RESUMO
Robust induction of cancer-antigen-specific CD8+ T cells is essential for the success of cancer peptide vaccines, which are composed of a peptide derived from a cancer-specific antigen and an immune-potentiating adjuvant, such as a Toll-like receptor (TLR) agonist. Efficient delivery of a vaccine antigen and an adjuvant to antigen-presenting cells in the draining lymph nodes (LNs) holds key to maximize vaccine efficacy. Here, we developed S-540956, a novel TLR9-agonistic adjuvant consisting of B-type CpG ODN2006 (also known as CpG7909), annealed to its complementary sequence oligodeoxynucleotide (ODN) conjugated to a lipid; it could target both a cancer peptide antigen and a CpG-adjuvant in the draining LNs. S-540956 accumulation in the draining LNs and activation of plasmacytoid dendritic cells (pDCs) were significantly higher than that of ODN2006. Mechanistic analysis revealed that S-540956 enhanced the induction of MHC class I peptide-specific CD8+ T cell responses via TLR9 in a CD4+ T cell-independent manner. In mice, the therapeutic effect of S-540956-adjuvanted with a human papillomavirus (HPV)-E7 peptide vaccine against HPV-E7-expressing TC-1 tumors was significantly better than that of an ODN2006-adjuvanted vaccine. Our findings demonstrate a novel adjuvant discovery with the complementary strand conjugated to a lipid, which enabled draining LN targeting and increased ODN2006 accumulation in draining LNs, thereby enhancing the adjuvant effect. Our findings imply that S-540956 is a promising adjuvant for cancer peptide vaccines and has a high potential for applications in various vaccines, including recombinant protein vaccines.
Assuntos
Adjuvantes de Vacinas/uso terapêutico , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Neoplasias Pulmonares/imunologia , Oligodesoxirribonucleotídeos/administração & dosagem , Proteínas E7 de Papillomavirus/imunologia , Linfonodo Sentinela/imunologia , Receptor Toll-Like 9/metabolismo , Adjuvantes de Vacinas/química , Animais , Diferenciação Celular , DNA/química , Feminino , Humanos , Imunização , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais , Oligodesoxirribonucleotídeos/química , Tensoativos/química , Receptor Toll-Like 9/agonistas , Receptor Toll-Like 9/genética , Vacinas de Subunidades AntigênicasRESUMO
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
Gapmer antisense oligonucleotides (gapmers) sometimes cleave nontarget pre-mRNAs by recognizing target-like intronic/exonic portions. This off-target RNA cleavage could be a major cause of the hepatotoxicity that is induced by gapmers. In line with these findings, we hypothesized that gapmers with higher specificity have less hepatotoxicity, and that those with lower specificity have greater toxicity. To examine this concept, we investigated various Malat1-targeting gapmers with various computationally evaluated target specificities. We had expected that higher specificity gapmers would have lower hepatotoxicity, but these factors were not significantly related. In silico analysis of gapmer sequences does not always contribute to mitigating the risk of hepatotoxicity. Transcriptome analysis indicated that nontoxic gapmers do not cleave off-target RNAs, although they have many target-like RNA sequences. The present results shed light on the mechanism of the hepatotoxicity of gapmers.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/genética , Oligonucleotídeos Antissenso/efeitos adversos , Precursores de RNA/efeitos adversos , RNA Longo não Codificante/genética , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/terapia , Linhagem Celular Tumoral , Doença Hepática Induzida por Substâncias e Drogas/patologia , Biologia Computacional , Simulação por Computador , Perfilação da Expressão Gênica/métodos , Humanos , Camundongos , Oligonucleotídeos Antissenso/administração & dosagem , Precursores de RNA/administração & dosagem , RNA Longo não Codificante/antagonistas & inibidores , RNA Mensageiro/genética , Ribonuclease H/antagonistas & inibidores , Ribonuclease H/genética , Transcriptoma/efeitos dos fármacos , Transcriptoma/genéticaRESUMO
Cholesterol (Chol) conjugation to the 5' or 3' end of antisense oligonucleotide (ASO) enables delivery to the liver, and Chol conjugation at the gap region can also be expected to improve delivery to the liver. In this study, we synthesized ASOs bearing the Chol-conjugated thiono triester and evaluated their activity and hepatic accumulation. We found that Chol conjugations at the gap region improved in vitro activity and hepatic accumulation when compared to unconjugated ASOs. However, Chol conjugation with phosphorothioate linkage did not improve in vivo activity in the liver, suggesting the importance of cleaving the phosphodiester between ASO and Chol. These results offer useful information for tuning the oligonucleotide structure to improve pharmaceutical properties and designing ASOs for multiple ligand conjugations and combinations with end modification.
Assuntos
Ésteres do Colesterol/farmacocinética , Fígado/metabolismo , Oligonucleotídeos Antissenso/farmacocinética , Animais , Apolipoproteínas B/genética , Apolipoproteínas B/metabolismo , Expressão Gênica , Técnicas de Silenciamento de Genes , Masculino , Camundongos Endogâmicos C57BL , Interferência de RNA , Distribuição TecidualRESUMO
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
Gapmer antisense oligonucleotides cleave target RNA effectively in vivo, and is considered as promising therapeutics. Especially, gapmers modified with locked nucleic acid (LNA) shows potent knockdown activity; however, they also cause hepatotoxic side effects. For developing safe and effective gapmer drugs, a deeper understanding of the mechanisms of hepatotoxicity is required. Here, we investigated the cause of hepatotoxicity derived from LNA-modified gapmers. Chemical modification of gapmer's gap region completely suppressed both knockdown activity and hepatotoxicity, indicating that the root cause of hepatotoxicity is related to intracellular gapmer activity. Gene silencing of hepatic ribonuclease H1 (RNaseH1), which catalyses gapmer-mediated RNA knockdown, strongly supressed hepatotoxic effects. Small interfering RNA (siRNA)-mediated knockdown of a target mRNA did not result in any hepatotoxic effects, while the gapmer targeting the same position on mRNA as does the siRNA showed acute toxicity. Microarray analysis revealed that several pre-mRNAs containing a sequence similar to the gapmer target were also knocked down. These results suggest that hepatotoxicity of LNA gapmer is caused by RNAseH1 activity, presumably because of off-target cleavage of RNAs inside nuclei.
Assuntos
Inativação Gênica , Fígado/metabolismo , Oligonucleotídeos Antissenso/toxicidade , Oligonucleotídeos/toxicidade , Ribonuclease H/genética , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oligonucleotídeos/genética , Oligonucleotídeos Antissenso/genética , Ribonuclease H/metabolismoRESUMO
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
TriantennaryN-acetyl galactosamine (GalNAc, GN3) and lipophilic ligands such as cholesterol andα-tocopherol conjugations dramatically improve the distribution and efficacy of second-generation antisense oligonucleotides (ASOs) in the whole liver. To characterize ligands for delivery to liver cells based on pharmacokinetics and efficacy, we used a locked nucleic acid gapmer of ASO targeting apolipoprotein B as a model compound and evaluated the amount of ASO and apolipoprotein B mRNA in the whole liver, hepatocytes, and nonparenchymal (NP) cells as well as plasma total cholesterol after administration of ASO conjugated with these ligands to mice. Compared with unconjugated ASO, GN3 conjugation increased the amount (7-fold) and efficacy (more than 10-fold) of ASO in hepatocytes only and showed higher efficacy than the increased rate of the amount of ASO. On the other hand, lipophilic ligand conjugations led to increased delivery (3- to 5-fold) and efficacy (5-fold) of ASO to both hepatocytes and NP cells. GN3 and lipophilic ligand conjugations increased the area under the curve of ASOs and the pharmacodynamic duration but did not change the half-life in hepatocytes and NP cells compared with unconjugated ASO. In the liver, the phosphodiester bond between ASO and these ligands was promptly cleaved to liberate unconjugated ASO. These ligand conjugations reduced plasma total cholesterol compared with unconjugated ASO, although these ASOs were well tolerated with no elevation in plasma transaminases. These findings could facilitate ligand selection tailored to liver cells expressed in disease-related genes and could contribute to the discovery and development of RNA interference-based therapy.
Assuntos
Acetilgalactosamina/química , Apolipoproteínas B/efeitos dos fármacos , Hepatócitos/metabolismo , Lipídeos/química , Fígado/metabolismo , Oligonucleotídeos Antissenso/farmacocinética , RNA Mensageiro/farmacocinética , Animais , Colesterol/sangue , Técnicas de Transferência de Genes , Meia-Vida , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/química , Interferência de RNA , RNA Mensageiro/administração & dosagem , RNA Mensageiro/química , Transaminases/metabolismoRESUMO
In the present study, we developed an assay to evaluate the kinetic binding properties of the unconjugated antisense oligonucleotide (ASO) and lipophilic and hydrophilic ligands conjugated ASOs to mouse and human serum albumin, and lipoproteins using surface plasmon resonance (SPR). The lipophilic ligands conjugated ASOs showed clear affinity to the albumins and lipoproteins, while the unconjugated and hydrophilic ligand conjugated ASOs showed no interaction. The SPR method showed reproducible immobilization of albumins and lipoproteins as ligands on the sensor chip, and reproducible affinity kinetic parameters of interaction of ASOs conjugated with the ligands could be obtained. The kinetic binding data of these ASOs to albumin and lipoproteins by SPR were related with the distributions in the whole liver in mice after administration of these conjugated ASOs. The results demonstrated that our SPR method could be a valuable tool for predicting the mechanism of the properties of delivery of conjugated ASOs to the organs.
Assuntos
Acetilgalactosamina/química , Lipoproteínas HDL/química , Lipoproteínas LDL/química , Oligonucleotídeos Antissenso/química , Albumina Sérica/química , Ressonância de Plasmônio de Superfície/métodos , Animais , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Camundongos , Ligação ProteicaRESUMO
Prostaglandin (PG) D2 elicits responses through either the DP1 and/or DP2 receptor. Experimental evidence suggests that stimulation of the DP1 receptor contributes to allergic responses, such that antagonists are considered to be directed therapies for allergic diseases. In this study, we demonstrate the activity of a novel synthetic DP1 receptor antagonist termed asapiprant (S-555739) for the DP1 receptor and other receptors in vitro, and assess the efficacy of asapiprant in several animal models of allergic diseases. We determined the affinity and selectivity of asapiprant for the DP1 receptor in binding assays. In the animal models of allergic rhinitis, changes in nasal resistance, nasal secretion, and cell infiltration in nasal mucosa were assessed after antigen challenge with and without asapiprant. Similarly, in the animal models of asthma, the effect of antigen challenge with and without asapiprant on antigen-induced bronchoconstriction, airway hyper-responsiveness, mucin production, and cell infiltration in lung were assessed. In binding studies, asapiprant exhibited high affinity and selectivity for the DP1 receptor. Significant suppression of antigen-induced nasal resistance, nasal secretion, and cell infiltration in nasal mucosa was observed with asapiprant treatment. In addition, treatment with asapiprant suppressed antigen-induced asthmatic responses, airway hyper-responsiveness, and cell infiltration and mucin production in lung. These results show that asapiprant is a potent and selective DP1 receptor antagonist, and exerts suppressive effects in the animal models of allergic diseases. Thus, asapiprant has potential as a novel therapy for allergic airway diseases.
Assuntos
Asma/tratamento farmacológico , Modelos Animais de Doenças , Receptores de Prostaglandina/antagonistas & inibidores , Rinite Alérgica/tratamento farmacológico , Tiofenos/uso terapêutico , Animais , Asma/imunologia , Asma/metabolismo , Cães , Feminino , Cobaias , Humanos , Masculino , Prostaglandinas/química , Prostaglandinas/farmacologia , Prostaglandinas/uso terapêutico , Ratos , Ratos Endogâmicos BN , Receptores de Prostaglandina/fisiologia , Hipersensibilidade Respiratória/tratamento farmacológico , Hipersensibilidade Respiratória/imunologia , Hipersensibilidade Respiratória/metabolismo , Rinite Alérgica/imunologia , Rinite Alérgica/metabolismo , Ovinos , Tiofenos/química , Tiofenos/farmacologia , Resultado do TratamentoRESUMO
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
Fatostatin, a recently discovered small molecule that inhibits activation of sterol regulatory element-binding protein (SREBP), blocks biosynthesis and accumulation of fat in obese mice. We synthesized and evaluated a series of fatostatin derivatives. Our structure-activity relationships led to the identification of N-(4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)phenyl)methanesulfonamide (24, FGH10019) as the most potent druglike molecule among the analogues tested. Compound 24 has high aqueous solubility and membrane permeability and may serve as a seed molecule for further development.
Assuntos
Proteínas de Ligação a Elemento Regulador de Esterol/antagonistas & inibidores , Sulfonamidas/síntese química , Tiazóis/síntese química , Animais , Glicemia/análise , Células CHO , Cricetinae , Cricetulus , Ingestão de Alimentos/efeitos dos fármacos , Hepatócitos/metabolismo , Masculino , Membranas Artificiais , Camundongos , Camundongos Obesos , Permeabilidade , Piridinas/síntese química , Piridinas/química , Piridinas/farmacologia , Solubilidade , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacologia , Tiazóis/química , Tiazóis/farmacologiaRESUMO
Sterol regulatory element binding proteins (SREBPs) are transcription factors that activate transcription of the genes involved in cholesterol and fatty acid biosynthesis. In the present study, we show that a small synthetic molecule we previously discovered to block adipogenesis is an inhibitor of the SREBP activation. The diarylthiazole derivative, now called fatostatin, impairs the activation process of SREBPs, thereby decreasing the transcription of lipogenic genes in cells. Our analysis suggests that fatostatin inhibits the ER-Golgi translocation of SREBPs through binding to their escort protein, the SREBP cleavage-activating protein (SCAP), at a distinct site from the sterol-binding domain. Fatostatin blocked increases in body weight, blood glucose, and hepatic fat accumulation in obese ob/ob mice, even under uncontrolled food intake. Fatostatin may serve as a tool for gaining further insights into the regulation of SREBP.
Assuntos
Ácidos Graxos/biossíntese , Piridinas/farmacologia , Proteínas de Ligação a Elemento Regulador de Esterol/antagonistas & inibidores , Tiazóis/farmacologia , Animais , Glicemia/metabolismo , Peso Corporal , Células CHO , Cricetinae , Cricetulus , Ácidos Graxos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Obesos , Ligação Proteica , Estrutura Terciária de Proteína , Piridinas/química , Proteínas de Ligação a Elemento Regulador de Esterol/química , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismo , Tiazóis/química , Transcrição GênicaAssuntos
Proteínas de Ligação a DNA/fisiologia , Lipogênese/genética , Receptores Citoplasmáticos e Nucleares/fisiologia , Animais , DNA , Proteínas de Ligação a DNA/agonistas , Proteínas de Ligação a DNA/classificação , Proteínas de Ligação a DNA/genética , Ácidos Graxos não Esterificados/metabolismo , Humanos , Hidrocarbonetos Fluorados , Ligantes , Receptores X do Fígado , Síndrome Metabólica/etiologia , Receptores Nucleares Órfãos , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/classificação , Receptores Citoplasmáticos e Nucleares/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Sulfonamidas , Transcrição GênicaRESUMO
Regioselective halogenations and Suzuki reactions ensure proper linkage of the aromatic rings in two total syntheses of terprenin (1). Both routes make it possible to prepare 1 efficiently and in large quantity.
RESUMO
We achieved a total synthesis of terprenin, a novel potent immunoglobulin E antibody suppressant which was obtained from the fermentation broth of Aspergillus candidus RF-5672 and has a highly oxygenated p-terphenyl skeleton with a prenyloxy side chain. The key steps relied on the Suzuki reaction to construct the terphenyl skeleton and on regioselective halogenations to selectively combine the aromatic rings. The highly efficient and practical production of this important natural product offers promise for the development of a new type of antiallergic drug.