RESUMO
Herein, we report the systematic investigation of stereopure phosphorothioate (PS) and phosphoryl guanidine (PN) linkages on siRNA-mediated silencing. The incorporation of appropriately positioned and configured stereopure PS and PN linkages to N-acetylgalactosamine (GalNAc)-conjugated siRNAs based on multiple targets (Ttr and HSD17B13) increased potency and durability of mRNA silencing in mouse hepatocytes in vivo compared with reference molecules based on clinically proven formats. The observation that the same modification pattern had beneficial effects on unrelated transcripts suggests that it may be generalizable. The effect of stereopure PN modification on silencing is modulated by 2'-ribose modifications in the vicinity, particularly on the nucleoside 3' to the linkage. These benefits corresponded with both an increase in thermal instability at the 5'-end of the antisense strand and improved Argonaute 2 (Ago2) loading. Application of one of our most effective designs to generate a GalNAc-siRNA targeting human HSD17B13 led to â¼80% silencing that persisted for at least 14 weeks after administration of a single 3 mg/kg subcutaneous dose in transgenic mice. The judicious use of stereopure PN linkages improved the silencing profile of GalNAc-siRNAs without disrupting endogenous RNA interference pathways and without elevating serum biomarkers for liver dysfunction, suggesting they may be suitable for therapeutic application.
Assuntos
Inativação Gênica , Interferência de RNA , RNA Mensageiro , Animais , Humanos , Camundongos , Camundongos Transgênicos , RNA Mensageiro/genética , RNA Interferente Pequeno/genéticaRESUMO
Technologies that recruit and direct the activity of endogenous RNA-editing enzymes to specific cellular RNAs have therapeutic potential, but translating them from cell culture into animal models has been challenging. Here we describe short, chemically modified oligonucleotides called AIMers that direct efficient and specific A-to-I editing of endogenous transcripts by endogenous adenosine deaminases acting on RNA (ADAR) enzymes, including the ubiquitously and constitutively expressed ADAR1 p110 isoform. We show that fully chemically modified AIMers with chimeric backbones containing stereopure phosphorothioate and nitrogen-containing linkages based on phosphoryl guanidine enhanced potency and editing efficiency 100-fold compared with those with uniformly phosphorothioate-modified backbones in vitro. In vivo, AIMers targeted to hepatocytes with N-acetylgalactosamine achieve up to 50% editing with no bystander editing of the endogenous ACTB transcript in non-human primate liver, with editing persisting for at least one month. These results support further investigation of the therapeutic potential of stereopure AIMers.
Assuntos
Oligonucleotídeos , Edição de RNA , Animais , Primatas/genética , Primatas/metabolismo , RNA , Edição de RNA/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Forty-four novel tricycles containing nonenolizable cyano enones (TCEs) were designed and synthesized on the basis of a semisynthetic pentacyclic triterpenoid, bardoxolone methyl, which is currently being developed in phase II clinical trials for the treatment of severe chronic kidney disease in diabetic patients. Most of the TCEs having two different kinds of nonenolizable cyano enones in rings A and C are highly potent suppressors of induction of inducible nitric oxide synthase stimulated with interferon-γ and are highly potent inducers of the cytoprotective enzymes heme oxygenase-1 and NAD(P)H:quinone oxidoreductase-1. Among these compounds, (±)-(4bS,8aR,10aS)-10a-ethynyl-4b,8,8-trimethyl-3,7-dioxo-3,4b,7,8,8a,9,10,10a-octahydrophenanthrene-2,6-dicarbonitrile ((±)-31) is the most potent in these bioassays in our pool of drug candidates including semisynthetic triterpenoids and synthetic tricycles. These facts strongly suggest that an essential factor for potency is not a triterpenoid skeleton but the cyano enone functionality. Notably, TCE 31 reduces hepatic tumorigenesis induced with aflatoxin in rats. Further preclinical studies and detailed mechanism studies on 31 are in progress.
Assuntos
Anti-Inflamatórios não Esteroides/síntese química , Antineoplásicos/síntese química , Nitrilas/síntese química , Fenantrenos/síntese química , Aflatoxina B1 , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Células Cultivadas , Citoproteção , Indução Enzimática , Heme Oxigenase-1/biossíntese , Interferon gama/farmacologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/prevenção & controle , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Masculino , Camundongos , NAD(P)H Desidrogenase (Quinona)/biossíntese , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/biossíntese , Nitrilas/química , Nitrilas/farmacologia , Triterpenos Pentacíclicos/química , Fenantrenos/química , Fenantrenos/farmacologia , Lesões Pré-Cancerosas/induzido quimicamente , Lesões Pré-Cancerosas/prevenção & controle , Ratos , Estereoisomerismo , Estômago/efeitos dos fármacos , Estômago/enzimologia , Relação Estrutura-AtividadeRESUMO
[reaction: see text] We have proposed a pathway for the base-catalyzed reverse vinylogous aldol reaction of (-)-(4abeta,5beta)-4,4a,5,6,7,8-hexahydro-5-hydroxy-1,4a-dimethylnaphthalen-2(3H)-one [(-)-8] under Robinson annulation conditions. For confirmation, 4-(2,6-dimethyl-3-oxocyclohex-1-enyl)butanal (11) and 4-(2,6-dimethyl-5-oxocyclohex-1-enyl)butanal (12), both of which potentially produce enolate I, were synthesized regioselectively. Unexpectedly, 11 gave a complex mixture, including only a trace amount of (+/-)-8 (less than 5% yield), under these basic conditions. To the contrary, 12 cleanly afforded (+/-)-8 in 66% yield. This result provides evidence for our proposed mechanism of the above reaction.
Assuntos
Naftalenos/química , Compostos Orgânicos/química , Compostos de Vinila/química , Catálise , Estrutura MolecularRESUMO
Novel tricyclic compounds with enone functionalities in rings A and C [tricyclic-bis-enone (TBE) compounds] were designed on the basis of the structure of a synthetic triterpenoid, 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO)(1), which is a promising drug candidate for prevention and/or treatment of cancer and inflammatory diseases whose pathogenesis may involve excessive production of nitric oxide (NO) and/or prostaglandins. A series of TBE compounds in racemic form shows high inhibitory activity against production of NO induced by interferon-[gamma](IFN-[gamma]) in mouse macrophages. One of these compounds, (+/-)-(4a[small beta],8a[small beta],10a[small alpha])-1,2,4a,6,8a,9,10,10a-octahydro-1,1,4a,8a-tetramethyl-2,6-dioxophenanthrene-3,7-dicarbonitrile ((+/-)-3), is orally active at 15 mg kg(-1)(single administration) in a preliminary study using mouse peritoneal inflammation induced by thioglycollate and IFN-[gamma]. Therefore, we desired to synthesize optically active TBE compounds for a comparison of the biological potency of both enantiomers. We now describe the synthesis of both enantiomers of (4a[small beta],8a[small beta],10a[small alpha])-1,2,4a,6,8a,9,10,10a-octahydro-1,1,4a,8a-tetramethyl-2,6-dioxophenanthrene-3-carbonitrile (2) and 3 from commercially available simple compounds. Interestingly, (+)-3 having the same configuration as the CDDO antipode shows about 10 times higher inhibitory activity than (-)-3 on NO production in mouse macrophages. In contrast, (-)-3 inhibits proliferation of MCF-7 breast cancer cells, whereas (+)-3 does not.
Assuntos
Anti-Inflamatórios não Esteroides/síntese química , Antineoplásicos/síntese química , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/síntese química , Administração Oral , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Interferon gama/química , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Modelos Químicos , Estrutura Molecular , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/biossíntese , Nitrilas/síntese química , Nitrilas/química , Nitrilas/farmacologia , Ácido Oleanólico/química , Ácido Oleanólico/farmacologia , Peritonite/induzido quimicamente , Peritonite/tratamento farmacológico , Fenantrenos/síntese química , Fenantrenos/química , Fenantrenos/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade , Tioglicolatos/químicaRESUMO
Novel tricyclic compounds with enone functionalities in rings A and C, which were designed on the basis of the structure of a synthetic triterpenoid, 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid, have been synthesized. Among them, 10 shows high inhibitory activity (IC(50) = 1 nM level) against production of nitric oxide induced by interferon-gamma in mouse macrophages and is orally active at 15 mg/kg (once) in a preliminary in vivo study using mouse peritoneal inflammation induced by thioglycollate and interferon-gamma.
Assuntos
Macrófagos/efeitos dos fármacos , Óxido Nítrico/antagonistas & inibidores , Nitrilas/síntese química , Fenantrenos/síntese química , Triterpenos/síntese química , Animais , Técnicas In Vitro , Interferon gama , Macrófagos/metabolismo , Camundongos , Óxido Nítrico/biossíntese , Nitrilas/química , Nitrilas/farmacologia , Peritonite/induzido quimicamente , Peritonite/tratamento farmacológico , Fenantrenos/química , Fenantrenos/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade , Tioglicolatos , Triterpenos/química , Triterpenos/farmacologiaRESUMO
New oleanane triterpenoids with various substituents at the C-17 position of 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) and methyl 2-carboxy-3,12-dioxooleana-1,9(11)-dien-28-oate were synthesized. Among them, 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-onitrile shows extremely high inhibitory activity (IC(50)=1 pM level) against production of nitric oxide induced by interferon-gamma in mouse macrophages. This potency is about 100 times and 30 times more potent than CDDO and dexamethasone, respectively.