RESUMO
ß-Site amyloid precursor protein cleaving enzyme 1 (BACE1) has been pursued as a prime target for the treatment of Alzheimer's disease (AD). In this report, we describe the discovery of BACE1 inhibitors with a 1-amino-3,4-dihydro-2,6-naphthyridine scaffold. Leveraging known inhibitors 2a and 2b, we designed the naphthyridine-based compounds by removing a structurally labile moiety and incorporating pyridine rings, which showed increased biochemical and cellular potency, along with reduced basicity on the amidine moiety. Introduction of a fluorine atom on the pyridine culminated in compound 11 which had improved cellular activity as well as further reduced basicity and demonstrated a robust and sustained cerebrospinal fluid (CSF) Aß reduction in dog. The crystal structure of compound 11 bound to BACE1 confirmed van der Waals interactions between the fluorine on the pyridine and Tyr71 in the flap.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Peptídeos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Naftiridinas/química , Inibidores de Proteases/química , Piridinas/química , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Animais , Ácido Aspártico Endopeptidases/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Cães , Avaliação Pré-Clínica de Medicamentos , Meia-Vida , Humanos , Microssomos/metabolismo , Simulação de Dinâmica Molecular , Naftiridinas/metabolismo , Naftiridinas/farmacologia , Inibidores de Proteases/metabolismo , Inibidores de Proteases/farmacologia , Ratos , Eletricidade Estática , Relação Estrutura-AtividadeRESUMO
Antisense oligonucleotides (ASOs) are classified into gapmer and non-gapmer types according to their chemical modification pattern and mechanism of action. Although gapmer ASOs effectively reduce target RNA expression through intracellular RNase H1, high-affinity gapmer ASOs also have hepatotoxic potential. Non-gapmer ASOs, which are mainly used for pre-mRNA splicing regulation or functional inhibition of microRNA through their steric effects, are also able to inhibit target RNA expression using nonsense-mediated decay. However, it was unknown if they induce high knockdown activity without showing hepatotoxicity. In this study, we investigated the modification pattern of non-gapmer ASOs and show that they have comparable knockdown potential if they have an appropriate melting temperature (Tm) range. We also demonstrated that non-gapmer ASOs show high knockdown effects without inducing hepatotoxicity in the mouse liver. These results indicated that non-gapmer ASOs have the potential to become an alternative inhibitor of target expression with a lower risk of hepatotoxicity.
Assuntos
Fígado/efeitos dos fármacos , Degradação do RNAm Mediada por Códon sem Sentido/efeitos dos fármacos , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos Fosforotioatos/farmacologia , Animais , Éxons/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Fígado/metabolismo , Camundongos , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Fosforotioatos/genética , RNA Mensageiro/efeitos dos fármacos , Ribonuclease H/antagonistas & inibidores , Ribonuclease H/genética , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/genéticaRESUMO
Accumulation of Aß peptides is a hallmark of Alzheimer's disease (AD) and is considered a causal factor in the pathogenesis of AD. ß-Secretase (BACE1) is a key enzyme responsible for producing Aß peptides, and thus agents that inhibit BACE1 should be beneficial for disease-modifying treatment of AD. Here we describe the discovery and optimization of novel oxazine-based BACE1 inhibitors by lowering amidine basicity with the incorporation of a double bond to improve brain penetration. Starting from a 1,3-dihydrooxazine lead 6 identified by a hit-to-lead SAR following HTS, we adopted a p Ka lowering strategy to reduce the P-gp efflux and the high hERG potential leading to the discovery of 15 that produced significant Aß reduction with long duration in pharmacodynamic models and exhibited wide safety margins in cardiovascular safety models. This compound improved the brain-to-plasma ratio relative to 6 by reducing P-gp recognition, which was demonstrated by a P-gp knockout mouse model.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Peptídeos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Oxazinas/química , Fragmentos de Peptídeos/metabolismo , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Secretases da Proteína Precursora do Amiloide/química , Animais , Ácido Aspártico Endopeptidases/química , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Cristalografia por Raios X , Cães , Desenho de Fármacos , Canal de Potássio ERG1/metabolismo , Cobaias , Humanos , Células Madin Darby de Rim Canino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxazinas/farmacologia , Inibidores de Proteases/farmacocinética , Relação Estrutura-AtividadeRESUMO
Antisense oligonucleotides are attractive therapeutic agents for several types of disease. One of the most promising modifications of antisense oligonucleotides is the introduction of bridged nucleic acids. As we report here, we designed novel bridged nucleic acids, triazole-bridged nucleic acid (TrNA), and tetrazole-bridged nucleic acid (TeNA), whose sugar conformations are restricted to N-type by heteroaromatic ring-bridged structures. We then successfully synthesized TrNA and TeNA and introduced these monomers into oligonucleotides. In UV-melting experiments, TrNA-modified oligonucleotides exhibited increased binding affinity toward complementary RNA and decreased binding affinity toward complementary DNA, although TeNA-modified oligonucleotides were decomposed under the annealing conditions. Enzymatic degradation experiments demonstrated that introduction of TrNA at the 3'-terminus rendered oligonucleotides resistant to nuclease digestion. Furthermore, we tested the silencing potencies of TrNA-modified antisense oligonucleotides using in vitro and in vivo assays. These experiments revealed that TrNA-modified antisense oligonucleotides induced potent downregulation of gene expression in liver. In addition, TrNA-modified antisense oligonucleotides showed a tendency for increased liver biodistribution. Taken together, our findings indicate that TrNA is a good candidate for practical application in antisense methodology.
Assuntos
DNA Complementar/química , Desoxirribonucleases/química , Ácidos Nucleicos/síntese química , Oligonucleotídeos Antissenso/química , RNA Complementar/química , Tetrazóis/síntese química , Desoxirribonucleases/metabolismo , Humanos , Conformação de Ácido Nucleico , Ácidos Nucleicos/química , Tetrazóis/químicaRESUMO
A sulfonamide-bridged nucleic acid without an N-substituent (SuNA[NH]) was successfully synthesized. A comparison of the SuNA[NMe]- and SuNA[NH]-modified oligonucleotides revealed that the duplex-forming abilities of the SuNA[NMe]-modified oligonucleotides with complementary DNA and RNA were higher than those of the SuNA[NH]-modified oligonucleotides. The crystal structures of DNA duplexes containing a SuNA[NR] revealed that the helical structures of the two duplexes and hydration patterns around the bridge moiety were different. These results provide insights into hydration patterns and rationale for the high RNA affinity of SuNA-modified oligonucleotides.
Assuntos
DNA/química , Nitrogênio/química , Conformação de Ácido Nucleico , RNA/química , Sulfonamidas/química , Sequência de Bases , DNA/genética , Modelos Moleculares , Hibridização de Ácido Nucleico , RNA/genéticaRESUMO
Mps1, also known as TTK, is a dual-specificity kinase that regulates the spindle assembly check point. Increased expression levels of Mps1 are observed in cancer cells, and the expression levels correlate well with tumor grade. Such evidence points to selective inhibition of Mps1 as an attractive strategy for cancer therapeutics. Starting from an aminopyridine-based lead 3a that binds to a flipped-peptide conformation at the hinge region in Mps1, elaboration of the aminopyridine scaffold at the 2- and 6-positions led to the discovery of 19c that exhibited no significant inhibition for 287 kinases as well as improved cellular Mps1 and antiproliferative activities in A549 lung carcinoma cells (cellular Mps1 IC50=5.3 nM, A549 IC50=26 nM). A clear correlation between cellular Mps1 and antiproliferative IC50 values indicated that the antiproliferative activity observed in A549 cells would be responsible for the cellular inhibition of Mps1. The X-ray structure of 19c in complex with Mps1 revealed that this compound retains the ability to bind to the peptide flip conformation. Finally, comparative analysis of the X-ray structures of 19c, a deamino analogue 33, and a known Mps1 inhibitor bound to Mps1 provided insights into the unique binding mode at the hinge region.
Assuntos
Aminopiridinas/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Aminopiridinas/síntese química , Aminopiridinas/química , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Estabilidade de Medicamentos , Humanos , Masculino , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Solubilidade , Relação Estrutura-Atividade , Distribuição TecidualRESUMO
2'-N,4'-C-(N-methylamino)sulfonylmethylene-bridged thymidine (SuNA), which has a six-membered linkage including a sulfonamide moiety, was synthesized and introduced into oligonucleotides. The oligonucleotides containing SuNA exhibited excellent nuclease resistance, a high affinity toward single-stranded RNA, and a low affinity toward single-stranded DNA compared to the natural oligonucleotide.
Assuntos
Hidrocarbonetos Aromáticos com Pontes/síntese química , Ácidos Nucleicos/química , Oligonucleotídeos/química , RNA/química , Ribonucleases/química , Ribonucleases/síntese química , Sulfonamidas/química , Timidina/análogos & derivados , Timidina/síntese química , Hidrocarbonetos Aromáticos com Pontes/química , Estrutura Molecular , Hibridização de Ácido Nucleico , Sulfonamidas/síntese química , Timidina/químicaRESUMO
Recently, we synthesized pyrimidine derivatives of the 2'-O,4'-C-methylenoxymethylene-bridged nucleic-acid (2',4'-BNA(COC)) monomer, the sugar conformation of which is restricted in N-type conformation by a seven-membered bridged structure. Oligonucleotides (BNA(COC)) containing this monomer show high affinity with complementary single-stranded RNA and significant resistance to nuclease degradation. Here, BNA(COC) consisting of 2',4'-BNA(COC) monomers bearing all four bases, namely thymine, 5-methylcytosine, adenine and guanine was efficiently synthesized and properties of duplexes containing the 2',4'-BNA(COC) monomers were investigated by UV melting experiments and circular dichroism (CD) spectroscopy. The UV melting curve analyses showed that the BNA(COC)/BNA(COC) duplex possessed excellent thermal stability and that the BNA(COC) increased thermal stability with a complementary RNA strand. On the other hand, BNA(COC)/DNA heteroduplexes showed almost the same thermal stability as RNA/DNA heteroduplexes. Furthermore, mismatched sequence studies showed that BNA(COC) generally improved the sequence selectivity with Watson-Crick base-pairing compared to the corresponding natural DNA and RNA. A CD spectroscopic analysis indicated that the BNA(COC) formed duplexes with complementary DNA and RNA in a manner similar to natural RNA.
Assuntos
Ácidos Nucleicos/química , Oligonucleotídeos/química , RNA/química , 5-Metilcitosina/química , Adenina/química , Pareamento Incorreto de Bases , Hidrocarbonetos Aromáticos com Pontes/síntese química , Hidrocarbonetos Aromáticos com Pontes/química , Dicroísmo Circular , DNA/química , Guanina/química , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Oligonucleotídeos/síntese química , Timina/químicaRESUMO
We newly designed and synthesized a 2'-deoxy type trans-3',4'-bridged nucleic acid (trans-3',4'-BNA) analogues bearing a 4,7-dioxabicyclo[4.3.0]nonane structure. The synthesis of the trans-3',4'-BNA was carried out successfully from thymidine over 21 steps. The structure of trans-3',4'-BNA was confirmed by x-ray crystallographic analysis, indicating that the furanose ring has a typical S-type conformation with C(3')-exo puckering.
Assuntos
Desoxirribonucleosídeos/síntese química , Compostos Bicíclicos com Pontes/síntese química , Compostos Bicíclicos com Pontes/química , Cristalografia por Raios X , Desoxirribonucleosídeos/química , Desenho de Fármacos , Métodos , Conformação Molecular , Estrutura Molecular , EstereoisomerismoRESUMO
We previously synthesized bridged nucleic acid (BNA) analogues possessing an S-type sugar conformation. Recently, we achieved the synthesis of a 2'-deoxy analogue of trans-3',4'-BNA. The structure of this compound was confirmed by X-ray crystallography, which indicated that its sugar ring had an S-type conformation. The hybridization ability of the 2'-deoxy-trans-3',4'-BNA-modified oligonucleotide with DNA and RNA complements was evaluated, and it was found that 2'-deoxy-trans-3',4'-BNA showed superior hybridization ability compared with other BNA analogues with S-type sugar conformation.
Assuntos
Hidrocarbonetos Aromáticos com Pontes/química , Nucleosídeos/química , Oligonucleotídeos/química , Timidina/análogos & derivados , Hidrocarbonetos Aromáticos com Pontes/síntese química , Configuração de Carboidratos , DNA/química , Hibridização de Ácido Nucleico , Nucleosídeos/síntese química , RNA/química , Timidina/síntese química , Timidina/químicaRESUMO
Recently, we have synthesized pyrimidine derivatives of 2',4'-BNA(COC) monomer, the sugar conformation of which was restricted in N-form by a seven-membered bridged structure. The oligonucleotides containing these monomers showed high affinity with complementary single-stranded RNA and significant resistance to nuclease degradation. For an application to antisense methodology, it is important to synthesize 2',4'-BNA(COC) monomers bearing a purine nucleobase. However, the formation of methyleneoxymethylene (COC) linkage in the purine derivatives failed under the acidic conditions used for the synthesis of 2',4'-BNA(COC) with pyrimidine nucleobases. After several examinations, we successfully achieved the synthesis of 2',4'-BNA(COC) monomers bearing a purine nucleobase via the formation of COC linkage using Pummerer-type reaction.
Assuntos
Oligonucleotídeos Antissenso/síntese química , Purinas/química , Hidrocarbonetos Aromáticos com Pontes/síntese química , Hidrocarbonetos Aromáticos com Pontes/química , Oligonucleotídeos Antissenso/químicaRESUMO
The synthesis of nucleoside analogues with a conformationally restricted sugar moiety is of great interest. The present research describes the synthesis of BNA (bridged nucleic acid) monomers 1 and 2 bearing a 4,7-dioxabicyclo[4.3.0]nonane skeleton and a methoxy group at the C2' position. Conformational analysis showed that the sugar moiety of these monomers is restricted in a typical S-type conformation. It was difficult to synthesize the phosphoramidite derivative of the ribo-type monomer 1, while the phosphoramidite of the arabino-type monomer 2 was successfully prepared and incorporated into oligodeoxynucleotides (ODNs). The hybridization ability of the obtained ODN derivatives containing 2 with complementary strands was evaluated by melting temperature (T(m)) measurements. As a result, the ODN derivatives hybridized with DNA and RNA complements in a sequence-selective manner, though the stability of the duplexes was lower than that of the corresponding natural DNA/DNA or DNA/RNA duplex.