RESUMO
The design and synthesis of macrocyclic inhibitors of human rhinovirus 3C protease is described. A macrocyclic linkage of the P1 and P3 residues, and the subsequent structure-based optimization of the macrocycle conformation and size led to the identification of a potent biochemical inhibitor 10 with sub-micromolar antiviral activity.
Assuntos
Antivirais/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Desenho de Fármacos , Compostos Macrocíclicos/farmacologia , Rhinovirus/efeitos dos fármacos , Proteínas Virais/antagonistas & inibidores , Proteases Virais 3C , Antivirais/síntese química , Antivirais/química , Cristalografia por Raios X , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/síntese química , Inibidores de Cisteína Proteinase/química , Relação Dose-Resposta a Droga , Humanos , Compostos Macrocíclicos/síntese química , Compostos Macrocíclicos/química , Testes de Sensibilidade Microbiana , Modelos Moleculares , Conformação Molecular , Rhinovirus/enzimologia , Relação Estrutura-Atividade , Proteínas Virais/metabolismoRESUMO
Lipoprotein(a) [Lp(a)] has recently been recognized as an independent risk factor for coronary heart disease. While plasma Lp(a) levels are correlated with cardiovascular risk, the mechanism by which this particle contributes to atherosclerosis is largely unknown. Although humanized transgenic mouse model has recently been described to study Lp(a) biology, non-human primates (NHP) are the only preclinical model available that allow study of the role of Lp(a) in atherosclerosis in an innate setting. We describe targeting of LPA using lipid nanoparticle formulated short interfering RNAs (siRNAs) in lean rhesus macaque monkeys. We show >90 % LPA mRNA lowering in the liver and >95 % Lp(a) plasma reduction for over 3 weeks after a single siRNA dose. Given the potency of LPA siRNAs, siRNA approach may enable chronic reduction of Lp(a) in atherosclerotic NHP and help to unmask the role for Lp(a) in the genesis and progression of atherosclerosis in man.
Assuntos
Aterosclerose/terapia , Lipoproteína(a)/genética , RNA Interferente Pequeno/genética , Terapêutica com RNAi , Animais , Aterosclerose/sangue , Aterosclerose/genética , Aterosclerose/patologia , Linhagem Celular , Modelos Animais de Doenças , Feminino , Técnicas de Transferência de Genes , Humanos , Lipídeos/química , Lipoproteína(a)/sangue , Fígado/metabolismo , Macaca mulatta , Masculino , Nanomedicina , Nanopartículas , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
Axitinib is a novel, oral, multitargeted tyrosine kinase inhibitor, which inhibits vascular endothelial growth factor receptors 1, 2, and 3 at subnanomolar concentrations in vitro. In the phase III clinical trial in patients with metastatic renal cell carcinoma, axitinib showed a high objective response rate, and significantly prolonged progression-free survival compared with sorafenib. Thus, it is the first drug that has proven the concept of sequencing tyrosine kinase inhibitors in second-line treatment in a phase III prospective randomized trial. Although generally well tolerated and associated with a low incidence of grade 3 or 4 toxicities, axitinib shows a distinct pattern of adverse events that require monitoring and management. The most common adverse events observed with axitinib include diarrhea, hypertension, fatigue, nausea, and vomiting. This article summarizes the most important adverse events observed and proposes recommendations for their monitoring, prevention, and treatment. The recommendations are based on the existing literature and discussion by an expert group of international physicians and nurses specialized in oncologic treatment of metastatic renal cell carcinoma, which gathered in July 2011 in London, UK. Proactive assessment and management of adverse events during axitinib therapy can minimize treatment interruptions and ensure optimal effect of treatment.
Assuntos
Carcinoma de Células Renais/tratamento farmacológico , Imidazóis/efeitos adversos , Imidazóis/uso terapêutico , Indazóis/efeitos adversos , Indazóis/uso terapêutico , Neoplasias Renais/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Axitinibe , Diarreia/induzido quimicamente , Diarreia/terapia , Disfonia/induzido quimicamente , Fadiga/induzido quimicamente , Humanos , Hipertensão/induzido quimicamente , Hipertensão/tratamento farmacológico , Imidazóis/administração & dosagem , Indazóis/administração & dosagem , Náusea/induzido quimicamente , Náusea/terapia , Inibidores de Proteínas Quinases/efeitos adversos , Proteinúria/induzido quimicamente , Proteinúria/tratamento farmacológico , Vômito/induzido quimicamente , Vômito/terapiaRESUMO
Angiotensinogen (AGT) is the precursor of active vasoconstrictive octapeptide angiotensin II (Ang II) in the renin-angiotensin-aldosterone system. Blocking the AGT-converting enzymes in the pathway and the Ang II receptor through pharmacological agents has been proven to be effective in lowering blood pressure (BP) in hypertensive patients. In this study, we developed chemically modified small interfering RNAs (siRNA) to target hepatic AGT mRNA in rats. Lipid nanoparticle encapsulated siRNAs were efficiently delivered to rat liver and resulted in significant reduction in hepatic Agt mRNA levels and plasma AGT concentration without impairing liver function. Single intravenous injection of Agt siRNA led to significant and sustained BP lowering in spontaneous hypertensive rats and in Sprague-Dawley rats, and the effect was maintained by weekly siRNA dosing. Data presented here provide proof-of-feasibility for the use of siRNA technology for inhibition of peripheral AGT levels via hepatic mRNA silencing with beneficial effects on BP in preclinical rat models. Similar approach could be used for validation of novel hypertension hepatic and extrahepatic targets.
Assuntos
Angiotensinogênio/metabolismo , Pressão Sanguínea/genética , Hipertensão/metabolismo , Fígado/metabolismo , Sistema Renina-Angiotensina/fisiologia , Angiotensinogênio/genética , Animais , Modelos Animais de Doenças , Hepatócitos/metabolismo , Hipertensão/genética , Hipertensão/fisiopatologia , Fígado/fisiopatologia , Masculino , Nanopartículas , RNA Interferente Pequeno , Ratos , Ratos Endogâmicos SHR , Ratos Sprague-DawleyRESUMO
Increased serum apolipoprotein (apo)B and associated LDL levels are well-correlated with an increased risk of coronary disease. ApoEâ»/â» and low density lipoprotein receptor (LDLr)â»/â» mice have been extensively used for studies of coronary atherosclerosis. These animals show atherosclerotic lesions similar to those in humans, but their serum lipids are low in apoB-containing LDL particles. We describe the development of a new mouse model with a human-like lipid profile. Ldlr CETPâº/â» hemizygous mice carry a single copy of the human CETP transgene and a single copy of a LDL receptor mutation. To evaluate the apoB pathways in this mouse model, we used novel short-interfering RNAs (siRNA) formulated in lipid nanoparticles (LNP). ApoB siRNAs induced up to 95% reduction of liver ApoB mRNA and serum apoB protein, and a significant lowering of serum LDL in Ldlr CETPâº/â» mice. ApoB targeting is specific and dose-dependent, and it shows lipid-lowering effects for over three weeks. Although specific triglycerides (TG) were affected by ApoB mRNA knockdown (KD) and the total plasma lipid levels were decreased by 70%, the overall lipid distribution did not change. Results presented here demonstrate a new mouse model for investigating additional targets within the ApoB pathways using the siRNA modality.
Assuntos
Apolipoproteínas B/genética , Aterosclerose/genética , Aterosclerose/metabolismo , Proteínas de Transferência de Ésteres de Colesterol/genética , LDL-Colesterol/sangue , Modelos Animais de Doenças , Receptores de LDL/genética , Animais , Apolipoproteínas B/sangue , Apolipoproteínas E/sangue , Apolipoproteínas E/genética , Aterosclerose/patologia , Linhagem Celular Tumoral , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Efeito Fundador , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Hemizigoto , Humanos , Metabolismo dos Lipídeos/genética , Lipossomos/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Nanopartículas/administração & dosagem , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/farmacologia , Receptores de LDL/metabolismo , Triglicerídeos/sangueAssuntos
Fiscalização e Controle de Instalações , Laboratórios/normas , Medicina na Literatura , Patologia Clínica/normas , Testes Genéticos/legislação & jurisprudência , Testes Genéticos/normas , Humanos , Consentimento Livre e Esclarecido/legislação & jurisprudência , Laboratórios/legislação & jurisprudência , Propriedade/legislação & jurisprudência , Patologia Clínica/legislação & jurisprudência , Doadores de Tecidos/legislação & jurisprudência , Estados UnidosAssuntos
Ética em Pesquisa , Genes , Pesquisa em Genética , Acessibilidade aos Serviços de Saúde/ética , Acessibilidade aos Serviços de Saúde/legislação & jurisprudência , Patentes como Assunto/ética , Patentes como Assunto/legislação & jurisprudência , Animais , Temas Bioéticos , Análise Ética , Teoria Ética , Europa (Continente) , Liberdade , Pesquisa em Genética/economia , Pesquisa em Genética/ética , Pesquisa em Genética/legislação & jurisprudência , Política de Saúde/legislação & jurisprudência , Humanos , Consentimento Livre e Esclarecido/ética , Consentimento Livre e Esclarecido/legislação & jurisprudência , Direitos do Paciente/ética , Direitos do Paciente/legislação & jurisprudência , Comportamento Reprodutivo , Nações Unidas , Estados UnidosAssuntos
Genes , Doenças Genéticas Inatas/genética , Patentes como Assunto , Doença de Alzheimer/genética , Asma/genética , Ataxia Telangiectasia/genética , Neoplasias da Mama/genética , Doença de Canavan/genética , Disautonomia Familiar/genética , Hemocromatose/genética , Humanos , Mutação , Obesidade/genética , Patentes como Assunto/legislação & jurisprudência , Polimorfismo Genético , Política Pública , Esquizofrenia/genética , Estados UnidosRESUMO
An allosteric ribozyme is an RNA-based enzyme (ribozyme) whose catalytic activity is modulated by molecular recognition of a protein. The direct coupling of a detectable catalytic event to molecular recognition by an allosteric ribozyme enables simple assays for quantitative protein detection. Most significantly, the mode of development and molecular recognition characteristics of allosteric ribozymes are fundamentally different from antibodies, providing them with functional characteristics that complement those of antibodies. Allosteric ribozymes can be developed using native proteins and, therefore, are often sensitive to protein conformation. In contrast, antibodies tend to recognize a series of adjacent amino acids as a consequence of antigen presentation and typically are not sensitive to protein conformation. Unlike antibody development, the development of allosteric ribozymes is a completely in vitro process that allows the specificity of an allosteric ribozyme to be tightly controlled. These significant differences from antibodies allow the pre-programmed development of conformation-state-specific protein detection reagents that can be used to investigate the activation-state of signal transduction components.
Assuntos
Proteínas/metabolismo , RNA Catalítico/metabolismo , Regulação Alostérica , Sítio Alostérico , Sequência de Bases , Catálise , Clonagem Molecular , DNA Complementar/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Evolução Molecular Direcionada/métodos , Transferência Ressonante de Energia de Fluorescência , Biblioteca Gênica , Cinética , Modelos Químicos , Modelos Moleculares , Conformação de Ácido Nucleico , Reação em Cadeia da Polimerase , Ligação Proteica , Conformação Proteica , Proteínas/química , RNA/química , RNA/genética , RNA/metabolismo , RNA Catalítico/química , RNA Catalítico/genética , Proteínas ViraisRESUMO
To establish peroxynitrite (ONOO(-)) as a mediator of acidic fibroblast growth factor (FGF-1) function, preparations of recombinant human FGF-1 were treated with the pro-oxidant in vitro and identified amino acid modifications were correlated with biologic activity. The sequence of FGF-1 amino acid modifications induced by increasing concentrations of ONOO(-) was from cysteine oxidation to dityrosine formation, and to tyrosine/tryptophan nitration. Low steady-state ONOO(-) concentrations (10-50 microM) induced formation of dityrosine, which involved less than 0.1% of the total tyrosines. Treatment of FGF-1 with ONOO(-) induced a dose-dependent (10-50 microM) loss of sulfhydryl groups that correlated with formation of reducible (dithiothreitol, arsenite) FGF-1 aggregates containing 50% latent biologic activity. Treatment with 0.1-0.5mM ONOO(-) induced increasing formation of non-reducible, inactivated FGF-1 structures. Combination of real-time spectral analysis and electrospray mass spectroscopy revealed that six residues (Y29, Y69, Y108, Y111, Y139, and W121) were nitrated by ONOO(-). ONOO(-) treatment (0.1mM) of an active FGF-1 mutant (cysteines converted to serines) induced dose-dependent, non-reversible inhibition of biologic activity that correlated with nitration of Y108 and Y111, both of which reside within a conserved domain encompassing the putative FGF-1 receptor binding site. Collectively, these observations predict a role for low levels of ONOO(-) during secretion of FGF-1 as an extracellular complex containing latent biologic activity. High steady-state levels of ONOO(-) may induce extensive cysteine oxidation, critical tyrosine nitration, and non-reversible inactivation of FGF-1, a potential inhibitory feedback mechanism restoring cellular homeostatis during the resolution of inflammation and repair.
Assuntos
Aminoácidos/química , Fator 1 de Crescimento de Fibroblastos/química , Fator 1 de Crescimento de Fibroblastos/farmacologia , Ácido Peroxinitroso/química , Ácido Peroxinitroso/farmacologia , Tirosina/análogos & derivados , Células 3T3 , Animais , Contagem de Células , Divisão Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Fator 1 de Crescimento de Fibroblastos/classificação , Humanos , Camundongos , Nitratos/química , Oxirredução , Tirosina/síntese químicaRESUMO
We describe a strategy for the ultra-sensitive detection of nucleic acids using "half" ribozymes that are devoid of catalytic activity unless completed by a trans-acting target nucleic acid. The half-ribozyme concept was initially demonstrated using a construct derived from a multiple turnover Class I ligase. Iterative RNA selection was carried out to evolve this half-ribozyme into one activated by a conserved sequence present in the hepatitis C virus (HCV) genome. Following sequence optimization of substrate RNAs, this HCV-activated half-ribozyme displayed a maximal turnover rate of 69 min(-1) (pH 8.3) and was induced in rate by approximately 2.6 x 10(9)-fold by the HCV target. It detected the HCV target oligonucleotide in the zeptomole range (6700 molecules), a sensitivity of detection roughly 2.6 x 10(6)-fold greater than that previously demonstrated by oligonucleotide-activated ribozymes, and one that is sufficient for molecular diagnostic applications.
Assuntos
Hepacivirus/genética , RNA Catalítico/metabolismo , RNA Viral/análise , Concentração de Íons de Hidrogênio , RNA Catalítico/genética , Fatores de TempoAssuntos
Predisposição Genética para Doença , Testes Genéticos , Herança Multifatorial/genética , Confidencialidade/ética , Confidencialidade/legislação & jurisprudência , Bases de Dados de Ácidos Nucleicos/ética , Bases de Dados de Ácidos Nucleicos/legislação & jurisprudência , Etnicidade/genética , Doenças Genéticas Inatas , Pesquisa em Genética , Testes Genéticos/economia , Testes Genéticos/ética , Testes Genéticos/legislação & jurisprudência , Humanos , Islândia , Consentimento Livre e Esclarecido/ética , Consentimento Livre e Esclarecido/legislação & jurisprudência , Marketing de Serviços de Saúde , Prontuários Médicos , Estresse Psicológico , Estados UnidosRESUMO
Concerns about human gene patents go beyond moral disquiet about creating a commodity from a part of the human body and also beyond legal questions about whether genes are unpatentable products of nature. New concerns are being raised about harm to public health and to research. In response to these concerns, various policy options, such as litigation, legislation, patent pools and compulsory licensing, are being explored to ensure that gene patents do not impede the practice of medicine and scientific progress.
Assuntos
Genes/ética , Propriedade Intelectual , Política Pública , Biotecnologia/economia , Biotecnologia/ética , Biotecnologia/legislação & jurisprudência , Empreendedorismo/ética , Europa (Continente) , Pesquisa em Genética/economia , Pesquisa em Genética/ética , Humanos , Licenciamento/economia , Licenciamento/legislação & jurisprudência , Patentes como Assunto/ética , Patentes como Assunto/legislação & jurisprudência , Estados UnidosRESUMO
An allosteric hammerhead ribozyme activated specifically by the unphosphorylated form of the protein kinase ERK2 was created through a rational design strategy that relies on molecular recognition of ERK2 to decrease the formation of an alternate, inactive ribozyme conformer. Neither closely related mitogen-activated protein kinases (MAPKs) nor the phosphorylated form of ERK2 induced ribozyme activity. The ribozyme quantitatively detected ERK2 added to mammalian cell lysates and also functioned quantitatively in a multiplexed solution-phase assay. This same strategy was used to construct a second ribozyme selectively activated by the phosphorylated (active) form of ERK2. This approach is generally applicable to the development of ribozymes capable of monitoring post-translational modification of specific proteins.