RESUMO
Glutamate plays a key role in cognition and mood, and it has been shown that inhibiting ionotropic glutamate receptors disrupts cognition, while enhancing ionotropic receptor activity is pro-cognitive. One approach to elevating glutamatergic tone has been to antagonize presynaptic metabotropic glutamate receptor 2 (mGluR2). A desire for selectivity over the largely homologous mGluR3 motivated a strategy to achieve selectivity through the identification of mGluR2 negative allosteric modulators (NAMs). Extensive screening and optimization efforts led to the identification of a novel series of 4-arylquinoline-2-carboxamides. This series was optimized for mGluR2 NAM potency, clean off-target activity, and desirable physical properties, which resulted in the identification of improved C4 and C7 substituents. The initial lead compound from this series was Ames-positive in a single strain with metabolic activation, indicating that a reactive metabolite was likely responsible for the genetic toxicity. Metabolic profiling and Ames assessment across multiple analogs identified key structure-activity relationships associated with Ames positivity. Further optimization led to the Ames-negative mGluR2 negative allosteric modulator MK-8768.
RESUMO
Modification of potent, selective metabotropic glutamate receptor 2 negative allosteric modulator (mGluR2 NAM) led to a series of analogues with excellent binding affinity, lipophilicity, and suitable physicochemical properties for a PET tracer with convenient chemical handles for incorporation of a 11C or 18F radiolabel. [11C]MK-8056 was synthesized and evaluated in vivo and demonstrated appropriate affinity, selectivity, and physicochemical properties to be used as a positron emission tomography tracer for mGluR2.
RESUMO
This paper presents a quantized Kalman filter implemented using unreliable memories. We consider that both the quantization and the unreliable memories introduce errors in the computations, and we develop an error propagation model that takes into account these two sources of errors. In addition to providing updated Kalman filter equations, the proposed error model accurately predicts the covariance of the estimation error and gives a relation between the performance of the filter and its energy consumption, depending on the noise level in the memories. Then, since memories are responsible for a large part of the energy consumption of embedded systems, optimization methods are introduced to minimize the memory energy consumption under the desired estimation performance of the filter. The first method computes the optimal energy levels allocated to each memory bank individually, and the second one optimizes the energy allocation per groups of memory banks. Simulations show a close match between the theoretical analysis and experimental results. Furthermore, they demonstrate an important reduction in energy consumption of more than 50%.
RESUMO
Matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry is an analytical method that detects macromolecules that can be used for proteomic fingerprinting and taxonomic identification in arthropods. The conventional MALDI approach uses fresh laboratory-reared arthropod specimens to build a reference mass spectra library with high-quality standards required to achieve reliable identification. However, this may not be possible to accomplish in some arthropod groups that are difficult to rear under laboratory conditions, or for which only alcohol preserved samples are available. Here, we generated MALDI mass spectra of highly abundant proteins from the legs of 18 Neotropical species of adult field-collected hard ticks, several of which had not been analyzed by mass spectrometry before. We then used their mass spectra as fingerprints to identify each tick species by applying machine learning and pattern recognition algorithms that combined unsupervised and supervised clustering approaches. Both Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA) classification algorithms were able to identify spectra from different tick species, with LDA achieving the best performance when applied to field-collected specimens that did have an existing entry in a reference library of arthropod protein spectra. These findings contribute to the growing literature that ascertains mass spectrometry as a rapid and effective method to complement other well-established techniques for taxonomic identification of disease vectors, which is the first step to predict and manage arthropod-borne pathogens.
Assuntos
Ixodidae/química , Proteômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/metabolismo , Vetores de Doenças/classificação , Ixodidae/classificação , Ixodidae/metabolismoRESUMO
Leucine-rich repeat kinase 2 (LRRK2) is a large, multidomain protein which contains a kinase domain and GTPase domain among other regions. Individuals possessing gain of function mutations in the kinase domain such as the most prevalent G2019S mutation have been associated with an increased risk for the development of Parkinson's disease (PD). Given this genetic validation for inhibition of LRRK2 kinase activity as a potential means of affecting disease progression, our team set out to develop LRRK2 inhibitors to test this hypothesis. A high throughput screen of our compound collection afforded a number of promising indazole leads which were truncated in order to identify a minimum pharmacophore. Further optimization of these indazoles led to the development of MLi-2 (1): a potent, highly selective, orally available, brain-penetrant inhibitor of LRRK2.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Indazóis/química , Indazóis/farmacologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/antagonistas & inibidores , Animais , Encéfalo/metabolismo , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacocinética , Humanos , Indazóis/administração & dosagem , Indazóis/farmacocinética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Masculino , Simulação de Acoplamento Molecular , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/enzimologia , Ratos , Ratos WistarRESUMO
Familial Parkinson's disease cases have recently been associated with the leucine rich repeat kinase 2 (LRRK2) gene. It has been hypothesized that inhibition of the LRRK2 protein may have the potential to alter disease pathogenesis. A dihydrobenzothiophene series of potent, selective, orally bioavailable LRRK2 inhibitors were identified from a high-throughput screen of the internal Merck sample collection. Initial SAR studies around the core established the series as a tractable small molecule lead series of LRRK2 inhibitors for potential treatment of Parkinson's disease. It was also found that incorporation of a lactam into the core drastically improved the CNS and DMPK properties of these small molecules.
Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Tiofenos/farmacologia , Administração Oral , Disponibilidade Biológica , Relação Dose-Resposta a Droga , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Simulação de Acoplamento Molecular , Estrutura Molecular , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade , Tiofenos/síntese química , Tiofenos/químicaRESUMO
Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are the most common genetic cause of Parkinson's disease (PD) and cause both autosomal dominant familial and sporadic PD. Currently, the physiological and pathogenic activities of LRRK2 are poorly understood. To decipher the biological functions of LRRK2, including the genes and pathways modulated by LRRK2 kinase activity in vivo, we assayed genome-wide mRNA expression in the brain and peripheral tissues from LRRK2 knockout (KO) and kinase hyperactive G2019S (G2019S) transgenic mice. Subtle but significant differences in mRNA expression were observed relative to wild-type (WT) controls in the cortex, striatum and kidney of KO animals, but only in the striatum in the G2019S model. In contrast, robust, consistent and highly significant differences were identified by the direct comparison of KO and G2019S profiles in the cortex, striatum, kidney and muscle, indicating opposite effects on mRNA expression by the two models relative to WT. Ribosomal and glycolytic biological functions were consistently and significantly up-regulated in LRRK2 G2019S compared with LRRK2 KO tissues. Genes involved in membrane-bound organelles, oxidative phosphorylation, mRNA processing and the endoplasmic reticulum were down-regulated in LRRK2 G2019S mice compared with KO. We confirmed the expression patterns of 35 LRRK2-regulated genes using quantitative reverse transcription polymerase chain reaction. These findings provide the first description of the transcriptional responses to genetically modified LRRK2 activity and provide preclinical target engagement and/or pharmacodynamic biomarker strategies for LRRK2 and may inform future therapeutic strategies for LRRK2-associated PD.
Assuntos
Doença de Parkinson/enzimologia , Doença de Parkinson/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transcrição Gênica , Animais , Encéfalo/enzimologia , Feminino , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Serina-Treonina Quinases/genéticaRESUMO
Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common forms of inheritable Parkinson's disease and likely play a role in sporadic disease as well. LRRK2 is a large multidomain protein containing two key groups, a Ras-like GTP binding domain and a serine, threonine kinase domain. Mutations in the LRRK2 gene that associate with Parkinson's disease reside primarily within the two functional domains of the protein, suggesting that LRRK2 function is critical to the pathogenesis of the disease. The most common LRRK2 mutation increases kinase activity, making LRRK2 kinase inhibition an attractive target for small molecule drug development. However, the physiological function of LRRK2 kinase as well as its endogenous protein substrates remains poorly understood and has hindered drug development efforts. Recent advances in LRRK2 biology have revealed several potential cellular roles, interacting proteins, and putative physiological substrates. Together, a picture emerges of a complex multifunctional protein that exists in multiple cellular compartments. Through unclear mechanisms, LRRK2 kinase regulates cytoskeleton architecture through control of protein translation, phosphorylation of cytoskeletal proteins, and response to cellular stressors. This article will briefly cover some interesting recent studies in LRRK2 cellular biology and highlight emerging cellular models of LRRK2 kinase function.
Assuntos
Neurônios/fisiologia , Doença de Parkinson/enzimologia , Doença de Parkinson/genética , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Animais , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/fisiologia , Proteínas do Citoesqueleto/ultraestrutura , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Neurônios/enzimologia , Doença de Parkinson/patologia , Biossíntese de Proteínas/genética , Modificação Traducional de Proteínas/genética , Proteínas Serina-Treonina Quinases/química , Especificidade por Substrato/genética , Especificidade por Substrato/fisiologiaAssuntos
Indústria Farmacêutica/tendências , Biologia Molecular/tendências , Doenças do Sistema Nervoso/tratamento farmacológico , Doenças do Sistema Nervoso/genética , Neurofarmacologia/tendências , Pesquisa Translacional Biomédica/tendências , Animais , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos/métodos , Avaliação Pré-Clínica de Medicamentos/tendências , Indústria Farmacêutica/métodos , Humanos , Biologia Molecular/métodos , Neurofarmacologia/métodos , Pesquisa Translacional Biomédica/métodosRESUMO
Linoleic acid is required for normal mammalian health and development, but is also prone to oxidation, yielding metabolites with biological effects. We screened linoleic acid, other fatty acids, and some of their derivatives and found that an epoxy-keto derivative of linoleic acid (but neither linoleic acid itself nor others of its oxidation products) strongly activates the antioxidant response element (ARE) in IMR-32 neuroblastoma cells and cerebro-cortical neurons. The active compound, 12,13-epoxy-9-keto-10(trans)-octadecenoic acid (EKODE), induces the expression of ARE-regulated cytoprotective genes such as NQO1 at the transcript and protein levels. EKODE requires transcription factor NRF2 and PI3-kinase for ARE activity. The results suggest that specific oxidation products of linoleic acid may initiate responses that lessen damage caused by oxidative stress.
Assuntos
Antioxidantes/metabolismo , Ácidos Linoleicos/farmacologia , Ácidos Oleicos/farmacologia , Elementos de Resposta/efeitos dos fármacos , Animais , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Humanos , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Células Tumorais CultivadasRESUMO
On the basis of growing evidence for G-quadruplex DNA structures in genomic DNA and the presumed need to resolve these structures for DNA replication, the G-quadruplex DNA unwinding ability of a prototypical replicative helicase, SV40 large T-antigen (T-ag), was investigated. Here, we demonstrate that this G-quadruplex helicase activity is robust and comparable to the duplex helicase activity of T-ag. Analysis of the SV40 genome demonstrates the presence of sequences that may form intramolecular G-quadruplexes, which are the presumed natural substrates for the G-quadruplex helicase activity of T-ag. A number of G-quadruplex-interactive agents as well as new perylene diimide (PDI) derivatives have been investigated as inhibitors of both the G-quadruplex and the duplex DNA helicase activities of T-ag. A unique subset of these G-quadruplex-interactive agents inhibits the G-quadruplex DNA unwinding activity of T-ag, relative to those reported to inhibit G-quadruplex DNA unwinding by RecQ-family helicases. We also find that certain PDIs are both potent and selective inhibitors of the G-quadruplex DNA helicase activity of T-ag. Surface plasmon resonance and fluorescence spectroscopic G-quadruplex DNA binding studies of these T-ag G-quadruplex helicase inhibitors have been carried out, demonstrating the importance of attributes in addition to binding affinity for G-quadruplex DNA that may be important for inhibition. The identification of potent and selective inhibitors of the G-quadruplex helicase activity of T-ag provides tools for probing the specific role of this activity in SV40 replication.
Assuntos
Antígenos Transformantes de Poliomavirus/imunologia , DNA Helicases/antagonistas & inibidores , DNA/metabolismo , Vírus 40 dos Símios/imunologia , Sequência de Bases , Primers do DNA , Espectrometria de FluorescênciaRESUMO
The transcription of antioxidant response element (ARE)-containing cytoprotective genes has been proposed as a means to combat oxidative stress-related disorders, such as cancer and Parkinson's disease. Transactivation of the ARE requires the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2). Cellular levels of Nrf2 protein are regulated by the Kelch-like ECH-associated protein 1 (Keap1), a substrate adaptor protein for the ubiquitin ligase machinery and subsequent proteasomal degradation. Recently, detailed studies have elucidated the structure and interactions of the Keap1-containing ubiquitin ligase complex. Here, we propose that small molecule modulation of Keap1 protein:protein interactions may permit Nrf2's nuclear accumulation and the transcription of ARE-dependent genes to enhance cellular resistance to oxidative insult.
Assuntos
Antioxidantes , Peptídeos e Proteínas de Sinalização Intracelular , Estresse Oxidativo/efeitos dos fármacos , Elementos de Resposta/genética , Complexos Ubiquitina-Proteína Ligase , Animais , Antioxidantes/química , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Ensaios Clínicos como Assunto , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteína 1 Associada a ECH Semelhante a Kelch , Modelos Moleculares , Estresse Oxidativo/genética , Ligação Proteica , Mapeamento de Interação de Proteínas , Complexos Ubiquitina-Proteína Ligase/antagonistas & inibidores , Complexos Ubiquitina-Proteína Ligase/química , Complexos Ubiquitina-Proteína Ligase/genéticaRESUMO
The antioxidant response element (ARE) is a cis-acting regulatory enhancer element found in the 5' flanking region of many phase II detoxification enzymes. Up-regulation of ARE-dependent target genes is known to have neuroprotective effects; yet, the mechanism of activation is largely unknown. By screening an arrayed collection of approximately 15,000 full-length expression cDNAs in the human neuroblastoma cell line IMR-32 with an ARE-luciferase reporter, we have identified several cDNAs not previously associated with ARE activation. A subset of cDNAs, encoding sequestosome 1 (SQSTM1) and dipeptidylpeptidase 3 (DPP3), activated the ARE in primary mouse-derived cortical neurons. Overexpression of SQSTM1 and DPP3 in IMR-32 cells stimulated NF-E2-related factor 2 (NRF2) nuclear translocation and led to increased levels of NAD(P)H:quinone oxidoreductase 1, a protein which is transcriptionally regulated by the ARE. When transfected into IMR-32 neuroblastoma cells that were depleted of transcription factor NRF2 by RNA interference, SQSTM1 and DPP3 were unable to activate the ARE or induce NAD(P)H:quinone oxidoreductase 1 expression, indicating that the ARE activation upon ectopic expression of these cDNAs is mediated by NRF2. Studies with pharmacological inhibitors indicated that 1-phosphatidylinositol 3-kinase and protein kinase C signaling are essential for activity. Overexpression of these cDNAs conferred partial resistance to hydrogen peroxide or rotenone-induced toxicity, consistent with the induction of antioxidant and phase II detoxification enzymes, which can protect from oxidative stress. This work and other such studies may provide mechanisms for activating the ARE in the absence of general oxidative stress and a yet-unexploited therapeutic approach to degenerative diseases and aging.
Assuntos
Antioxidantes/metabolismo , Genômica/métodos , Elementos de Resposta/genética , Transativadores/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , DNA Complementar/metabolismo , Indução Enzimática/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Biblioteca Gênica , Genoma/efeitos dos fármacos , Proteínas de Choque Térmico/biossíntese , Camundongos , NAD(P)H Desidrogenase (Quinona) , NADPH Desidrogenase/biossíntese , Fator 2 Relacionado a NF-E2/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Elementos de Resposta/efeitos dos fármacos , Proteína Sequestossoma-1 , Transativadores/genéticaRESUMO
Electrospray ionization mass spectrometry (ESI-MS) and spectroscopic studies in solution were used to evaluate the self-association, G-quadruplex DNA binding, and selectivity of a series of perylene diimides (PDIs) (PIPER, Tel01, Tel11, Tel12, and Tel18) or benzannulated perylene diimide ligands (Tel34 and Tel32). Fluorescence and resonance light scattering spectra of Tel01, Tel12, Tel32, and Tel34 reveal that these analogs undergo self-association in solution. UV-Vis and fluorescence titrations with G-quadruplex, duplex, or single-stranded DNA demonstrate that all the analogs, with the exception of Tel32, bind to G-quadruplex DNA, with those PDIs that are self-associated in solution showing the highest degree of selectivity for binding G-quadruplex DNA. Parallel ESI-MS analysis of the stoichiometries demonstrates the ability of the ligands, with the exception of Tel32, to bind to G-quadruplex DNA. While most ligands show major 1:1 and 2:1 binding stoichiometries as expected in the case of end-stacking, interestingly, three of the most quadruplex-selective ligands show a different behavior. Tel01 forms 3:1 complexes, while Tel12 and Tel32 only form 1:1 complexes. Collisional activation dissociation patterns are compatible with ligand binding to G-quadruplex DNA via stacking on the ends of the terminal G-tetrads. Experiments with duplex and single strand DNA were performed to assess the binding selectivities of the ligands. PIPER, Tel11, and Tel18 demonstrated extensive complexation with duplex DNA, while Tel11 and Tel18 bound to single strand DNA, confirming the lack of selectivity of these two ligands. Our results indicate that Tel01, Tel12, and Tel34 are the most selective for G-quadruplex DNA.
Assuntos
DNA/metabolismo , Imidas/química , Imidas/metabolismo , Perileno/análogos & derivados , Espectrometria de Massas por Ionização por Electrospray/métodos , Sequência de Bases , Sítios de Ligação , DNA/química , Ligantes , Estrutura Molecular , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/metabolismo , Perileno/química , Perileno/metabolismo , Espectrometria de Fluorescência , Espectrofotometria UltravioletaRESUMO
Parkinson's disease, a progressive neurodegenerative disorder, is characterized by loss of midbrain dopaminergic neurons. The etiology of sporadic Parkinson's disease is unknown; however, oxidative stress is thought to play a major role in disease pathogenesis. Little is known regarding the transcriptional changes that occur in Parkinson's disease. The antioxidant response element is a cis-acting enhancer sequence that is upstream of many phase II detoxification and antioxidant genes. Here we show that 6-hydroxydopamine, a mitochondrial inhibitor used to model Parkinson's disease, activates the antioxidant response element both in cultured neurons and in the striatum and brainstem of 6-OHDA-lesioned mice. Pretreatment with antioxidants or NMDA receptor antagonists reduced but did not abolish activation. Further induction of this pathway with tert-butylhydroquinone was able to significantly reduce cell death due to 6-OHDA in vitro. These observations indicate that 6-OHDA activates the antioxidant response element through components of oxidative stress, excitotoxicity, and potential structural factors. Further induction of this endogenous defense mechanism may suggest a novel therapeutic venue in Parkinson's disease.
Assuntos
Antioxidantes/farmacologia , Oxidopamina/toxicidade , Elementos de Resposta/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Astrócitos/efeitos dos fármacos , Tronco Encefálico/efeitos dos fármacos , Corpo Estriado/efeitos dos fármacos , Proteínas de Ligação a DNA/fisiologia , Feminino , Hidroquinonas/farmacologia , Camundongos , Fator 2 Relacionado a NF-E2 , Estresse Oxidativo , Doença de Parkinson/etiologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Transativadores/fisiologiaRESUMO
Two N,N'-disubstituted perylene diimide G-quadruplex DNA ligands, Tel11 (N,N'-bis-[3-(4-methyl-morpholin-4-yl)-propyl]-3,4,9,10-perylenetetracarboxylic acid diimide diiodide) and Tel12 (N,N'-bis-[(3-phosphono)-propyl]-3,4,9,10-perylenetetracarboxylic acid diimide tetrapotassium salt) were synthesized and studied. Visible absorbance spectroscopy, resonance light scattering, and fluorescence spectroscopy were utilized to explore the aggregation state, affinity for various DNA structures, and G-quadruplex selectivity of these ligands. The water-soluble ligands exist in a monomer-dimer equilibrium with the cationic Tel11 exhibiting a greater affinity for various DNA structures than the anionic Tel12. Tel12 has greater selectivity for G-quadruplex DNA over double-stranded DNA than Tel11.
Assuntos
DNA/química , Imidas/química , Perileno/análogos & derivados , Composição de Bases , Sequência de Bases , Quadruplex G , Guanina , Conformação de Ácido Nucleico , Espalhamento de Radiação , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Especificidade por SubstratoRESUMO
Human telomeres are comprised of d(TTAGGG) repeats that are capable of forming G-quadruplex DNA structures. Ligands that bind to and stabilize these G-quadruplex DNA structures are potential inhibitors of the cancer cell-associated enzyme telomerase. Other potential biological uses of G-quadruplex targeting ligands have been proposed. One particularly challenging aspect of the contemplated uses of G-quadruplex targeting ligands is their selectivity for G-quadruplex DNA versus double-stranded DNA structures. We have previously reported the observation that two structurally related 3,4,9,10-perylenetetracarboxylic acid diimide-based G-quadruplex DNA ligands, PIPER [N,N'-bis(2-(1-piperidino)ethyl)-3,4,9,10-perylenetetracarboxylic acid diimide] and Tel01 [N,N'-bis(3-(4-morpholino)propyl)-3,4,9,10-perylenetetracarboxylic acid diimide], have different levels of G-quadruplex DNA binding selectivity at pH 7 as determined by absorbance changes in the presence of different DNA structures [Kerwin, S. M., Chen, G., Kern, J. T., and Thomas, P. W. (2002) Bioorg. Med. Chem. Lett. 12, 447-450]. Here we report that the less G-quadruplex DNA selective ligand PIPER can unwind double-stranded, closed circular plasmid DNA, as determined by a topoisomerase I assay. A model for the interaction of Tel01 with the G-quadruplex DNA structure formed by d(TAGGGTTA) was determined from NMR experiments. This model is similar to the previously published model for PIPER bound to the same G-quadruplex DNA and failed to provide a structural basis for the observed increased selectivity of Tel01 interaction with G-quadruplex DNA. In contrast, investigation into the aggregation state of Tel01 and PIPER as well as other 3,4,9,10-perylenetetracarboxylic acid diimide analogues bearing basic side chains demonstrates that ligand aggregation is correlated with G-quadruplex DNA binding selectivity. For all six analogues examined, those ligands that were aggregated at pH 7 in 70 mM potassium phosphate, 100 mM KCl, 1 mM EDTA buffer also demonstrated G-quadruplex DNA binding selectivity under these buffer conditions. Ligands that were not aggregated under these conditions display much lower levels of G-quadruplex DNA selectivity. The aggregation state of these ligands is extremely sensitive to the buffer pH. Tel01, which is aggregated at pH 7, is not aggregated at pH 6.4, where it demonstrates only modest G-quadruplex DNA binding selectivity, and PIPER in pH 8.5 buffer is both aggregated and highly G-quadruplex DNA-selective. To our knowledge, these studies demonstrate the first DNA structure selectivity as achieved through pH-mediated ligand aggregation. The potential impact of these findings on the selectivity of other classes of G-quadruplex DNA ligands is discussed.
Assuntos
Antracenos/química , DNA/química , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Piperidinas/química , Telomerase/antagonistas & inibidores , Sequência de Bases , DNA/efeitos dos fármacos , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Perileno/análogos & derivados , EspectrofotometriaRESUMO
Two N,N'-disubstituted perylene diimide G-quadruplex DNA ligands, PIPER [N,N'-bis-(2-(1-piperidino)ethyl)-3,4,9,10-perylene tetracarboxylic acid diimide] and Tel01 [N,N'-bis-(3-(4-morpholino)-propyl)-3,4,9,10-perylene tetracarboxylic acid diimide] were studied. Visible absorbance, resonance light scattering, and fluorescence spectroscopy were used to characterize the pH-dependent aggregation of these ligands. The G-quadruplex DNA binding selectivity of these ligands as monitored by absorption spectroscopy is also pH-dependent. The ligands bind to both duplex and G-quadruplex DNA under low pH conditions, where the ligands are not aggregated. At higher pH, where the ligands are extensively aggregated, the apparent G-quadruplex DNA binding selectivity is high.