genética , Lamina Tipo B/genética , Microscopía Confocal , Proteínas Mutantes/química , Proteínas Mutantes/genética , Unión Proteica , Conformación ProteicaRESUMEN
Chelerythrine (CHL), a plant alkaloid, possesses antimicrobial, anti-inflammatory, and antitumor properties. Although CHL influences several key signal transduction pathways, its ability to interact directly with nucleoprotein complex chromatin, in eukaryotic cells has so far not been looked into. Here we have demonstrated its association with hierarchically assembled chromatin components, viz. long chromatin, chromatosome, nucleosome, chromosomal DNA, and histone H3 and the consequent effect on chromatin structure. CHL was found to repress acetylation at H3K9. It is more target-specific in terms of gene expression alteration and less cytotoxic compared to its structural analog sanguinarine.
Asunto(s)
Antineoplásicos/farmacología , Benzofenantridinas/farmacología , Eucromatina/efectos de los fármacos , Histonas/metabolismo , Nucleosomas/efectos de los fármacos , Procesamiento Proteico-Postraduccional , Acetilación/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ensamble y Desensamble de Cromatina , ADN/química , ADN/metabolismo , Epigénesis Genética , Eucromatina/química , Eucromatina/metabolismo , Células HeLa , Histonas/genética , Humanos , Isoquinolinas/farmacología , Nucleosomas/química , Nucleosomas/metabolismo , Regiones Promotoras GenéticasRESUMEN
Triplet-triplet (T-T) absorption spectroscopy has been used successfully as a molecular ruler to understand the actual release process of sanguinarine as a drug molecule from a gold nanoparticle surface in the presence of cell components, that is, DNA and chromatin. The obtained results have been verified by fluorescence and surface-enhanced Raman spectroscopy (SERS), and a plausible explanation has been put forward to describe the underestimation and overestimation of the percentage (%) of the release of drug molecules measured by fluorescence- and SERS-based techniques, respectively, over the highlighted T-T absorption spectroscopy. Because of the intrinsic nature of absorption, the reported T-T absorption spectroscopic assay overpowers fluorescence- and SERS-based assays, which are limited by the long-range interaction and nonlinear dependence of the concentration of analytes, respectively.
Asunto(s)
Cromatina/química , ADN/química , Nanopartículas del Metal/química , Preparaciones Farmacéuticas/química , Animales , Benzofenantridinas/química , Benzofenantridinas/metabolismo , Bovinos , Cromatina/metabolismo , ADN/metabolismo , Liberación de Fármacos , Oro/química , Isoquinolinas/química , Isoquinolinas/metabolismo , Microscopía Electrónica de Transmisión , Preparaciones Farmacéuticas/metabolismo , Espectrometría de Fluorescencia , Espectrometría RamanRESUMEN
Combating Type-2 diabetes mellitus is a pivotal challenge in front of the present world. Several lines of therapy are in practice for resisting this deadly disease which often culminates with cardiovascular complexities, neuropathy and retinopathy. Among various therapies, administration of alpha glucosidase inhibitors is common and widely practiced. Sulfonylurea category of anti diabetic drug often suffers from cross reactivity with sulfamethoxazole (SMX), a common drug in use to treat a handful of microbial infections. However the specific cellular target generating postprandial hypoglycemia on SMX administration is till date unraveled. The present work has been initiated to elucidate the effects of a group of sulfonamide drugs inclusive of SMX for their amylase inhibitory role. SMX inhibits porcine pancreatic amylase (PPA) in a noncompetitive mode with an average IC50 value 0.94 mM respectively. Interaction of SMX with PPA is manifested with gradual quenching of tryptophan fluorescence with concomitant shift in lambda max value (λmax). Binding is governed by entropy driven factor (24.8 cal mol(-1) K(-1)) with unfavorable contribution from enthalpy change. SMX interferes with the activity of acarbose in a synergistic mode to reduce the effective dose of acarbose as evident from the in vitro PPA inhibition study. In summary, loss of PPA activity in presence of SMX is indicative of structural changes of PPA which is further augmented in the presence of acarbose as explained in the schematic model and docking study.
Asunto(s)
Antiinfecciosos/farmacología , Inhibidores Enzimáticos/farmacología , alfa-Amilasas Pancreáticas/antagonistas & inhibidores , Sulfametoxazol/farmacología , Animales , Antiinfecciosos/química , Inhibidores Enzimáticos/química , Simulación del Acoplamiento Molecular , alfa-Amilasas Pancreáticas/química , alfa-Amilasas Pancreáticas/metabolismo , Sulfametoxazol/química , PorcinosRESUMEN
Aurora kinases are the most commonly targeted mitotic kinases in the intervention of cancer progression. Here, we report a resorcinol derivative, 5-methyl-4-(2-thiazolylazo) resorcinol (PTK66), a dual inhibitor of Aurora A and Aurora B kinases. PTK66 is a surface binding non-ATP analogue inhibitor that shows a mixed pattern of inhibition against both of Aurora A and B kinases. The in vitro IC50 is approximately 47 and 40 µm for Aurora A and Aurora B kinases, respectively. In cellular systems, PTK66 exhibits a substantially low cytotoxicity at micromolar concentrations but it can induce aneuploidy under similar dosages as a consequence of Aurora kinase inhibition. This result was corroborated by a drop in the histone H3 (S10) phosphorylation level detected via Western blot analysis using three different cell types. Altogether, our findings indicate that the ligand containing resorcinol backbone is one of the novel scaffolds targeting the Aurora family of kinases, which could be a target for antineoplastic drug development.
Asunto(s)
Adenosina Trifosfato , Aurora Quinasa A , Aurora Quinasa B , Inhibidores de Proteínas Quinasas , Resorcinoles , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/química , Adenosina Trifosfato/farmacología , Aurora Quinasa A/antagonistas & inhibidores , Aurora Quinasa A/química , Aurora Quinasa A/metabolismo , Aurora Quinasa B/antagonistas & inhibidores , Aurora Quinasa B/química , Aurora Quinasa B/metabolismo , Línea Celular , Humanos , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Resorcinoles/química , Resorcinoles/farmacologíaRESUMEN
Currently, considerable interest exists with regard to the dissociation of close packed aminoacids within proteins, in the course of unfolding, which could result in either wet or dry moltenglobules. The progressive disjuncture of residues constituting the hydrophobic core ofcyclophilin from L. donovani (LdCyp) has been studied during the thermal unfolding of the molecule, by molecular dynamics simulations. LdCyp has been represented as a surface contactnetwork (SCN) based on the surface complementarity (Sm) of interacting residues within themolecular interior. The application of Sm to side chain packing within proteins make it a very sensitive indicator of subtle perturbations in packing, in the thermal unfolding of the protein. Network based metrics have been defined to track the sequential changes in the disintegration ofthe SCN spanning the hydrophobic core of LdCyp and these metrics prove to be highly sensitive compared to traditional metrics in indicating the increased conformational (and dynamical) flexibility in the network. These metrics have been applied to suggest criteria distinguishing DMG, WMG and transition state ensembles and to identify key residues involved in crucial conformational/topological events during the unfolding process.
Asunto(s)
Ciclofilinas/química , Proteínas Protozoarias/química , Interacciones Hidrofóbicas e Hidrofílicas , Leishmania donovani/química , Simulación de Dinámica Molecular , Conformación Proteica , Estructura Secundaria de Proteína , Desplegamiento Proteico , Propiedades de SuperficieRESUMEN
Lamin B1 is one of the major constituents of the nuclear lamina, a filamentous network underlying the nucleoplasmic side of the inner nuclear membrane. Homopolymerization of lamin B1, coupled to the homotypic and heterotypic association of other lamin types, is central to building the higher order network pattern inside the nucleus. This in turn maintains the mechanical and functional integrity of the lamina. We have characterized the molecular basis of the self-association of lamin B1 using spectroscopic and calorimetric methods. We report that concentration dependent lamin B1 oligomerization involves significant alterations in secondary and tertiary structures of the protein resulting in fairly observable compaction in size. Comparison of the energetics of the homotypic association of lamin B1 with that of lamin A reported earlier led to the finding that lamin A oligomers had higher thermodynamic stability. This leads us to conjecture that lamin B1 has less stress bearing ability compared to lamin A.
Asunto(s)
Lamina Tipo B/química , Multimerización de Proteína , Calorimetría , Dicroismo Circular , Clonación Molecular , Dispersión Dinámica de Luz , Humanos , Lamina Tipo B/genética , Lamina Tipo B/aislamiento & purificación , Espectrometría de Fluorescencia , TermodinámicaRESUMEN
The role of Mithramycin as an anticancer drug has been well studied. Sarcoma is a type of cancer arising from cells of mesenchymal origin. Though incidence of sarcoma is not of significant percentage, it becomes vital to understand the role of Mithramycin in controlling tumor progression of sarcoma. In this article, we have analyzed the global gene expression profile changes induced by Mithramycin in two different sarcoma lines from whole genome gene expression profiling microarray data. We have found that the primary mode of action of Mithramycin is by global repression of key cellular processes and gene families like phosphoproteins, kinases, alternative splicing, regulation of transcription, DNA binding, regulation of histone acetylation, negative regulation of gene expression, chromosome organization or chromatin assembly and cytoskeleton.
RESUMEN
Recognition of core histone components of chromatin along with chromosomal DNA by a class of small molecule modulators is worth examining to evaluate their intracellular mode of action. A plant alkaloid ellipticine (ELP) which is a putative anticancer agent has so far been reported to function via DNA intercalation, association with topoisomerase II and binding to telomere region. However, its effect upon the potential intracellular target, chromatin is hitherto unreported. Here we have characterized the biomolecular recognition between ELP and different hierarchical levels of chromatin. The significant result is that in addition to DNA, it binds to core histone(s) and can be categorized as a 'dual binder'. As a sequel to binding with histone(s) and core octamer, it alters post-translational histone acetylation marks. We have further demonstrated that it has the potential to modulate gene expression thereby regulating several key biological processes such as nuclear organization, transcription, translation and histone modifications.
Asunto(s)
Cromatina/efectos de los fármacos , Elipticinas/farmacología , Acilación , Cromatina/metabolismo , Dicroismo Circular , Histonas/metabolismo , Unión Proteica , Espectrometría de FluorescenciaRESUMEN
Guanine rich sequences present in the promoter region of oncogenes could fold into G-quadruplexes and modulate transcription. Equilibrium between folding and unfolding of the quadruplexes in these regions play important role in disease processes. We have studied the effect of a putative anticancer agent chelerythrine on G-rich NHE III1 present in the promoter region of c-myc oncogene. We have demonstrated the ability of chelerythrine, a telomerase inhibitor, to block the hybridization of Pu27 with its complementary strand via folding it into a quadruplex structure. Calorimetry shows that the association of Pu27 with chelerythrine is primarily enthalpy driven with high binding affinity (â¼10(5) M(-1)). The association does not lead to any major structural perturbation of Pu27. The resulting 2:1 complex has enhanced stability as compared to free Pu27. Another notable feature is that the presence of molecular crowding agent like ficoll 70 does not change the mode of recognition though the binding affinity decreases. We suggest that the anticancer activity of chelerythrine could be ascribed to its ability to stabilize the quadruplex structure in the c-myc promoter region thereby downregulating its transcription.
Asunto(s)
Benzofenantridinas/farmacología , Genes myc , Regiones Promotoras Genéticas/efectos de los fármacos , Benzofenantridinas/metabolismo , Rastreo Diferencial de Calorimetría , Dicroismo Circular , Entropía , Ficoll/farmacología , G-Cuádruplex , Terapia Molecular DirigidaRESUMEN
Small molecules that interact with G-quadruplex structures formed by the human telomeric region and stabilize them have the potential to evolve as anticancer therapeutic agents. Herein we report the interaction of a putative anticancer agent from a plant source, chelerythrine, with the human telomeric DNA sequence. It has telomerase inhibitory potential as demonstrated from telomerase repeat amplification assay in cancer cell line extract. We have attributed this to the quadruplex binding potential of the molecule and characterized the molecular details of the interaction by means of optical spectroscopy such as absorbance and circular dichroism and calorimetric techniques such as isothermal titration calorimetry and differential scanning calorimetry. The results show that chelerythrine binds with micromolar dissociation constant and 2:1 binding stoichiometry to the human telomeric DNA sequence. Chelerythrine association stabilizes the G-quadruplex. Nuclear magnetic resonance spectroscopy ((1)H and (31)P) shows that chelerythrine binds to both G-quartet and phosphate backbone of the quadruplex leading to quadruplex aggregation. Molecular dynamics simulation studies support the above inferences and provide further insight into the mechanism of ligand binding. The specificity toward quartet binding for chelerythrine is higher compared to that of groove binding. MM-PBSA calculation mines out the energy penalty for quartet binding to be -4.7 kcal/mol, whereas that of the groove binding is -1.7 kcal/mol. We propose that the first chelerythrine molecule binds to the quartet followed by a second molecule which binds to the groove. This second molecule might bring about aggregation of the quadruplex structure which is evident from the results of nuclear magnetic resonance.
Asunto(s)
Secuencia de Bases/fisiología , Benzofenantridinas/química , Benzofenantridinas/metabolismo , Agregado de Proteínas/fisiología , Telómero/química , Telómero/metabolismo , Alcaloides/metabolismo , Cristalografía por Rayos X , G-Cuádruplex , Células HeLa , Humanos , Unión Proteica/fisiología , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Pathogenic bacteria that are resistant to ß-lactam antibiotics mostly utilize serine ß-lactamases to degrade the antibiotics. Current studies have shown that different subclasses of metallo ß-lactamases (E[MBL]) are involved in the defense mechanism of drug resistant bacteria. Here we report that the Zn(2+) containing subclass B1 E[MBL] from Bacillus cereus binds to a naturally occurring anti-cancer drug mithramycin (MTR). Spectroscopic (CD and fluorescence) and isothermal titration calorimetry studies show that MTR forms a high affinity complex with the Zn(2+) ion containing E[MBL]. Abolished interaction of MTR with apo E[MBL] suggests that the formation of this high affinity complex occurs due to the potential of MTR to bind bivalent metal ions like Zn(2+). Furthermore, CD spectroscopy, dynamic light scattering and differential scanning calorimetry studies indicate that the strong association with sub-micromolar dissociation constant leads to an alteration in the enzyme conformation at both secondary and tertiary structural levels. The enzyme activity decreases as a consequence to this conformational disruption arising from the formation of a ternary complex involving MTR, catalytic Zn(2+) and the enzyme. Our results suggest that the naturally occurring antibiotic MTR, a generic drug, has the potential as an E[MBL] inhibitor.
Asunto(s)
Antibióticos Antineoplásicos/química , Bacillus cereus/enzimología , Proteínas Bacterianas/química , Plicamicina/química , Zinc/química , beta-Lactamasas/químicaRESUMEN
Mithramycin (MTR), an aureolic acid group of antitumor antibiotic is used for the treatment of several types of tumors. We have reported here the association of MTR with an essential micronutrient, manganese (Mn(2+)). Spectroscopic methods have been used to characterize and understand the kinetics and mechanism of complex formation between them. MTR forms a single type of complex with Mn(2+) in the mole ratio of 2:1 [MTR: Mn(2+)] via a two step kinetic process. Circular dichroism (CD) spectroscopic study indicates that the complex [(MTR)2 Mn(2+)] has a right-handed twist conformation similar in structure with the complexes reported for Mg(2+) and Zn(2+). This conformation allows binding via minor groove of DNA with (G, C) base preference during the interaction with double-stranded B-DNA. Using absorbance, fluorescence, and CD spectroscopy we have shown that [(MTR)2 Mn(2+)] complex binds to double-stranded DNA with an apparent dissociation constant of 32 µM and binding site size of 0.2 (drug/nucleotide). It binds to chicken liver chromatin with apparent dissociation constant value 298 µM. Presence of histone proteins in chromatin inhibits the accessibility of the complex for chromosomal DNA. We have also shown that MTR binds to Mn(2+) containing metalloenzyme manganese superoxide dismutase from Escherichia coli.
Asunto(s)
Antibióticos Antineoplásicos/química , Manganeso/química , Plicamicina/química , Animales , Pollos , Cromatina/química , ADN/química , Escherichia coli , Proteínas de Escherichia coli/química , Cinética , Superóxido Dismutasa/química , TermodinámicaRESUMEN
Mithramycin (MTR) is a clinically approved DNA-binding antitumor antibiotic currently in Phase 2 clinical trials at National Institutes of Health for treatment of osteosarcoma. In view of the resurgence in the studies of this generic antibiotic as a human medicine, we have examined the binding properties of MTR with the integral component of chromatin - histone proteins - as a part of our broad objective to classify DNA-binding molecules in terms of their ability to bind chromosomal DNA alone (single binding mode) or both histones and chromosomal DNA (dual binding mode). The present report shows that besides DNA, MTR also binds to core histones present in chromatin and thus possesses the property of dual binding in the chromatin context. In contrast to the MTR-DNA interaction, association of MTR with histones does not require obligatory presence of bivalent metal ion like Mg(2+). As a consequence of its ability to interact with core histones, MTR inhibits histone H3 acetylation at lysine 18, an important signature of active chromatin, in vitro and ex vivo. Reanalysis of microarray data of Ewing sarcoma cell lines shows that upon MTR treatment there is a significant down regulation of genes, possibly implicating a repression of H3K18Ac-enriched genes apart from DNA-binding transcription factors. Association of MTR with core histones and its ability to alter post-translational modification of histone H3 clearly indicates an additional mode of action of this anticancer drug that could be implicated in novel therapeutic strategies.
RESUMEN
Cyclophilin from Leishmania donovani (LdCyp) is a ubiquitous peptidyl-prolyl cis-trans isomerase involved in a host of important cellular activities, such as signaling, heat shock response, chaperone activity, mitochondrial pore maintenance and regulation of HIV-1 infectivity. It also acts as the prime cellular target for the auto-immune drug cyclosporine A (CsA). LdCyp is composed of a beta barrel encompassing the unique hydrophobic core of the molecule and is flanked by two helices (H1, H2) on either end of the barrel. The protein contains a lone partially exposed tryptophan. In the present work the equilibrium unfolding of LdCyp has been studied by fluorescence, circular dichroism and the non-coincidence of their respective Cm's, indicates a non-two state transition. This fact was further corroborated by binding studies of the protein with bis-ANS and the lack of an isochromatic point in far UV CD. The thermal stability of the possible intermediates was characterized by differential scanning calorimetry. Further, MD simulations performed at 310, 400 and 450K exhibited the tendency of both helices to partially unwind and adopt non-native geometries with respect to the core, quite early in the unfolding process, in contrast to the relatively stable beta barrel.
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Ciclofilinas/química , Leishmania donovani/enzimología , Desplegamiento Proteico , Guanidina/farmacología , Simulación de Dinámica Molecular , Estructura Secundaria de Proteína , Desplegamiento Proteico/efectos de los fármacosRESUMEN
Eukaryotic DNA is compacted in the form of chromatin, in a complex with histones and other non-histone proteins. The intimate association of DNA and histones in chromatin raises the possibility that DNA-interactive small molecules may bind to chromatin-associated proteins such as histones. Employing biophysical and biochemical techniques we have characterized the interaction of a classical intercalator, ethidium bromide (EB) and its structural analogue propidium iodide (PI) with hierarchical genomic components: long chromatin, chromatosome, core octamer and chromosomal DNA. Our studies show that EB and PI affect both chromatin structure and function, inducing chromatin compaction and disruption of the integrity of the chromatosome. Calorimetric studies and fluorescence measurements of the ligands demonstrated and characterized the association of these ligands with core histones and the intact octamer in absence of DNA. The ligands affect acetylation of histone H3 at lysine 9 and acetylation of histone H4 at lysine 5 and lysine 8 ex vivo. PI alters the post-translational modifications to a greater extent than EB. This is the first report showing the dual binding (chromosomal DNA and core histones) property of a classical intercalator, EB, and its longer analogue, PI, in the context of chromatin.