RESUMO
The DNA sequence 5'-ACGCGT-3' is in the core site of the Mlu 1 cell-cycle box, a transcriptional element in the promoter region of human Dbf4 gene that is highly correlated with a large number of aggressive solid cancers. The polyamide formamido-imidazole-pyrrole-imidazole-amine(+) (f-Im-Py-Im-Am(+) ) can target the minor groove of 5'-ACGCGT-3' as an antiparallel stacked dimer and has shown good activity in inhibiting transcription factor binding. Recently, f-Im-Py-Im-Am(+) derivatives that involve different orthogonally positioned substituents were synthesized to target the same binding site, and some of them have displayed improved binding and pharmacological properties. In this study, the gel electrophoresis-ligation ladders assay was used to evaluate the conformational effects of f-Im-Py-Im-Am(+) and derivatives on the target DNA, an essential factor for establishing the molecular basis of polyamide-DNA complexes and their transcription factor inhibition. The results show that the ACGCGT site in DNA has a relatively wide minor groove and a B-form like overall structure. After binding with f-Im-Py-Im-Am(+) derivatives, the DNA conformation is changed as indicated by the different mobilities in the gel. These conformational effects on DNA will at least help to point to the mechanism for the observed Mlu 1 inhibition activity of these polyamides. Therefore, modulating DNA transcription by locking the DNA shape or altering the minor groove geometry to affect the binding affinity of certain transcription factors is an attractive possible therapeutic mechanism for polyamides. Some of the substituents are charged with electrostatic interactions with DNA phosphate groups, and their charge effects on DNA gel mobility have been observed.
Assuntos
DNA/química , DNA/metabolismo , Formamidas/química , Imidazóis/química , Conformação de Ácido Nucleico , Nylons/metabolismo , Pirróis/química , Aminas/química , Aminas/metabolismo , Sequência de Bases , Benzamidas/química , Benzamidas/metabolismo , Eletroforese em Gel de Poliacrilamida , Formamidas/metabolismo , Humanos , Imidazóis/metabolismo , Modelos Moleculares , Nylons/química , Pirróis/metabolismoRESUMO
Rules for polyamide-DNA recognition have proved invaluable for the design of sequence-selective DNA binding agents in cell-free systems. However, these rules are not fully transferrable to predicting activity in cells, tissues or animals, and additional refinements to our understanding of DNA recognition would help biomedical studies. Similar complexities are encountered when using internal ß-alanines as polyamide building blocks in place of N-methylpyrrole; ß-alanines were introduced in polyamide designs to maintain good hydrogen bonding registry with the target DNA, especially for long polyamides or those with several GC bp (P.B. Dervan, A.R. Urbach, Essays Contemp. Chem. (2001) 327-339). Thus, to clarify important subtleties of molecular recognition, we studied the effects of replacing a single pyrrole with ß-alanine in 8-ring polyamides designed against the Ets-1 transcription factor. Replacement of a single internal N-methylpyrrole with ß-alanine to generate a ß/Im pairing in two 8-ring polyamides causes a decrease in DNA binding affinity by two orders of magnitude and decreases DNA binding selectivity, contrary to expectations based on the literature. Measurements were made by fluorescence spectroscopy, quantitative DNA footprinting and surface plasmon resonance, with these vastly different techniques showing excellent agreement. Furthermore, results were validated for a range of DNA substrates from small hairpins to long dsDNA sequences. Docking studies helped show that ß-alanine does not make efficient hydrophobic contacts with the rest of the polyamide or nearby DNA, in contrast to pyrrole. These results help refine design principles and expectations for polyamide-DNA recognition.
Assuntos
Ciclo-Oxigenase 2/química , DNA/química , Papillomavirus Humano 16/química , Nylons/química , Proteína Proto-Oncogênica c-ets-1/química , Pirróis/química , beta-Alanina/química , Sequência de Bases , Ciclo-Oxigenase 2/genética , Pegada de DNA , Papillomavirus Humano 16/genética , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Sequências Repetidas Invertidas/genética , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Nylons/síntese química , Regiões Promotoras Genéticas , Proteína Proto-Oncogênica c-ets-1/genética , Espectrometria de Fluorescência , Relação Estrutura-Atividade , Ressonância de Plasmônio de SuperfícieRESUMO
Direct modulation of gene expression by targeting oncogenic transcription factors is a new area of research for cancer treatment. ERG, an ETS-family transcription factor, is commonly over-expressed or translocated in leukaemia and prostate carcinoma. In this work, we selected the di-(thiophene-phenyl-amidine) compound DB1255 as an ERG/DNA binding inhibitor using a screening test of synthetic inhibitors of the ERG/DNA interaction followed by electrophoretic mobility shift assays (EMSA) validation. Spectrometry, footprint and biosensor-surface plasmon resonance analyses of the DB1255/DNA interaction evidenced sequence selectivity and groove binding as dimer. Additional EMSA evidenced the precise DNA-binding sequence required for optimal DB1255/DNA binding and thus for an efficient ERG/DNA complex inhibition. We further highlighted the structure activity relationships from comparison with derivatives. In cellulo luciferase assay confirmed this modulation both with the constructed optimal sequences and the Osteopontin promoter known to be regulated by ERG and which ERG-binding site was protected from DNaseI digestion on binding of DB1255. These data showed for the first time the ERG/DNA complex modulation, both in vitro and in cells, by a heterocyclic diamidine that specifically targets a portion of the ERG DNA recognition site.
Assuntos
Amidinas/farmacologia , Antineoplásicos/farmacologia , Tiofenos/farmacologia , Transativadores/antagonistas & inibidores , Ativação Transcricional/efeitos dos fármacos , Amidinas/química , Amidinas/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , DNA/química , DNA/metabolismo , Avaliação Pré-Clínica de Medicamentos , Humanos , Tiofenos/química , Tiofenos/metabolismo , Transativadores/metabolismo , Regulador Transcricional ERGRESUMO
The objective of this study was to detect and characterize latent equine herpes virus (EHV)-1 and -4 from the submandibular (SMLN) and bronchial lymph (BLN) nodes, as well as from the trigeminal ganglia (TG) of 70 racing Thoroughbred horses submitted for necropsy following sustaining serious musculoskeletal injuries while racing. A combination of nucleic acid precipitation and pre-amplification steps was used to increase analytical sensitivity. Tissues were deemed positive for latent EHV-1 and/or -4 infection when found PCR positive for the corresponding glycoprotein B (gB) gene in the absence of detectable late structural protein gene (gB gene) mRNA. The EHV-1 genotype was also determined using a discriminatory real-time PCR assay targeting the DNA polymerase gene (ORF 30). Eighteen (25.7%) and 58 (82.8%) horses were PCR positive for the gB gene of EHV-1 and -4, respectively, in at least one of the three tissues sampled. Twelve horses were dually infected with EHV-1 and -4, two carried a latent neurotropic strain of EHV-1, six carried a non-neurotropic genotype of EHV-1 and 10 were dually infected with neurotropic and non-neurotropic EHV-1. The distribution of latent EHV-1 and -4 infection varied in the samples, with the TG found to be most commonly infected. Overall, non-neurotropic strains were more frequently detected than neurotropic strains, supporting the general consensus that non-neurotropic strains are more prevalent in horse populations, and hence the uncommon occurrence of equine herpes myeloencephalopathy.
Assuntos
Infecções por Herpesviridae/veterinária , Herpesvirus Equídeo 1/fisiologia , Herpesvirus Equídeo 4/fisiologia , Doenças dos Cavalos/epidemiologia , Linfonodos/virologia , Latência Viral , Animais , Brônquios/virologia , California/epidemiologia , DNA Viral/genética , Feminino , Infecções por Herpesviridae/epidemiologia , Infecções por Herpesviridae/virologia , Herpesvirus Equídeo 1/isolamento & purificação , Herpesvirus Equídeo 4/isolamento & purificação , Doenças dos Cavalos/virologia , Cavalos , Masculino , Mandíbula/virologia , Linhagem , Prevalência , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Gânglio Trigeminal/virologiaRESUMO
Pyrrole- and imidazole-containing polyamides are widely investigated as DNA sequence selective binding agents that have potential use as gene control agents. The key challenges that must be overcome to realize this goal is the development of polyamides with low molar mass so the molecules can readily diffuse into cells and concentrate in the nucleus. In addition, the molecules must have appreciable water solubility, bind DNA sequence specifically, and with high affinity. It is on this basis that the orthogonally positioned diamino/dicationic polyamide Ph-ImPy*Im 5 was designed to target the sequence 5'-ACGCGT-3'. Py* denotes the pyrrole unit that contains a N-substituted aminopropyl pendant group. The DNA binding properties of diamino polyamide 5 were determined using a number of techniques including CD, ΔT(M), DNase I footprinting, SPR and ITC studies. The effects of the second amino moiety in Py* on DNA binding affinity over its monoamino counterpart Ph-ImPyIm 3 were assessed by conducting DNA binding studies of 3 in parallel with 5. The results confirmed the minor groove binding and selectivity of both polyamides for the cognate sequence 5'-ACGCGT-3'. The diamino/dicationic polyamide 5 showed enhanced binding affinity and higher solubility in aqueous media over its monoamino/monocationic counterpart Ph-ImPyIm 3. The binding constant of 5, determined from SPR studies, was found to be 1.5 × 10(7)M(-1), which is â¼3 times higher than that for its monoamino analog 3 (4.8 × 10(6)M(-1)). The affinity of 5 is now approaching that of the parent compound f-ImPyIm 1 and its diamino equivalent 4. The advantages of the design of diamino polyamide 5 over 1 and 4 are its sequence specificity and the ease of synthesis compared to the N-terminus pyrrole analog 2.
Assuntos
Benzamidas/síntese química , DNA/metabolismo , Distamicinas/química , Imidazóis/síntese química , Nylons/química , Pirróis/química , Sequência de Bases , Benzamidas/química , Calorimetria , Dicroísmo Circular , DNA/química , Desoxirribonuclease I/metabolismo , Imidazóis/química , Nylons/síntese química , Ressonância de Plasmônio de SuperfícieRESUMO
Equine herpes myeloencephalopathy (EHM), although a relatively uncommon manifestation of equine herpesvirus-1 (EHV-1) infection, can cause devastating losses on individual farms or boarding stables. Although outbreaks of EHM have been recognized for centuries in domestic horse populations, many aspects of this disease remained poorly characterized. In recent years, an improved understanding of EHM has emerged from experimental studies and from data collected during field outbreaks at riding schools, racetracks and veterinary hospitals throughout North America and Europe. These outbreaks have highlighted the contagious nature of EHV-1 and have prompted a re-evaluation of diagnostic procedures, treatment modalities, preventative measures and biosecurity protocols for the disease. This review concentrates on these and other selected, clinically relevant aspects of EHM.
Assuntos
Infecções por Herpesviridae/veterinária , Herpesvirus Equídeo 1/patogenicidade , Doenças dos Cavalos/virologia , Doenças do Sistema Nervoso/veterinária , Animais , Surtos de Doenças/veterinária , Infecções por Herpesviridae/epidemiologia , Infecções por Herpesviridae/virologia , Doenças dos Cavalos/epidemiologia , Cavalos , Doenças do Sistema Nervoso/epidemiologia , Doenças do Sistema Nervoso/virologiaRESUMO
The purpose of this study was to determine the effect of blood contamination of cerebrospinal fluid (CSF) on the results of indirect fluorescent antibody tests (IFATs) for Sarcocystis neurona and Neospora hughesi. The in vitro study used antibody-negative CSF collected from non-neurologic horses immediately after euthanasia and blood samples from 40 healthy horses that had a range of IFAT antibody titers against S. neurona and N. hughesi. Serial dilutions of whole blood were made in seronegative CSF to generate blood-contaminated CSF with red blood cell (RBC) concentrations ranging from 10 to 100,000 RBCs/microl. The blood-contaminated CSF samples were then tested for antibodies against both pathogens using IFAT. Blood contamination of CSF had no detectable effect on IFAT results for S. neurona or N. hughesi at any serologic titer when the RBC concentration in CSF was <10,000 RBCs/microl. At concentrations of 10,000-100,000 RBCs/microl of CSF, positive CSF results (IFAT titer >or=5) for S. neurona and N. hughesi were detected only when the corresponding serum titers were >or=160 and >or=80, respectively. The IFAT performed on CSF is reliable for testing horses for equine protozoal myeloencephalitis caused by S. neurona or N. hughesi, even when blood contamination causes the RBC concentration in CSF to be up to 10,000 RBCs/microl.
Assuntos
Coccidiose/veterinária , Encefalomielite/veterinária , Doenças dos Cavalos/parasitologia , Neospora/isolamento & purificação , Sarcocystis/isolamento & purificação , Sarcocistose/veterinária , Animais , Anticorpos Antiprotozoários/líquido cefalorraquidiano , Coccidiose/sangue , Coccidiose/líquido cefalorraquidiano , Coccidiose/parasitologia , Encefalomielite/sangue , Encefalomielite/líquido cefalorraquidiano , Encefalomielite/parasitologia , Técnica Indireta de Fluorescência para Anticorpo/normas , Técnica Indireta de Fluorescência para Anticorpo/veterinária , Doenças dos Cavalos/sangue , Doenças dos Cavalos/líquido cefalorraquidiano , Doenças dos Cavalos/diagnóstico , Cavalos , Sarcocistose/sangue , Sarcocistose/líquido cefalorraquidiano , Sarcocistose/parasitologia , Manejo de Espécimes/veterináriaRESUMO
Replicon particles derived from a vaccine strain of Venezuelan equine encephalitis (VEE) virus were used as vectors for expression in vivo of the major envelope proteins (G(L) and M) of equine arteritis virus (EAV), both individually and in heterodimer form (G(L)/M). The immunogenicity of the different replicons was evaluated in horses, as was their ability to protectively immunize horses against intranasal and intrauterine challenge with a virulent strain of EAV (EAV KY84). Horses immunized with replicons that express both the G(L) and M proteins in heterodimer form developed neutralizing antibodies to EAV, shed little or no virus, and developed only mild or inapparent signs of equine viral arteritis (EVA) after challenge with EAV KY84. In contrast, unvaccinated horses and those immunized with replicons expressing individual EAV envelope proteins (M or G(L)) shed virus for 6-10 days in their nasal secretions and developed severe signs of EVA after challenge. These data confirm that replicons that co-express the G(L) and M envelope proteins effectively, induce EAV neutralizing antibodies and protective immunity in horses.