RESUMO
Modulation of N-glycosylation using human Golgi α-mannosidase II (α-hGMII) inhibitors is a potential anticancer approach, but the clinical utility of current α-hGMII inhibitors is limited by their co-inhibition of human lysosomal α-mannosidase (α-hLM), resulting in abnormal storage of oligomannoses. We describe the synthesis and screening of a small library of novel bicyclic iminosugar-based scaffolds, prepared via natural product-inspired combinatorial chemistry (NPICC), which resulted in the identification of a primary α-hGMII inhibitor with 13.5-fold selectivity over α-hLM. Derivatization of this primary inhibitor using computation-guided synthesis (CGS) yielded an advanced α-hGMII inhibitor with nanomolar potency and 106-fold selectivity over α-hLM. In vitro studies demonstrated its N-glycan modulation and inhibitory effect on hepatocellular carcinoma (HCC) cells. In vivo studies confirmed its encouraging anti-HCC activity, without evidence of oligomannose accumulation.
RESUMO
Infectious diseases cause millions of deaths annually in the developing world. Recently, microfluidic paper-based analytical devices (µPADs) have been developed to diagnose such diseases, as these tests are low cost, biocompatible, and simple to fabricate. However, current µPADs are difficult to use in resource-limited areas due to their reliance on external instrumentation to measure and analyze the test results. In this work, we propose an electricity and external instrumentation-free µPAD sensor based on the colorimetric enzyme-linked immunosorbent assay (ELISA) for the diagnosis of infectious disease (3D-tPADs). Designed based on the principle of origami, the proposed µPAD enables the sequential steps of the colorimetric ELISA test to be completed in just â¼10 min. In addition, in order to obtain an accurate ELISA result without using any instrument, we have integrated an electricity-free "timer" within the µPAD that can be controlled by the buffer viscosity and fluid path volume to indicate the appropriate times for washing and color development steps, which can avoid false positive or false negative results caused by an extended or shortened amount of washing and development times. Due to the low background noise and high positive signal intensity of the µPAD, positive and negative detection results can be distinguished by just the naked eye. Furthermore, the ELISA result can be semi-quantified by comparing the results shown on the µPAD with a color chart diagram with a detection limit of HIV type 1(HIV-1) p24 antigen as low as 0.03 ng mL-1. These results demonstrate the proposed sensor can perform infectious disease diagnosis without external instrumentation or electricity, extending the application of the µPAD test for on-site detection and use in resource-limited settings.
Assuntos
Doenças Transmissíveis , Técnicas Analíticas Microfluídicas , Eletricidade , Humanos , Dispositivos Lab-On-A-Chip , PapelRESUMO
In this study, we report a new reductive etherification procedure for protection of carbohydrate substrates and its application for one-pot preparation of glycosyl building blocks. The reported procedure features the use of polymethylhydrosiloxane (PMHS) as a sub-stoichiometric reducing agent, which prevents the transilylation side reaction and improves the efficiency of the reductive etherification method. Application of the PMHS reductive etherification procedure for one-pot protecting group manipulation are described.
RESUMO
The Wnt/ß-catenin signaling pathway is aberrantly activated in colorectal (CRC) and many other cancers, and novel strategies for effectively targeting it may be needed due to its complexity. In this report, SM08502, a novel small molecule in clinical development for the treatment of solid tumors, was shown to reduce Wnt pathway signaling and gene expression through potent inhibition of CDC-like kinase (CLK) activity. SM08502 inhibited serine and arginine rich splicing factor (SRSF) phosphorylation and disrupted spliceosome activity, which was associated with inhibition of Wnt pathway-related gene and protein expression. Additionally, SM08502 induced the generation of splicing variants of Wnt pathway genes, suggesting that its mechanism for inhibition of gene expression includes effects on alternative splicing. Orally administered SM08502 significantly inhibited growth of gastrointestinal tumors and decreased SRSF phosphorylation and Wnt pathway gene expression in xenograft mouse models. These data implicate CLKs in the regulation of Wnt signaling and represent a novel strategy for inhibiting Wnt pathway gene expression in cancers. SM08502 is a first-in-class CLK inhibitor being investigated in a Phase 1 clinical trial for subjects with advanced solid tumors (NCT03355066).
Assuntos
Neoplasias Colorretais/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Fatores de Processamento de Serina-Arginina/metabolismo , Neoplasias Gástricas/tratamento farmacológico , Via de Sinalização Wnt/efeitos dos fármacos , Processamento Alternativo/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Humanos , Concentração Inibidora 50 , Camundongos , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Ratos , Neoplasias Gástricas/patologia , Via de Sinalização Wnt/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A series of 3S,4S,5S-trihydroxylated piperidines bearing structural diversity at C-2 or C-6 positions has been synthesized and tested to determine their ability to stabilize the activity of recombinant human α-Galactosidase A (rh-α-Gal A). Hit molecules were identified by rapid inhibitory activity screening, and then further investigated for their ability to protect this enzyme from thermo-induced denaturation and enhance its activity in Fabry patient cell lines. Our study resulted in the identification of a new class of small molecules as enzyme stabilizers for the potential treatment of Fabry disease. Of these, stabilizer 21 was the most effective, showing a 12-fold increase in rh-α-Gal A activity in Fabry disease cell lines.
Assuntos
Inibidores Enzimáticos/farmacologia , Doença de Fabry/tratamento farmacológico , Piperidinas/farmacologia , alfa-Galactosidase/antagonistas & inibidores , Linhagem Celular , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Estabilidade Enzimática , Doença de Fabry/metabolismo , Doença de Fabry/patologia , Humanos , Estrutura Molecular , Piperidinas/síntese química , Piperidinas/química , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , alfa-Galactosidase/metabolismoRESUMO
Pro-inflammatory cytokines play a critical role in the development of autoimmune and inflammatory diseases. Targeting the cytokine environment has proven efficient for averting inflammation. In this study, we reported that 6-[4-(aminomethyl)-2-chlorophenoxyl]benzo[c][1,2]oxaborol-1(3H)-ol (AN3485), a benzoxaborole analog, inhibited TLR2-, TLR3-, TLR4-, and TLR5-mediated TNF-α, IL-1ß, and IL-6 release from human PBMCs and isolated monocytes with IC(50) values ranging from 18 to 580 nM, and the inhibition was mediated at the transcriptional level. Topical administration of AN3485 significantly reduced PMA-induced contact dermatitis and oxazolone-induced delayed-type hypersensitivity in mice, indicating its capability of penetrating skin and potential topical application in skin inflammation. Oral administration of AN3485 showed dose-dependent suppression of LPS-induced TNF-α and IL-6 production in mice with an ED(90) of 30 mg/kg. Oral AN3485, 35 mg/kg, twice a day, suppressed collagen-induced arthritis in mice over a 20-day period. The potent anti-inflammatory activity in in vitro and in vivo disease models makes AN3485 an attractive therapeutic lead for a variety of cutaneous and systemic inflammatory diseases.
Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Artrite/tratamento farmacológico , Compostos de Boro/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Dermatite Alérgica de Contato/tratamento farmacológico , Hipersensibilidade a Drogas/tratamento farmacológico , Hipersensibilidade Tardia/tratamento farmacológico , Receptores Toll-Like/antagonistas & inibidores , Administração Oral , Animais , Anti-Inflamatórios não Esteroides/administração & dosagem , Anti-Inflamatórios não Esteroides/farmacocinética , Anti-Inflamatórios não Esteroides/toxicidade , Artrite/imunologia , Artrite/metabolismo , Compostos de Boro/administração & dosagem , Compostos de Boro/farmacocinética , Compostos de Boro/toxicidade , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Compostos Bicíclicos Heterocíclicos com Pontes/farmacocinética , Compostos Bicíclicos Heterocíclicos com Pontes/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/biossíntese , Citocinas/metabolismo , Dermatite Alérgica de Contato/etiologia , Dermatite Alérgica de Contato/imunologia , Dermatite Alérgica de Contato/metabolismo , Relação Dose-Resposta a Droga , Hipersensibilidade a Drogas/etiologia , Hipersensibilidade a Drogas/imunologia , Hipersensibilidade a Drogas/metabolismo , Feminino , Humanos , Hipersensibilidade Tardia/induzido quimicamente , Hipersensibilidade Tardia/imunologia , Hipersensibilidade Tardia/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
There is an increasing interest in in vivo metabolite identification in early drug discovery in order to (i) give a more complete picture of metabolic profile in investigational animal models, (ii) propose phase I and phase II metabolites using the same pharmacokinetic/toxicokinetic study samples, (iii) expose metabolically labile groups where chemical modifications could improve stability, and (iv) enable early safety assessment of metabolites. In the early discovery stage of our anti-inflammatory program, one novel benzoxaborole, AN6414, exhibiting both PDE4 enzyme and TNFα inhibition activities, became our primary candidate for further investigation. The traditional metabolite identifications usually require high dosed samples with long data scans and analysis. In this study, we conducted quick and more selective core-structure related precursor scans followed by daughter ion scans and identified a total of 10 major phase I and phase II metabolites using rat plasma samples from a toxicokinetic study at an oral dosing of 30 mg/kg. Plasma samples were treated with solid phase extraction (SPE) prior to LC/MS/MS. An AB SCIEX API 4000 QTRAP mass spectrometer coupled with a Shimadzu LC system was used for LC/MS/MS analysis. We found the major metabolites of AN6414 to be oxidative deboronation, protodeboronation, oxidation products and their sulfate-conjugated species. This analysis drove analoging efforts which improved the pharmacokinetic profile, namely, lowering clearance and increasing exposure relative to AN6414. Toxicity predictions by the software program DEREK suggest the identified potential metabolites to be safe.
Assuntos
Anti-Inflamatórios/farmacocinética , Compostos de Boro/farmacocinética , Cromatografia Líquida , Descoberta de Drogas/métodos , Inibidores da Fosfodiesterase 4/farmacologia , Piridinas/farmacocinética , Espectrometria de Massas em Tandem , Administração Oral , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/sangue , Anti-Inflamatórios/urina , Área Sob a Curva , Biotransformação , Compostos de Boro/administração & dosagem , Compostos de Boro/sangue , Compostos de Boro/toxicidade , Feminino , Masculino , Taxa de Depuração Metabólica , Modelos Biológicos , Oxirredução , Inibidores da Fosfodiesterase 4/administração & dosagem , Inibidores da Fosfodiesterase 4/sangue , Inibidores da Fosfodiesterase 4/toxicidade , Piridinas/administração & dosagem , Piridinas/sangue , Piridinas/toxicidade , Ratos , Ratos Sprague-Dawley , Medição de Risco , Software , Extração em Fase Sólida , Sulfatos/farmacocinéticaRESUMO
Antrodia cinnamomea is a Taiwanese medicinal mushroom with high antioxidant and polysaccharide content. The objective of this study is to investigate developmental toxicity of A. cinnamomea in pregnant Sprague-Dawley rats. Animals were daily gavaged with A. cinnamomea mycelium at dosage levels of 0 (reverse osmosis water), 50, 150, and 500 mg/kg from gestation day (GD) 6 to 15. All dams were sacrificed on GD 20 and were subjected to cesarean section. Fetuses were examined for external, visceral, and skeletal abnormalities. All copulated females survived until the end of the study. No significant differences were recorded in body weight change, food consumption, and maternal gestational parameters. Only two fetal malformations were noted in 970 fetuses from the treatment groups. Some variations, such as enlarged fontanel, split sternebrae, absent sacral, absent caudal vertebral centra, absent thoracic centra, absent 13th-14th ribs, and fused ribs, were found during the skeletal examination, but no treatment-induced abnormalities occurred. No dose dependency was observed in any of the developmental variations. Overall observation of foetal malformations from rats given A. cinnamomea mycelium during pregnancy demonstrates that this material is not teratogenic at doses up to 500 mg/kg. It is concluded that A. cinnamomea BCRC 35398 mycelium has no teratogenic effects in female rats and is safe to be used as a functional food ingredient.