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1.
J Chin Med Assoc ; 83(3): 266-271, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31990819

RESUMO

BACKGROUND: Acute Stanford type A aortic dissection is a lethal disease requiring surgery. Evidence regarding the effects of preoperative creatinine in mortality is limited, and few studies have evaluated the effect of postoperative dialysis treatment on it. METHODS: In this cohort study, we continuously recruited 632 surgical patients who were treated for acute type A aortic dissection in our hospital between January 2015 and May 2017. The preoperative level of serum creatinine was measured. All patients were followed up after surgery for 30 days to determine early mortality. RESULTS: The 30-day mortality after surgery increased with elevated levels of preoperative serum creatinine. Median (interquartile range) serum creatinine levels in survivors were 9.61 µmol/dL (7.28-12.62 µmol/dL) versus 13.41 µmol/dL (10.28-20.63 µmol/dL) in death (p < 0.01). Adjusted odds ratios for increasing per µmol/dL serum creatinine were 1.09 (95% confidence interval, 1.03-1.15). We also found that the effect of preoperative creatinine on 30-day mortality was diminished by dialysis treatment after surgery. CONCLUSION: Preoperative serum creatinine predicts outcome in patients undergoing surgery for Stanford type A aortic dissection, and postoperative dialysis treatment can reduce its hazard.

2.
Mol Genet Genomic Med ; 8(1): e1041, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31830381

RESUMO

BACKGROUND: Marfan syndrome (MFS) is an inherited connective tissue disease that mainly involves Fibrillin-1 (FBN1) mutations and aortic manifestations. In this study, we investigated the correlations between the FBN1 genotype-phenotype and aortic events (aortic dissection and aortic aneurysm) in patients with Marfan syndrome. METHODS: Genotype and phenotype information was evaluated in 180 patients with MFS. DNA sequencing was performed on each patient. According to the clinical manifestation, these patients were split into two groups: the aortic dissection group and the aortic aneurysm group. Aortic wall tissue was obtained from Marfan patients who underwent surgery and was used for staining. RESULTS: A total of 180 patients with FBN1 mutations were grouped into four categories: 90 with missense mutations, 32 with splicing mutations, 29 with frameshift mutations, and 29 with nonsense mutations. There was a significantly higher frequency of frameshift and nonsense mutations observed in aortic dissection than in aortic aneurysm (25.58% vs. 4.35%, p = .005; 25.58% vs. 8.70%, p = .033, respectively;), while missense mutations showed a higher frequency in aortic aneurysm than in aortic dissection (69.57% vs. 32.56%, respectively; p < .001) and a higher rate of lens dislocation (34.78% vs. 13.95%, respectively; p = .008). Pathological staining showed that elastic fibers were sparser in patients with a frameshift and nonsense mutations, and the smooth muscle cells were sparser and more disorganized than those observed in patients with missense mutations. CONCLUSION: This study showed that FBN1 gene frameshift and nonsense mutations are more common in patients with aortic dissection and may have meaningful guidance for the treatment of Marfan syndrome patients.

3.
Pharmacol Res Perspect ; 7(6): e00547, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31832205

RESUMO

Activation of MrgX2, an orphan G protein-coupled receptor expressed on mast cells, leads to degranulation and histamine release. Human MrgX2 binds promiscuously to structurally diverse peptides and small molecules that tend to have basic properties (basic secretagogues), resulting in acute histamine-like adverse drug reactions of injected therapeutic agents. We set out to identify MrgX2 orthologues from other mammalian species used in nonclinical stages of drug development. Previously, the only known orthologue of human MrgX2 was from mouse, encoded by Mrgprb2. MrgX2 genes of rat, dog (beagle), minipig, pig, and Rhesus and cynomolgus monkey were identified by bioinformatic approaches and verified by their ability to mediate calcium mobilization in transfected cells in response to the classical MrgX2 agonist, compound 48/80. The peptide GSK3212448 is an inhibitor of the PRC2 epigenetic regulator that caused profound anaphylactoid reactions upon intravenous infusion to rat. We showed GSK3212448 to be a potent MrgX2 agonist particularly at rat MrgX2. We screened sets of drug-like molecules and peptides to confirm the highly promiscuous nature of MrgX2. Approximately 20% of drug-like molecules activated MrgX2 (pEC50 ranging from 4.5 to 6), with the principle determinant being basicity. All peptides tested of net charge +3 or greater exhibited agonist activity, including the cell penetrating peptides polyarginine (acetyl-Arg9-amide) and TAT (49-60), a fragment of HIV-1 TAT protein. Finally, we showed that the glycopeptide antibiotic vancomycin, which is associated with clinical pseudo-allergic reactions known as red man syndrome, is an agonist of MrgX2.

4.
Medicine (Baltimore) ; 98(43): e17023, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31651834

RESUMO

Stanford type A aortic dissection (AD) is a lethal disease requiring surgery. Evidence regarding the prognostic ability of perioperative myocardiac markers on long-term outcome is limited.In this cohort study, we measured perioperative myocardiac markers level in 583 surgical patients with type A AD in our hospital between 2015 and 2017. All patients were followed up after surgery for a median period of 864 days to determine short- and long-term mortality.About one-fifth of patients has a positive preoperative myocardial markers, which was increased significantly after operation. Increase log10 post-creatine kinase MB isoenzyme (CK-MB) (hazard ratio [HR], 4.64; 95% confidence interval [CI] 1.89-11.43; P = .0008), log10 post-TnI (HR, 3.11; 95% CI 1.56-6.21; P = .0013), log10 post-Mb (HR, 3.00; 95% CI 1.40-6.43; P = .0048), log10 pre-CK-MB (HR,1.82; 95% CI 1.03-3.21; P = .0377), and upper tertile of post-CK-MB (HR,1.52; 95% CI 1.05-2.20; P = .0261) were the independent risk factor for 30 days mortality adjusted for potential confounders. None of cardiac markers was significantly associated with long-term outcome independent of other factors.Perioperative myocardiac predicts early outcome in type A AD patients undergoing surgery. Increasing perioperative myocardial markers do not appear to be a predictor for long-term all-cause mortality.


Assuntos
Aneurisma Dissecante/sangue , Aneurisma Dissecante/mortalidade , Creatina Quinase Forma MB/sangue , Troponina I/sangue , Adulto , Aneurisma Dissecante/cirurgia , Biomarcadores/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Período Pós-Operatório , Valor Preditivo dos Testes , Período Pré-Operatório , Prognóstico , Modelos de Riscos Proporcionais , Estudos Retrospectivos , Fatores de Risco , Resultado do Tratamento
5.
Mol Med Rep ; 20(1): 549-558, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31180540

RESUMO

Hypertrophic cardiomyopathy (HCM) is a complex inherited cardiovascular disease. The present study investigated the long noncoding (lnc)RNA/microRNA (mi)RNA/mRNA expression pattern of patients with HCM and aimed to identify key molecules involved in the development of this condition. An integrated strategy was conducted to identify differentially expressed miRNAs (DEmiRs), differentially expressed lncRNAs (DElncs) and differentially expressed genes (DEGs) based on the GSE36961 (mRNA), GSE36946 (miRNA), GSE68316 (lncRNA/mRNA) and GSE32453 (mRNA) expression profiles downloaded from the Gene Expression Omnibus datasets. Bioinformatics tools were employed to perform function and pathway enrichment analysis, protein­protein interaction, lncRNA­miRNA­mRNA and hub gene networks. Subsequently, DEGs were used as targets to predict drugs. The results indicated that a total of 2,234 DElncs (1,120 upregulated and 1,114 downregulated), 5 DEmiRs (2 upregulated and 3 downregulated) and 42 DEGs (35 upregulated and 7 downregulated) were identified in 4 microarray profiles. Gene ontology analysis revealed that DEGs were mainly involved in actin filament and stress fiber formation and in calcium ion binding, whereas Kyoto Encyclopedia of Genes and Genomes pathway analysis identified the hypoxia inducible factor­1, transforming growth factor­ß and tumor necrosis factor signaling pathways as the main pathways involved in these processes. The hub genes were screened using cytoHubba. A total of 1,086 lncRNA­miRNA­mRNA interactions including 67 lncRNAs, 5 miRNAs and 25 mRNAs were mined in the present study based on prediction websites. Drug prediction indicated that the targeted drugs mainly included angiotensin converting enzyme inhibitors or ß­blockers. A comprehensive bioinformatics analysis of the molecular regulatory lncRNA­miRNA­mRNA network was performed and potential therapeutic applications of drugs were predicted in HCM patients. The data may unravel the future molecular mechanism of HCM.


Assuntos
Cardiomiopatia Hipertrófica/tratamento farmacológico , Descoberta de Drogas/métodos , MicroRNAs/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/metabolismo , Biologia Computacional/métodos , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Terapia de Alvo Molecular/métodos , Mapas de Interação de Proteínas/efeitos dos fármacos
6.
Biomed Res Int ; 2019: 1420216, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31119151

RESUMO

Background: Continued debates exist regarding the optimal temperature during hypothermic circulatory arrest in aortic arch repair for patients with type A aortic dissection. This study seeks to examine whether the use of moderate hypothermic circulatory arrest in a pig model provides comparable vital organ protection outcomes to the use of deep hypothermic circulatory arrest. Methods: Thirteen pigs were randomly assigned to 30 minutes of hypothermic circulatory arrest without cerebral perfusion at 15°C (n = 5), 25°C (n = 5), and a control group (n = 3). The changes in standard laboratory tests and capacity for protection against apoptosis in different vital organs were monitored with different temperatures of hypothermic circulatory arrest management in pig model to determine which temperature was optimal for hypothermic circulatory arrest. Results: There were no significant differences in the capacity for protection against apoptosis in vital organs between 2 groups (p > 0.05, respectively). Compared with the moderate hypothermic circulatory arrest group, the deep hypothermic circulatory arrest group had no significant advantages in terms of the biologic parameters of any other organs (p > 0.05). Conclusions: Compared with deep hypothermic circulatory arrest, moderate hypothermic circulatory arrest is a moderate technique that has similar advantages with regard to the levels of biomarkers of injury and capacity for protection against apoptosis in vital organs.


Assuntos
Apoptose/genética , Parada Circulatória Induzida por Hipotermia Profunda , Hipotermia Induzida , Miocárdio/metabolismo , Aneurisma Dissecante/genética , Aneurisma Dissecante/patologia , Aneurisma Dissecante/terapia , Animais , Aorta Torácica/metabolismo , Aorta Torácica/patologia , Encéfalo/irrigação sanguínea , Encéfalo/metabolismo , Caspase 3/genética , Circulação Cerebrovascular/genética , Parada Cardíaca Induzida/métodos , Humanos , Rim/irrigação sanguínea , Rim/metabolismo , Fígado/irrigação sanguínea , Fígado/metabolismo , Miocárdio/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Suínos , Proteína X Associada a bcl-2/genética
7.
J Cardiothorac Surg ; 14(1): 90, 2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31064409

RESUMO

BACKGROUND: Thoracic aortic surgery and cardiopulmonary bypass are both associated with development of postoperative acute kidney injury. In this study, we undertook to investigate the relationship between cardiopulmonary bypass time and postoperative acute kidney injury in patients undergoing thoracic aortic surgery for acute DeBakey Type I aortic dissection. METHODS: All patients receiving thoracic aortic surgery for acute DeBakey Type I aortic dissection in Beijing Anzhen hospital from December 2015 to April 2017 were included. Cardiopulmonary bypass time was recorded during surgery. Acute kidney injury was defined based on the Kidney Disease Improving Global Outcomes criteria. A total of 115 consecutive patients were eventually analyzed. RESULTS: The overall incidence of acute kidney injury was 53.0% (n = 61). The average age was 47.8 ± 10.7 years; 74.8% were male. Mean cardiopulmonary bypass time was 211 ± 56 min. In-hospital mortality was 7.8%. Multivariate logistic regression revealed that cardiopulmonary bypass time was independently associated with the occurrence of postoperative acute kidney injury after adjust confounding factors (odds ratio = 1.171; 95% confidence interval: 1.002-1.368; P = 0.047). CONCLUSIONS: Cardiopulmonary bypass time is independently associated with an increased hazard of acute kidney injury after thoracic aortic surgery for acute DeBakey Type I aortic dissection. Further understanding of the mechanism of this association is crucial to the design of preventative strategies.


Assuntos
Lesão Renal Aguda/etiologia , Aneurisma Dissecante/cirurgia , Aorta Torácica/cirurgia , Aneurisma da Aorta Torácica/cirurgia , Ponte Cardiopulmonar/efeitos adversos , Procedimentos Cirúrgicos Torácicos/efeitos adversos , Lesão Renal Aguda/diagnóstico , Lesão Renal Aguda/mortalidade , Adulto , Feminino , Mortalidade Hospitalar , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Fatores de Risco , Fatores de Tempo , Resultado do Tratamento
8.
J Cardiothorac Surg ; 14(1): 81, 2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-31023343

RESUMO

BACKGROUND: The purpose of this study is to identify the risk factors for postoperative severe hypoxemia after surgery for acute type A aortic dissection. METHODS: This was a single-center retrospective study including 112 consecutive patients undergoing urgent aortic arch surgery for acute type A aortic dissection between December 2016 and April 2017 at Beijing Anzhen Hospital. RESULTS: Multivariate logistic regression analysis identified female (OR, 12.978; 95% CI, 3.332 to 50.546; p < 0.001) and increased body mass index (OR, 1.473; 95% CI, 1.213 to 1.789; p < 0.001) as independent predictors of postoperative severe hypoxemia in patients with acute type A aortic dissection. CONCLUSIONS: Obesity and female were independent risk factors for postoperative severe hypoxemia in patients with acute type A aortic dissection. More attention should be paid to preventing postoperative severe hypoxemia in obese women with acute type A aortic dissection.


Assuntos
Aneurisma Dissecante/cirurgia , Aneurisma da Aorta Torácica/cirurgia , Implante de Prótese Vascular/efeitos adversos , Hipóxia/fisiopatologia , Obesidade/fisiopatologia , Adulto , Idoso , Aneurisma Dissecante/complicações , Aneurisma Dissecante/diagnóstico por imagem , Aneurisma Dissecante/fisiopatologia , Aneurisma da Aorta Torácica/complicações , Aneurisma da Aorta Torácica/diagnóstico por imagem , Aneurisma da Aorta Torácica/fisiopatologia , Índice de Massa Corporal , Feminino , Humanos , Hipóxia/etiologia , Masculino , Pessoa de Meia-Idade , Obesidade/complicações , Estudos Retrospectivos , Fatores de Risco , Adulto Jovem
9.
J Med Chem ; 62(9): 4683-4702, 2019 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-30973731

RESUMO

The KEAP1-NRF2-mediated cytoprotective response plays a key role in cellular homoeostasis. Insufficient NRF2 signaling during chronic oxidative stress may be associated with the pathophysiology of several diseases with an inflammatory component, and pathway activation through direct modulation of the KEAP1-NRF2 protein-protein interaction is being increasingly explored as a potential therapeutic strategy. Nevertheless, the physicochemical nature of the KEAP1-NRF2 interface suggests that achieving high affinity for a cell-penetrant druglike inhibitor might be challenging. We recently reported the discovery of a highly potent tool compound which was used to probe the biology associated with directly disrupting the interaction of NRF2 with the KEAP1 Kelch domain. We now present a detailed account of the medicinal chemistry campaign leading to this molecule, which included exploration and optimization of protein-ligand interactions in three energetic "hot spots" identified by fragment screening. In particular, we also discuss how consideration of ligand conformational stabilization was important to its development and present evidence for preorganization of the lead compound which may contribute to its high affinity and cellular activity.

10.
SLAS Discov ; 24(2): 175-189, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30383469

RESUMO

Nrf2, a master regulator of the phase II gene response to stress, is kept at low concentrations in the cell through binding to Keap1, an adaptor protein for the Cul3 ubiquitin ligase complex. To identify Nrf2 activators, two separate time-resolved fluorescence resonance energy transfer (TR-FRET) assays were developed to monitor the binding of Nrf2-Keap1 and Cul3-Keap1, respectively. The triterpenoid, 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl] imidazole (CDDO-Im) and its analogs, exhibited approximately 100-fold better potency in the Cul3-Keap1 assay than in the Nrf2-Keap1 assay, and this difference was more profound at 37 °C than at room temperature in the Nrf2-Keap1 assay, but this phenomenon was not observed in the Cul3-Keap1 assay. A full diversity screen of approximately 2,200,000 GSK compounds was run with the Cul3-Keap1 TR-FRET assay and multiple chemical series were identified and characterized.

12.
Bioorg Med Chem Lett ; 28(23-24): 3676-3680, 2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30554630

RESUMO

Beta-hemoglobinopathies such as sickle cell disease represent a major global unmet medical need. De-repression of fetal hemoglobin in erythrocytes is a clinically validated approach for the management of sickle cell disease, but the only FDA-approved medicine for this purpose has limitations to its use. We conducted a phenotypic screen in human erythroid progenitor cells to identify molecules with the ability to de-repress fetal hemoglobin, which resulted in the identification of the benzoxaborole-containing hit compound 1. This compound was found to have modest cellular potency and lead-like pharmacokinetics, but no identifiable SAR to enable optimization. Systematic deconstruction of a closely related analog of 1 revealed the fragment-like carboxylic acid 12, which was then optimized to provide tetrazole 31, which had approximately 100-fold improved cellular potency compared to 1, high levels of oral exposure in rats, and excellent solubility.


Assuntos
Benzoxazóis/química , Hemoglobina Fetal/metabolismo , Animais , Benzoxazóis/farmacocinética , Benzoxazóis/farmacologia , Disponibilidade Biológica , Ácidos Borônicos/química , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Meia-Vida , Humanos , Ratos , Ratos Sprague-Dawley , Solubilidade
13.
PLoS One ; 13(12): e0207140, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30540745

RESUMO

Atonal homolog 1 (Atoh1) is a basic helix-loop-helix 9 (bHLH) transcription factor acting downstream of Notch and is required for the differentiation of sensory hair cells in the inner ear and the specification of secretory cells during the intestinal crypt cell regeneration. Motivated by the observations that the upregulation of Atoh1 gene expression, through genetic manipulation or pharmacological inhibition of Notch signaling (e.g. γ-secretase inhibitors, GSIs), induces ectopic hair cell growth in the cochlea of the inner ear and partially restores hearing after injuries in experimental models, we decided to identify small molecule modulators of the Notch-Atoh1 pathway, which could potentially regenerate hair cells. However, the lack of cellular models of the inner ear has precluded the screening and characterization of such modulators. Here we report using a colon cancer cell line LS-174T, which displays Notch inhibition-dependent Atoh1 expression as a surrogate cellular model to screen for inducers of Atoh1 expression. We designed an Atoh1 promoter-driven luciferase assay to screen a target-annotated library of ~6000 compounds. We further developed a medium throughput, real-time quantitative RT-PCR assay measuring the endogenous Atoh1 gene expression to confirm the hits and eliminate false positives from the reporter-based screen. This strategy allowed us to successfully recover GSIs of known chemotypes. This LS-174T cell-based assay directly measures Atoh1 gene expression induced through Notch-Hes1 inhibition, and therefore offers an opportunity to identify novel cellular modulators along the Notch-Atoh1 pathway.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Receptores Notch/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Benzodiazepinas/farmacologia , Linhagem Celular Tumoral , Expressão Gênica/efeitos dos fármacos , Genes Reporter , Humanos , Microscopia de Fluorescência , Regiões Promotoras Genéticas , Receptores Notch/antagonistas & inibidores , Transdução de Sinais , Bibliotecas de Moléculas Pequenas/farmacologia , Fatores de Transcrição HES-1/genética , Fatores de Transcrição HES-1/metabolismo
14.
Methods Mol Biol ; 1439: 207-26, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27316998

RESUMO

Luciferase reporter gene assays have long been used for drug discovery due to their high sensitivity and robust signal. A dual reporter gene system contains a gene of interest and a control gene to monitor non-specific effects on gene expression. In our dual luciferase reporter gene system, a synthetic promoter of γ-globin gene was constructed immediately upstream of the firefly luciferase gene, followed downstream by a synthetic ß-globin gene promoter in front of the Renilla luciferase gene. A stable cell line with the dual reporter gene was cloned and used for all assay development and HTS work. Due to the low activity of the control Renilla luciferase, only the firefly luciferase activity was further optimized for HTS. Several critical factors, such as cell density, serum concentration, and miniaturization, were optimized using tool compounds to achieve maximum robustness and sensitivity. Using the optimized reporter assay, the HTS campaign was successfully completed and approximately 1000 hits were identified. In this chapter, we also describe strategies to triage hits that non-specifically interfere with firefly luciferase.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Genes Reporter , Regiões Promotoras Genéticas/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , gama-Globinas/genética , Animais , Linhagem Celular , Vaga-Lumes/genética , Humanos , Luciferases de Vaga-Lume/genética , Luciferases de Renilla/genética , Renilla/genética , Transfecção/métodos
15.
J Biomol Screen ; 21(3): 260-8, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26656867

RESUMO

Abnormal accumulation of ß-catenin protein, a key transcriptional activator required for Wnt signaling, is the hallmark of many tumor types, including colon cancer. In normal cells, ß-catenin protein level is tightly controlled by a multiprotein complex through the proteosome pathway. Mutations in the components of the ß-catenin degradation complex, such as adenomatous polyposis coli (APC) and Axin, lead to ß-catenin stabilization and the constitutive activation of target genes. Since the signal transduction of Wnt/ß-catenin is mainly mediated by protein-protein interactions, this pathway has been particularly refractory to conventional target-based small-molecule screening. Here we designed a cellular high-content imaging assay to detect ß-catenin protein through immunofluorescent staining in the SW480 colon cancer cell line, which has elevated ß-catenin endogenously. We demonstrate that the assay is robust and specific to screen a focused biologically diverse chemical library set against known targets that play diverse cellular functions. We identified a number of hits that reduce ß-catenin levels without causing cell death. These hits may serve as tools to understand the dynamics of ß-catenin degradation. This study demonstrates that detecting cell-based ß-catenin protein stability is a viable approach to identifying novel mechanisms of ß-catenin regulation as well as small molecules of therapeutic potential.


Assuntos
Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Ensaios de Triagem em Larga Escala , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismo , Polipose Adenomatosa do Colo/tratamento farmacológico , Anticorpos Monoclonais/farmacologia , Especificidade de Anticorpos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Concentração Inibidora 50 , Bibliotecas de Moléculas Pequenas , beta Catenina/antagonistas & inibidores
16.
ACS Comb Sci ; 17(12): 722-31, 2015 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-26562224

RESUMO

DNA-encoded small-molecule library technology has recently emerged as a new paradigm for identifying ligands against drug targets. To date, this technology has been used with soluble protein targets that are produced and used in a purified state. Here, we describe a cell-based method for identifying small-molecule ligands from DNA-encoded libraries against integral membrane protein targets. We use this method to identify novel, potent, and specific inhibitors of NK3, a member of the tachykinin family of G-protein coupled receptors (GPCRs). The method is simple and broadly applicable to other GPCRs and integral membrane proteins. We have extended the application of DNA-encoded library technology to membrane-associated targets and demonstrate the feasibility of selecting DNA-tagged, small-molecule ligands from complex combinatorial libraries against targets in a heterogeneous milieu, such as the surface of a cell.


Assuntos
Acetatos/farmacologia , DNA/química , Quinolinas/farmacologia , Receptores da Neurocinina-3/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Acetatos/química , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Ligantes , Estrutura Molecular , Quinolinas/química , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade
17.
Bioorg Med Chem Lett ; 25(14): 2739-43, 2015 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-26022841

RESUMO

Nod-like receptors (NLRs) are cytoplasmic pattern recognition receptors that are promising targets for the development of anti-inflammatory therapeutics. Drug discovery efforts targeting NLRs have been hampered by their inherent tendency to form aggregates making protein generation and the development of screening assays very challenging. Herein we report the results of an HTS screen of NLR family member NLRP1 (NLR family, pyrin domain-containing 1) which was achieved through the large scale generation of recombinant GST-His-Thrombin-NLRP1 protein. The screen led to the identification of a diverse set of ATP competitive inhibitors with micromolar potencies. Activity of these hits was confirmed in a FP binding assay, and two homology models were employed to predict the possible binding mode of the leading series and facilitate further lead-optimization. These results highlight a promising strategy for the identification of inhibitors of NLR family members which are rapidly emerging as key drivers of inflammation in human disease.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Trifosfato de Adenosina/química , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Inflamassomos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Sítios de Ligação , Ligação Competitiva , Ensaios de Triagem em Larga Escala , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Pirazóis/química , Pirazóis/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Relação Estrutura-Atividade
18.
PLoS One ; 9(5): e96737, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24806487

RESUMO

NOD1 is an intracellular pattern recognition receptor that recognizes diaminopimelic acid (DAP), a peptidoglycan component in gram negative bacteria. Upon ligand binding, NOD1 assembles with receptor-interacting protein (RIP)-2 kinase and initiates a signaling cascade leading to the production of pro-inflammatory cytokines. Increased NOD1 signaling has been associated with a variety of inflammatory disorders suggesting that small-molecule inhibitors of this signaling complex may have therapeutic utility. We utilized a cell-based screening approach with extensive selectivity profiling to search for small molecule inhibitors of the NOD1 signaling pathway. Via this process we identified three distinct chemical series, xanthines (SB711), quinazolininones (GSK223) and aminobenzothiazoles (GSK966) that selectively inhibited iE-DAP-stimulated IL-8 release via the NOD1 signaling pathway. All three of the newly identified compound series failed to block IL-8 secretion in cells following stimulation with ligands for TNF receptor, TLR2 or NOD2 and, in addition, none of the compound series directly inhibited RIP2 kinase activity. Our initial exploration of the structure-activity relationship and physicochemical properties of the three series directed our focus to the quinazolininone biarylsulfonamides (GSK223). Further investigation allowed for the identification of significantly more potent analogs with the largest boost in activity achieved by fluoro to chloro replacement on the central aryl ring. These results indicate that the NOD1 signaling pathway, similarly to activation of NOD2, is amenable to modulation by small molecules that do not target RIP2 kinase. These compounds should prove useful tools to investigate the importance of NOD1 activation in various inflammatory processes and have potential clinical utility in diseases driven by hyperactive NOD1 signaling.


Assuntos
Benzotiazóis/farmacologia , Proteína Adaptadora de Sinalização NOD1/metabolismo , Quinazolinonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Xantinas/farmacologia , Animais , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Fosforilação , Ligação Proteica , Relação Estrutura-Atividade
19.
Mol Biosyst ; 10(2): 251-7, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24257700

RESUMO

Epigenetic regulation by histone methylation is crucial for proper programming of the genome during development. Homeostasis of histone methylation is balanced by the activities of histone methyltransferases and demethylases. Although these methyltransferases and demethylases represent logical targets for potential drug discovery, the activities of methyltransferases and demethylases regulated in response to a complex biological stimulus are also important and not yet clear. To manipulate and study histone methylation in biological systems, we screened a Biologically Diverse Compound Set (BDCS) utilizing a phenotypic assay system that directly measures the Histone 3 K27 tri-methylation (H3K27me3) level in cells. The BDCS is a unique set of target-annotated chemical probes, containing a total of 5853 compounds targeting 736 unique proteins with multiple maximally selective compounds for each target. A number of targets, with multiple hits against each target, were identified in the screen. This gave us confidence that these targets and pathways may be relevant, and included the identification of non-methyltransferase/demethylase targets as potential upstream regulators of H3K27me3. Our study suggests that a systematically designed chemical probe library can serve as a powerful drug discovery tool when combined with phenotypic screening. Follow-up studies using these findings may reveal novel therapeutically useful pathways and targets of H3K27me3 regulation.


Assuntos
Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Histonas/metabolismo , Metiltransferases/metabolismo , Peptídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Bases de Dados de Produtos Farmacêuticos , Epigênese Genética , Humanos , Metilação , Fenótipo
20.
J Biomol Screen ; 18(10): 1212-22, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24163393

RESUMO

Sickle cell anemia (SCA) is a genetic disorder of the ß-globin gene. SCA results in chronic ischemia with pain and tissue injury. The extent of SCA symptoms can be ameliorated by treatment with drugs, which result in increasing the levels of γ-globin in patient red blood cells. Hydroxyurea (HU) is a Food and Drug Administration-approved drug for SCA, but it has dose-limiting toxicity, and patients exhibit highly variable treatment responses. To identify compounds that may lead to the development of better and safer medicines, we have established a method using primary human bone marrow day 7 erythroid progenitor cells (EPCs) to screen for compounds that induce γ-globin production. First, human marrow CD34(+) cells were cultured and expanded for 7 days and characterized for the expression of erythroid differentiation markers (CD71, CD36, and CD235a). Second, fresh or cryopreserved EPCs were treated with compounds for 3 days in 384-well plates followed by γ-globin quantification by an enzyme-linked immunosorbent assay (ELISA), which was validated using HU and decitabine. From the 7408 compounds screened, we identified at least one new compound with confirmed γ-globin-inducing activity. Hits are undergoing analysis in secondary assays. In this article, we describe the method of generating fit-for-purpose EPCs; the development, optimization, and validation of the ELISA and secondary assays for γ-globin detection; and screening results.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Células Precursoras Eritroides/metabolismo , Ativação Transcricional/efeitos dos fármacos , gama-Globinas/genética , Anemia Falciforme/tratamento farmacológico , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Ácido Butírico/farmacologia , Sobrevivência Celular , Metilases de Modificação do DNA/antagonistas & inibidores , Metilases de Modificação do DNA/metabolismo , Decitabina , Ensaio de Imunoadsorção Enzimática , Epigênese Genética/efeitos dos fármacos , Células Precursoras Eritroides/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Humanos , Cultura Primária de Células , gama-Globinas/metabolismo
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