RESUMEN
Individual oncogenic KRAS mutants confer distinct differences in biochemical properties and signaling for reasons that are not well understood. KRAS activity is closely coupled to protein dynamics and is regulated through two interconverting conformations: state 1 (inactive, effector binding deficient) and state 2 (active, effector binding enabled). Here, we use 31P NMR to delineate the differences in state 1 and state 2 populations present in WT and common KRAS oncogenic mutants (G12C, G12D, G12V, G13D, and Q61L) bound to its natural substrate GTP or a commonly used nonhydrolyzable analog GppNHp (guanosine-5'-[(ß,γ)-imido] triphosphate). Our results show that GppNHp-bound proteins exhibit significant state 1 population, whereas GTP-bound KRAS is primarily (90% or more) in state 2 conformation. This observation suggests that the predominance of state 1 shown here and in other studies is related to GppNHp and is most likely nonexistent in cells. We characterize the impact of this differential conformational equilibrium of oncogenic KRAS on RAF1 kinase effector RAS-binding domain and intrinsic hydrolysis. Through a KRAS G12C drug discovery, we have identified a novel small-molecule inhibitor, BBO-8956, which is effective against both GDP- and GTP-bound KRAS G12C. We show that binding of this inhibitor significantly perturbs state 1-state 2 equilibrium and induces an inactive state 1 conformation in GTP-bound KRAS G12C. In the presence of BBO-8956, RAF1-RAS-binding domain is unable to induce a signaling competent state 2 conformation within the ternary complex, demonstrating the mechanism of action for this novel and active-conformation inhibitor.
Asunto(s)
Proteínas Proto-Oncogénicas p21(ras) , Proteínas ras , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas ras/metabolismo , Guanosina Trifosfato/metabolismo , Espectroscopía de Resonancia Magnética , Transducción de Señal , MutaciónRESUMEN
Development of new targeted inhibitors for oncogenic KRAS mutants may benefit from insight into how a given mutation influences the accessibility of protein residues and how compounds interact with mutant or wild-type KRAS proteins. Targeted proteomic analysis, a key validation step in the KRAS inhibitor development process, typically involves both intact mass- and peptide-based methods to confirm compound localization or quantify binding. However, these methods may not always provide a clear picture of the compound binding affinity for KRAS, how specific the compound is to the target KRAS residue, and how experimental conditions may impact these factors. To address this, we have developed a novel top-down proteomic assay to evaluate in vitro KRAS4B-compound engagement while assessing relative quantitation in parallel. We present two applications to demonstrate the capabilities of our assay: maleimide-biotin labeling of a KRAS4BG12D cysteine mutant panel and treatment of three KRAS4B proteins (WT, G12C, and G13C) with small molecule compounds. Our results show the time- or concentration-dependence of KRAS4B-compound engagement in context of the intact protein molecule while directly mapping the compound binding site.
Asunto(s)
Proteómica , Proteínas Proto-Oncogénicas p21(ras) , Proteínas Proto-Oncogénicas p21(ras)/genética , Mutación , Sitios de UniónRESUMEN
Control over the simultaneous delivery of different functionalities and their synchronized intracellular activation can greatly benefit the fields of RNA and DNA biomedical nanotechnologies and allow for the production of nanoparticles and various switching devices with controllable functions. We present a system of multiple split functionalities embedded in the cognate pairs of RNA-DNA hybrids which are programmed to recognize each other, re-associate and form a DNA duplex while also releasing the split RNA fragments which upon association regain their original functions. Simultaneous activation of three different functionalities (RNAi, Förster resonance energy transfer and RNA aptamer) confirmed by multiple in vitro and cell culture experiments prove the concept. To automate the design process, a novel computational tool that differentiates between the thermodynamic stabilities of RNA-RNA, RNA-DNA and DNA-DNA duplexes was developed. Moreover, here we demonstrate that besides being easily produced by annealing synthetic RNAs and DNAs, the individual hybrids carrying longer RNAs can be produced by RNA polymerase II-dependent transcription of single-stranded DNA templates.
Asunto(s)
ADN/química , ARN/química , Aptámeros de Nucleótidos/química , Línea Celular Tumoral , Transferencia Resonante de Energía de Fluorescencia , Humanos , Modelos Moleculares , Interferencia de ARN , ARN Polimerasa II/metabolismo , Termodinámica , Transcripción GenéticaRESUMEN
PABA/NO [O(2)-{2,4-dinitro-5-[4-(N-methylamino)benzoyloxy]phenyl} 1-(N,N-dimethylamino) diazen-1-ium-1,2-diolate] is a nitric oxide (NO)-releasing arylating agent designed to be selectively activated by reaction with glutathione (GSH) on catalysis by glutathione S-transferase P1 (GSTP1), an enzyme frequently overexpressed in cancer cells. PABA/NO has proven active in several cancer models in vitro and in vivo, but its tendency to be metabolized via a variety of pathways, some that generate inactive metabolites and hydrolysis products, limits its potential as a drug. Here we show that a simple replacement of cyano for nitro at the 4 position to give compound 4b ('p-cyano-PABA/NO') has the dual effect of slowing the undesired side reactions while enhancing the proportion of NO release and arylating activity on catalysis by GSTP1. Compound 4b showed increased resistance to hydrolysis and uncatalyzed reaction with GSH, along with a more favorable product distribution in the presence of GSTP1. It also showed significant proapoptotic activity. The data suggest p-cyano-PABA/NO to be a more promising prodrug than PABA/NO, with better selectivity toward cancer cells.
Asunto(s)
Ácido 4-Aminobenzoico/química , Gutatión-S-Transferasa pi/metabolismo , Óxido Nítrico/química , Ácido 4-Aminobenzoico/metabolismo , Ácido 4-Aminobenzoico/farmacología , Biocatálisis , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Glutatión/química , Glutatión/metabolismo , Gutatión-S-Transferasa pi/química , Células HL-60 , Humanos , Isomerismo , Cinética , Conformación Molecular , Óxido Nítrico/metabolismo , Óxido Nítrico/toxicidadRESUMEN
MALDI-TOF mass spectrometry enables high-throughput screening of covalent fragment libraries and SAR compound progressions of selective KRAS G12C inhibitors. Using the MALDI-TOF platform instead of the more traditional ESI-MS TOF/orbitrap instrumentation can radically shorten sample acquisition time, allowing up to 384 samples to be screened in 30 min. The typical throughput for a covalent library screen is 1152 samples per 8 h, including processing, calculation, and reporting steps. The throughput can be doubled without any significant assay modification.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento , Proteínas Proto-Oncogénicas p21(ras) , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Proteínas Proto-Oncogénicas p21(ras)/genética , Ensayos Analíticos de Alto Rendimiento/métodos , MutaciónRESUMEN
With recent advances proving that effective inhibition of KRAS is possible, there have been significant efforts made to develop inhibitors of specific mutant alleles. Here we describe a detailed protocol that employs homogeneous time-resolved fluorescence (HTRF) to identify compounds acting on KRAS signaling in malignant cell lines. This method allows for high-throughput, cell-based screens of large compound libraries for the development of RAS-targeted therapeutics.
Asunto(s)
Antineoplásicos , Proteínas Proto-Oncogénicas p21(ras) , Proteínas Proto-Oncogénicas p21(ras)/genética , Antineoplásicos/farmacología , Línea Celular , Transducción de Señal , Ensayos Analíticos de Alto Rendimiento/métodos , Línea Celular TumoralRESUMEN
We present a novel method to determine engagement and specificity of KRAS4B-targeting compounds in vitro. By employing top-down mass spectrometry (MS), which analyzes intact and modified protein molecules (proteoforms), we can directly visualize and confidently characterize each KRAS4B species within compound-treated samples. Moreover, by employing targeted MS2 fragmentation, we can precisely localize each compound molecule to a specific residue on a given KRAS4B proteoform. This method allows us to comprehensively evaluate compound specificity, clearly detect nonspecific binding events, and determine the order and frequency with which they occur. We provide two proof-of-concept examples of our method employing publicly available compounds, along with detailed protocols for sample preparation, top-down MS data acquisition, targeted proteoform MS2 fragmentation, and analysis of the resulting data. Our results demonstrate the concentration dependence of KRAS4B-compound engagement and highlight the ability of top-down MS to directly map compound binding location(s) without disrupting the KRAS4B primary structure. Our hope is that this novel method may help accelerate the identification of new successful targeted inhibitors for KRAS4B and other RAS isoforms.
Asunto(s)
Proteínas Proto-Oncogénicas p21(ras) , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Humanos , Espectrometría de Masas/métodos , Unión Proteica , Espectrometría de Masas en Tándem/métodosRESUMEN
Prior analysis of intact and modified protein forms (proteoforms) of KRAS4B isolated from cell lines and tumor samples by top-down mass spectrometry revealed the presence of novel posttranslational modifications (PTMs) and potential evidence of context-specific KRAS4B modifications. However, low endogenous proteoform signal resulted in ineffective characterization, making it difficult to visualize less abundant PTMs or perform follow-up PTM validation using standard proteomic workflows. The NCI RAS Initiative has developed a model system, whereby KRAS4B bearing an N-terminal FLAG tag can be stably expressed within a panel of cancer cell lines. Herein, we present a method for combining immunoprecipitation with complementary proteomic methods to directly analyze N-terminally FLAG-tagged KRAS4B proteoforms and PTMs. We provide detailed protocols for FLAG-KRAS4B purification, proteoform analysis by targeted top-down LC-MS/MS, and validation of abundant PTMs by bottom-up LC-MS/MS with example results.
Asunto(s)
Proteómica , Espectrometría de Masas en Tándem , Cromatografía Liquida , Espectrometría de Masas en Tándem/métodos , Proteómica/métodos , Procesamiento Proteico-Postraduccional , Cromatografía Líquida con Espectrometría de MasasRESUMEN
Attachment of glutathione (GSH) to cysteine residues in proteins (S-glutathionylation) is a reversible post-translational modification that can profoundly alter protein structure and function. Often serving in a protective role, for example, by temporarily saving protein thiols from irreversible oxidation and inactivation, glutathionylation can be identified and semiquantitatively assessed using anti-GSH antibodies, thought to be specific for recognition of the S-glutathionylation modification. Here, we describe an alternate mechanism of protein glutathionylation in which the sulfur atoms of the GSH and the protein's thiol group are covalently bound via a cross-linking agent, rather than through a disulfide bond. This form of thiol cross-linking has been shown to occur and has been confirmed by mass spectrometry at the solution chemistry level, as well as in experiments documenting the potent antiproliferative activity of the bis-diazeniumdiolate Double JS-K in H1703 cells in vitro and in vivo. The modification is recognized by the anti-GSH antibody as if it were authentic S-glutathionylation, requiring mass spectrometry to distinguish between them.
Asunto(s)
Antineoplásicos/farmacología , Compuestos Azo/farmacología , Glutatión/metabolismo , Piperazinas/farmacología , Acetilcisteína/química , Actinas/metabolismo , Animales , Antineoplásicos/química , Compuestos Azo/química , Línea Celular Tumoral , Dinitrofluorobenceno/análogos & derivados , Dinitrofluorobenceno/química , Femenino , Glutatión/química , Humanos , Ratones , Ratones Desnudos , Nanopartículas/química , Óxido Nítrico/metabolismo , Oxígeno/metabolismo , Piperazinas/químicaRESUMEN
We report an indirect method for synthesis of previously inaccessible diazeniumdiolated carbamates. Synthesis involves use of previously reported triisopropylsilyloxymethylated isopropylamine diazeniumdiolate (TOM-ylated IPA/NO). These novel diazeniumdiolated carbamate prodrugs upon activation release nitric oxide (NO) similar to their secondary amine counterparts. They are also efficient sources of intracellular NO. These prodrugs may have potential applications as therapeutic NO-donors.
Asunto(s)
Carbamatos/química , Carbamatos/farmacología , Donantes de Óxido Nítrico/química , Donantes de Óxido Nítrico/farmacología , Profármacos/química , Profármacos/farmacología , Compuestos Azo/síntesis química , Compuestos Azo/química , Compuestos Azo/farmacología , Carbamatos/síntesis química , Línea Celular , Técnicas de Química Sintética/métodos , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Óxido Nítrico/metabolismo , Donantes de Óxido Nítrico/síntesis química , Profármacos/síntesis químicaRESUMEN
JS-K, a diazeniumdiolate-based nitric oxide (NO)-releasing prodrug, is currently in late pre-clinical development as an anti-cancer drug candidate. This prodrug was designed to be activated by glutathione (GSH) to release NO. To increase the potency of JS-K, we are investigating the effect of slowing the reaction of the prodrugs with GSH. Herein, we report the effect of replacement of nitro group(s) by other electron-withdrawing group(s) in JS-K and its homo-piperazine analogues on GSH activation and the drugs' biological activity. We show that nitro-to-cyano substitution increases the half-life of the prodrug in the presence of GSH without compromising the compound's in vivo antitumor activity.
Asunto(s)
Antineoplásicos/química , Compuestos Azo/química , Glutatión/metabolismo , Profármacos/química , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Compuestos Azo/farmacología , Estabilidad de Medicamentos , Semivida , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Cinética , Ratones , Neoplasias/tratamiento farmacológico , Óxido Nítrico/metabolismo , Profármacos/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
RAS proteins cycling between the active-form (GTP-bound) and inactive-form (GDP-bound) play a key role in cell signaling pathways that control cell survival, proliferation, and differentiation. Mutations at codon 12, 13, and 61 in RAS are known to attenuate its GTPase activity favoring the RAS active state and constitutively active downstream signaling. This hyperactivation accounts for various malignancies including pancreatic, lung, and colorectal cancers. Active KRAS is found to exist in equilibrium between two rapidly interconverting conformational states (State1-State2) in solution. Due to this dynamic feature of the protein, the 1H-15N correlation cross-peak signals of several amino acid (AA) residues of KRAS belonging to the flexible loop regions are absent from its 2D 1H-15N HSQC spectrum within and near physiological solution pH. A threonine to serine mutation at position 35 (T35S) shifts the interconverting equilibrium to State1 conformation and enables the emergence of such residues in the 2D 1H-15N HSQC spectrum due to gained conformational rigidity. We report here the 1HN, 15N, and 13C backbone resonance assignments for the 19.2 kDa (AA 1-169) protein constructs of KRAS-GppNHp harboring T35S mutation (KRAST35S/C118S-GppNHp) and of its oncogenic counterpart harboring the Q61L mutation (KRAST35S/Q61L/C118S-GppNHp) using heteronuclear, multidimensional NMR spectroscopy at 298 K. High resolution NMR data allowed the unambiguous assignments of 1H-15N correlation cross-peaks for all the residues except for Met1. Furthermore, 2D 1H-15N HSQC overlay of two proteins assisted in determination of Q61L mutation-induced chemical shift perturbations for select residues in the regions of P-loop, Switch-II, and helix α3.
Asunto(s)
Proteínas Proto-Oncogénicas p21(ras) , Guanosina Trifosfato/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Proteínas Proto-Oncogénicas p21(ras)/química , Proteínas Proto-Oncogénicas p21(ras)/genéticaRESUMEN
Non-small-cell lung cancer is among the most common and deadly forms of human malignancies. Early detection is unusual, and there are no curative therapies in most cases. Diazeniumdiolate-based nitric oxide (NO)-releasing prodrugs are a growing class of promising NO-based therapeutics. Here, we show that O(2)-(2,4-dinitrophenyl)-1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) is a potent cytotoxic agent against a subset of human non-small-cell lung cancer cell lines both in vitro and as xenografts in mice. JS-K treatment led to 75% reduction in the growth of H1703 lung adenocarcinoma cells in vivo. Differences in sensitivity to JS-K in different lung cancer cell lines seem to be related to their endogenous levels of reactive oxygen species (ROS)/reactive nitrogen species (RNS). Other related factors, levels of peroxiredoxin 1 (PRX1) and 8-oxo-deoxyguanosine glycosylase (OGG1), also correlated with drug sensitivity. Treatment of the lung adenocarcinoma cells with JS-K resulted in oxidative/nitrosative stress in cells with high basal levels of ROS/RNS, which, combined with the arylating properties of the compound, was reflected in glutathione depletion and alteration in cellular redox potential, mitochondrial membrane permeabilization, and cytochrome c release. Inactivation of manganese superoxide dismutase by nitration was associated with increased superoxide and significant DNA damage. Apoptosis followed these events. Taken together, the data suggest that diazeniumdiolate-based NO-releasing prodrugs may have application as a personalized therapy for lung cancers characterized by high levels of ROS/RNS. PRX1 and OGG1 proteins, which can be easily measured, could function as biomarkers for identifying tumors sensitive to the therapy.
Asunto(s)
Antineoplásicos/farmacología , Compuestos Azo/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Piperazinas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Animales , Apoptosis/efectos de los fármacos , Compuestos Azo/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Daño del ADN , Femenino , Glutatión/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Mitocondrias/metabolismo , Piperazinas/uso terapéutico , Especies de Nitrógeno Reactivo/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We report the stabilization of the nitric oxide (NO) prodrugs and anticancer lead compounds, PABA/NO (O(2)-{2,4-dinitro-5-[4-(N-methylamino)benzoyloxy]phenyl} 1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate) and "Double JS-K" 1,5-bis-{1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diol-2-ato}-2,4-dinitrobenzene, through their incorporation into polymer-protected nanoparticles. The prodrugs were formulated in block copolymer-stabilized nanoparticles with sizes from 220 to 450 nm by a novel rapid precipitation process. The block copolymers, with polyethylene glycol (PEG) soluble blocks, provide a steric barrier against NO prodrug activation by glutathione. Too rapid activation and NO release has been a major barrier to effective administration of this class of compounds. The nanoparticle stabilized PABA/NO are protected from attack by glutathione as evidenced by a significant increase in time taken for 50% decomposition from 15 min (unformulated) to 5 h (formulated); in the case of Double JS-K, the 50% decomposition time was extended from 4.5 min (unformulated) to 40 min (formulated). The more hydrophobic PABA/NO produced more stable nanoparticles and correspondingly more extended release times in comparison with Double JS-K. The hydrophobic blocks of the polymer were either polystyrene or polylactide. Both blocks produced nanoparticles of approximately the same size and release kinetics. This combination of PEG-protected nanoparticles with sizes appropriate for cancer targeting by enhanced permeation and retention (EPR) and delayed release of NO may afford enhanced therapeutic benefit.
Asunto(s)
Antineoplásicos/administración & dosificación , Donantes de Óxido Nítrico/administración & dosificación , Profármacos/administración & dosificación , Ácido 4-Aminobenzoico/administración & dosificación , Ácido 4-Aminobenzoico/química , Compuestos Azo/administración & dosificación , Compuestos Azo/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Preparaciones de Acción Retardada , Estabilidad de Medicamentos , Humanos , Nanopartículas , Donantes de Óxido Nítrico/química , Tamaño de la Partícula , Piperazinas/administración & dosificación , Piperazinas/química , Polietilenglicoles , Profármacos/química , para-AminobenzoatosRESUMEN
Here we report a number of novel JS-K structural analogues with sub-micromolar anti-proliferative activities against human leukemia cell lines HL-60 and U937; JS-K is the anti-cancer lead compound O(2)-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate. The ability of these compounds to generate intracellular nitric oxide correlated well with their observed anti-proliferative effects: analogues that had potent inhibitory activity against leukemia cells formed elevated levels of intracellular nitric oxide.
Asunto(s)
Antineoplásicos/síntesis química , Compuestos Azo/química , Piperazinas/química , Antineoplásicos/química , Antineoplásicos/farmacología , Compuestos Azo/síntesis química , Compuestos Azo/farmacología , Línea Celular Tumoral , Humanos , Óxido Nítrico/metabolismo , Piperazinas/síntesis química , Piperazinas/farmacologíaRESUMEN
Nitric oxide (NO) prodrugs such as O(2)-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) are a growing class of promising NO-based therapeutics. Nitric oxide release from the anti-cancer lead compound, JS-K, is proposed to occur through a nucleophilic aromatic substitution by glutathione (GSH) catalyzed by glutathione S-transferase (GST) to form a diazeniumdiolate anion that spontaneously releases NO. In this study, a number of structural analogues of JS-K were synthesized and their chemical and biological properties were compared with those of JS-K. The homopiperazine analogue of JS-K showed anti-cancer activity that is comparable with that of JS-K but with a diminished reactivity towards both GSH and GSH/GST; both the aforementioned compounds displayed no cytotoxic activity towards normal renal epithelial cell line at concentrations where they significantly diminished the proliferation of a panel of renal cancer cell lines. These properties may prove advantageous in the further development of this class of nitric oxide prodrugs as cancer therapeutic agents.
Asunto(s)
Antineoplásicos/farmacología , Compuestos Azo/química , Glutatión Transferasa/metabolismo , Glutatión/metabolismo , Óxido Nítrico/metabolismo , Piperazinas/química , Profármacos/farmacología , Animales , Antineoplásicos/química , Compuestos Azo/síntesis química , Compuestos Azo/farmacología , Línea Celular Tumoral , Células HL-60 , Humanos , Ratones , Donantes de Óxido Nítrico , Piperazinas/síntesis química , Piperazinas/farmacología , Profármacos/síntesis química , Profármacos/química , Células U937RESUMEN
We report the antitumor effects of nitric oxide (NO) releasing derivatives of the PARP-1 inhibitor olaparib (1). Compound 5b was prepared by coupling the carboxyl group of 3b and the free amino group of arylated diazeniumdiolated piperazine 4. Analogue 5a has the same structure except that the F is replaced by H. Compound 13 is the same as 5b except that a Me2N-N(O)âNO- group was added para and ortho to the nitro groups of the dinitrophenyl ring. The resulting prodrugs are activated by glutathione in a reaction accelerated by glutathione S-transferase P1 (GSTP1), an enzyme frequently overexpressed in cancers. This metabolism generates NO plus a PARP-1 inhibitor simultaneously, consuming reducing equivalents, leading to DNA damage concomitant with inhibition of DNA repair, and in the case of 13 inducing cross-linking glutathionylation of proteins. Compounds 5b and 13 reduced the growth rates of A549 human lung adenocarcinoma xenografts with no evidence of systemic toxicity.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Gutatión-S-Transferasa pi/metabolismo , Neoplasias/enzimología , Óxido Nítrico/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Apoptosis/efectos de los fármacos , Ácidos Borónicos/farmacología , Bortezomib , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayo Cometa , Daño del ADN , Diseño de Fármacos , Sinergismo Farmacológico , Inhibidores Enzimáticos/farmacología , Humanos , Isoenzimas/efectos de los fármacos , Modelos Moleculares , Neoplasias/tratamiento farmacológico , Poli(ADP-Ribosa) Polimerasa-1 , Profármacos/síntesis química , Profármacos/farmacología , Pirazinas/farmacología , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
JS-K is a nitric oxide (NO)-releasing prodrug of the O (2)-arylated diazeniumdiolate family that has demonstrated pronounced cytotoxicity and antitumor properties in a variety of cancer models both in vitro and in vivo. The current study of the metabolic actions of JS-K was undertaken to investigate mechanisms of its cytotoxicity. Consistent with model chemical reactions, the activating step in the metabolism of JS-K in the cell is the dearylation of the diazeniumdiolate by glutathione (GSH) via a nucleophilic aromatic substitution reaction. The resulting product (CEP/NO anion) spontaneously hydrolyzes, releasing two equivalents of NO. The GSH/GSSG redox couple is considered to be the major redox buffer of the cell, helping maintain a reducing environment under basal conditions. We have quantified the effects of JS-K on cellular GSH content, and show that JS-K markedly depletes GSH, due to JS-K's rapid uptake and cascading release of NO and reactive nitrogen species. The depletion of GSH results in alterations in the redox potential of the cellular environment, initiating MAPK stress signaling pathways, and inducing apoptosis. Microarray analysis confirmed signaling gene changes at the transcriptional level and revealed alteration in the expression of several genes crucial for maintenance of cellular redox homeostasis, as well as cell proliferation and survival, including MYC. Pre-treating cells with the known GSH precursor and nucleophilic reducing agent N-acetylcysteine prevented the signaling events that lead to apoptosis. These data indicate that multiplicative depletion of the reduced glutathione pool and deregulation of intracellular redox balance are important initial steps in the mechanism of JS-K's cytotoxic action.
Asunto(s)
Compuestos Azo/farmacología , Leucemia/metabolismo , Donantes de Óxido Nítrico/farmacología , Piperazinas/farmacología , Profármacos/síntesis química , Acetilcisteína/farmacología , Compuestos Azo/síntesis química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glutatión/metabolismo , Humanos , Leucemia/patología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Óxido Nítrico/metabolismo , Donantes de Óxido Nítrico/síntesis química , Oxidación-Reducción/efectos de los fármacos , Piperazinas/síntesis química , Profármacos/farmacología , Especies de Nitrógeno Reactivo/metabolismoRESUMEN
Split-protein systems, an approach that relies on fragmentation of proteins with their further conditional re-association to form functional complexes, are increasingly used for various biomedical applications. This approach offers tight control of protein functions and improved detection sensitivity. Here we report a similar technique based on a pair of RNA-DNA hybrids that can be used generally for triggering different split functionalities. Individually, each hybrid is inactive but when two cognate hybrids re-associate, different functionalities are triggered inside mammalian cells. As a proof of concept, this work mainly focuses on the activation of RNA interference. However, the release of other functionalities (such as resonance energy transfer and RNA aptamer) is also shown. Furthermore, in vivo studies demonstrate a significant uptake of the hybrids by tumours together with specific gene silencing. This split-functionality approach presents a new route in the development of 'smart' nucleic acid-based nanoparticles and switches for various biomedical applications.
Asunto(s)
ADN/metabolismo , Ácidos Nucleicos Heterodúplex/metabolismo , ARN/metabolismo , Animales , Aptámeros de Nucleótidos/metabolismo , Línea Celular Tumoral , Fluorescencia , Transferencia Resonante de Energía de Fluorescencia , Técnicas de Silenciamiento del Gen , Proteínas Fluorescentes Verdes/metabolismo , VIH-1/metabolismo , Humanos , Espacio Intracelular/metabolismo , Cinética , Ratones , Microscopía Confocal , ARN Interferente Pequeño/metabolismo , Temperatura , Factores de Tiempo , Distribución Tisular , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Promising drug candidates of the diazeniumdiolate (NONOate) chemical family include several types of thiol modification among their mechanisms of action: 1) drugs designed to release nitric oxide (NO) on reaction with the thiol group of glutathione (GSH) arylate the GSH, a step that removes reducing equivalents from the cell; (2) a similar reaction of the drug with the thiol group of a protein changes its structure, leading to potentially impaired function and cell death; (3) the NO generated as a byproduct in the above reactions can undergo oxidation, leading to S-nitrosylation and S-glutathionylation; and (4) diazeniumdiolates can also generate nitroxyl, which reacts with thiol groups to form disulfides or sulfinamides.