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1.
J Org Chem ; 86(5): 4281-4289, 2021 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-33618515

RESUMO

The development of a multigram synthesis of 3-exo-isopropylbicyclo[2.2.1]heptan-2-endo-amine hydrochloride (1) (also known as BRD4780 and AGN-192403) is described. The process involves protection of the amine as 4-nitrobenzyl carbamate, pNZ, which enables chiral SFC chromatography. The absolute configuration (AC) of the individual enantiomers has been determined by Mosher's amide method, VCD spectroscopy, and X-ray crystallography. We highlight the VCD approach as a rapid and effective means of AC determination that can be deployed directly on the target compounds.


Assuntos
Amidas , Dicroísmo Circular , Cristalografia por Raios X , Estereoisomerismo
2.
Cell Rep Med ; 1(8): 100137, 2020 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-33294858

RESUMO

Drug repurposing has the advantage of identifying potential treatments on a shortened timescale. In response to the pandemic spread of SARS-CoV-2, we took advantage of a high-content screen of 3,713 compounds at different stages of clinical development to identify FDA-approved compounds that reduce mucin-1 (MUC1) protein abundance. Elevated MUC1 levels predict the development of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) and correlate with poor clinical outcomes. Our screen identifies fostamatinib (R788), an inhibitor of spleen tyrosine kinase (SYK) approved for the treatment of chronic immune thrombocytopenia, as a repurposing candidate for the treatment of ALI. In vivo, fostamatinib reduces MUC1 abundance in lung epithelial cells in a mouse model of ALI. In vitro, SYK inhibition by the active metabolite R406 promotes MUC1 removal from the cell surface. Our work suggests fostamatinib as a repurposing drug candidate for ALI.

3.
J Org Chem ; 85(22): 14592-14609, 2020 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-33125847

RESUMO

Nucleoside 5'-triphosphate (dNTP) analogues in which the ß,γ-oxygen is mimicked by a CXY group (ß,γ-CXY-dNTPs) have provided information about DNA polymerase catalysis and fidelity. Definition of CXY stereochemistry is important to elucidate precise binding modes. We previously reported the (R)- and (S)-ß,γ-CHX-dGTP diastereomers (X = F, Cl), prepared via P,C-dimorpholinamide CHCl (6a, 6b) and CHF (7a, 7b) bisphosphonates (BPs) equipped with an (R)-mandelic acid as a chiral auxiliary, with final deprotection using H2/Pd. This method also affords the ß,γ-CHCl-dTTP (11a, 11b), ß,γ-CHF (12a, 12b), and ß,γ-CHCl (13a, 13b) dATP diastereomers as documented here, but the reductive deprotection step is not compatible with dCTP or the bromo substituent in ß,γ-CHBr-dNTP analogues. To complete assembly of the toolkit, we describe an alternative synthetic strategy featuring ethylbenzylamine or phenylglycine-derived chiral BP synthons incorporating a photolabile protecting group. After acid-catalyzed removal of the (R)-(+)-α-ethylbenzylamine auxiliary, coupling with activated dCMP and photochemical deprotection, the individual diastereomers of ß,γ-CHBr- (33a, 33b), ß,γ-CHCl- (34a, 34b), ß,γ-CHF-dCTP (35a, 35b) were obtained. The ß,γ-CH(CH3)-dATPs (44a, 44b) were obtained using a methyl (R)-(-)-phenylglycinate auxiliary. 31P and 19F NMR Δδ values are correlated with CXY stereochemistry and pKa2-4 values for 13 CXY-bisphosphonic acids and imidodiphosphonic acid are tabulated.


Assuntos
DNA Polimerase Dirigida por DNA , Nucleotídeos de Desoxicitosina , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética
4.
bioRxiv ; 2020 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-32637960

RESUMO

Drug repurposing is the only method capable of delivering treatments on the shortened time-scale required for patients afflicted with lung disease arising from SARS-CoV-2 infection. Mucin-1 (MUC1), a membrane-bound molecule expressed on the apical surfaces of most mucosal epithelial cells, is a biochemical marker whose elevated levels predict the development of acute lung injury (ALI) and respiratory distress syndrome (ARDS), and correlate with poor clinical outcomes. In response to the pandemic spread of SARS-CoV-2, we took advantage of a high content screen of 3,713 compounds at different stages of clinical development to identify FDA-approved compounds that reduce MUC1 protein abundance. Our screen identified Fostamatinib (R788), an inhibitor of spleen tyrosine kinase (SYK) approved for the treatment of chronic immune thrombocytopenia, as a repurposing candidate for the treatment of ALI. In vivo , Fostamatinib reduced MUC1 abundance in lung epithelial cells in a mouse model of ALI. In vitro , SYK inhibition by Fostamatinib promoted MUC1 removal from the cell surface. Our work reveals Fostamatinib as a repurposing drug candidate for ALI and provides the rationale for rapidly standing up clinical trials to test Fostamatinib efficacy in patients with COVID-19 lung injury.

5.
Org Lett ; 17(14): 3608-11, 2015 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-26144210

RESUMO

An analogue 2 of Brasilicardin A, 1 (BraA), a potent immunosuppressive and cytotoxic agent, was synthesized in which the natural tricyclic skeleton was replaced with a synthetically more accessible substituted tetrahydronaphthalene core. BraA, this analogue (BraL), and cyclosporine A were tested for their ability to inhibit the proliferation of human T cells upon CD3/CD28 activation. Although BraL did not impact T cell activation over the dose range tested, this study shows the inhibitory activity of BraA on human T cells for the first time.


Assuntos
Aminoglicosídeos/síntese química , Aminoglicosídeos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Ciclosporina/química , Imunossupressores/farmacologia , Naftalenos/química , Linfócitos T/efeitos dos fármacos , Aminoglicosídeos/química , Antineoplásicos/química , Antígenos CD28 , Complexo CD3 , Humanos , Imunossupressores/química , Estrutura Molecular
6.
Cell Metab ; 20(5): 910-918, 2014 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-25440061

RESUMO

The LXR-regulated E3 ubiquitin ligase IDOL controls LDLR receptor stability independent of SREBP and PCSK9, but its relevance to plasma lipid levels is unknown. Here we demonstrate that the effects of the LXR-IDOL axis are both tissue and species specific. In mice, LXR agonist induces Idol transcript levels in peripheral tissues but not in liver, and does not change plasma LDL levels. Accordingly, Idol-deficient mice exhibit elevated LDLR protein levels in peripheral tissues, but not in the liver. By contrast, LXR activation in cynomolgus monkeys induces hepatic IDOL expression, reduces LDLR protein levels, and raises plasma LDL levels. Knockdown of IDOL in monkeys with an antisense oligonucleotide blunts the effect of LXR agonist on LDL levels. These results implicate IDOL as a modulator of plasma lipid levels in primates and support further investigation into IDOL inhibition as a potential strategy for LDL lowering in humans.


Assuntos
LDL-Colesterol/sangue , Fígado/metabolismo , Receptores Nucleares Órfãos/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismo , Animais , Células Cultivadas , LDL-Colesterol/metabolismo , Haplorrinos , Humanos , Receptores X do Fígado , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores de LDL/metabolismo , Especificidade da Espécie
7.
ACS Med Chem Lett ; 5(4): 363-7, 2014 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-24900841

RESUMO

EGA, 1, prevents the entry of multiple viruses and bacterial toxins into mammalian cells by inhibiting vesicular trafficking. The cellular target of 1 is unknown, and a structure-activity relationship study was conducted in order to develop a strategy for target identification. A compound with midnanomolar potency was identified (2), and three photoaffinity labels were synthesized (3-5). For this series, the expected photochemistry of the phenyl azide moiety is a more important factor than the IC50 of the photoprobe in obtaining a successful photolabeling event. While 3 was the most effective reversible inhibitor of the series, it provided no protection to cells against anthrax lethal toxin (LT) following UV irradiation. Conversely, 5, which possessed weak bioactivity in the standard assay, conferred robust irreversible protection vs LT to cells upon UV photolysis.

8.
Biochemistry ; 53(11): 1842-8, 2014 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-24580380

RESUMO

Kinetics studies of dNTP analogues having pyrophosphate-mimicking ß,γ-pCXYp leaving groups with variable X and Y substitution reveal striking differences in the chemical transition-state energy for DNA polymerase ß that depend on all aspects of base-pairing configurations, including whether the incoming dNTP is a purine or pyrimidine and if base-pairings are right (T•A and G•C) or wrong (T•G and G•T). Brønsted plots of the catalytic rate constant (log(kpol)) versus pKa4 for the leaving group exhibit linear free energy relationships (LFERs) with negative slopes ranging from -0.6 to -2.0, consistent with chemical rate-determining transition-states in which the active-site adjusts to charge-stabilization demand during chemistry depending on base-pair configuration. The Brønsted slopes as well as the intercepts differ dramatically and provide the first direct evidence that dNTP base recognition by the enzyme-primer-template complex triggers a conformational change in the catalytic region of the active-site that significantly modifies the rate-determining chemical step.


Assuntos
DNA Polimerase beta/química , DNA Polimerase beta/metabolismo , Pareamento de Bases/genética , Catálise , Dano ao DNA/genética , DNA Polimerase beta/genética , Estabilidade Enzimática , Ligação de Hidrogênio , Conformação Proteica
9.
J Lipid Res ; 55(6): 1120-30, 2014 06.
Artigo em Inglês | MEDLINE | ID: mdl-24671012

RESUMO

The liver X receptors (LXRs) are members of the nuclear receptor superfamily that regulate sterol metabolism and inflammation. We sought to identify previously unknown genes regulated by LXRs in macrophages and to determine their contribution to atherogenesis. Here we characterize a novel LXR target gene, the lipopolysaccharide binding protein (LBP) gene. Surprisingly, the ability of LXRs to control LBP expression is cell-type specific, occurring in macrophages but not liver. Treatment of macrophages with oxysterols or loading with modified LDL induces LBP in an LXR-dependent manner, suggesting a potential role for LBP in the cellular response to cholesterol overload. To investigate this further, we performed bone marrow transplant studies. After 18 weeks of Western diet feeding, atherosclerotic lesion burden was assessed revealing markedly smaller lesions in the LBP(-/-) recipients. Furthermore, loss of bone marrow LBP expression increased apoptosis in atherosclerotic lesions as determined by terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Supporting in vitro studies with isolated macrophages showed that LBP expression does not affect cholesterol efflux but promotes the survival of macrophages in the setting of cholesterol loading. The LBP gene is a macrophage-specific LXR target that promotes foam cell survival and atherogenesis.


Assuntos
Proteínas de Fase Aguda/metabolismo , Apoptose , Aterosclerose/metabolismo , Proteínas de Transporte/metabolismo , Células Espumosas/metabolismo , Receptores X do Fígado/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Fase Aguda/genética , Animais , Aterosclerose/genética , Aterosclerose/patologia , Proteínas de Transporte/genética , Sobrevivência Celular/genética , Células Espumosas/patologia , Lipoproteínas LDL/genética , Lipoproteínas LDL/metabolismo , Receptores X do Fígado/genética , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout
10.
Cell Metab ; 18(5): 685-97, 2013 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-24206663

RESUMO

The fatty acyl composition of phospholipids determines the biophysical character of membranes and impacts the function of membrane proteins. Here, we define a nuclear receptor pathway for the dynamic modulation of membrane composition in response to changes in cellular lipid metabolism. Ligand activation of liver X receptors (LXRs) preferentially drives the incorporation of polyunsaturated fatty acids into phospholipids through induction of the remodeling enzyme Lpcat3. Promotion of Lpcat3 activity ameliorates endoplasmic reticulum (ER) stress induced by saturated free fatty acids in vitro or by hepatic lipid accumulation in vivo. Conversely, Lpcat3 knockdown in liver exacerbates ER stress and inflammation. Mechanistically, Lpcat3 modulates inflammation both by regulating inflammatory kinase activation through changes in membrane composition and by affecting substrate availability for inflammatory mediator production. These results outline an endogenous mechanism for the preservation of membrane homeostasis during lipid stress and identify Lpcat3 as an important mediator of LXR effects on metabolism.


Assuntos
Membrana Celular/metabolismo , Estresse do Retículo Endoplasmático , Inflamação/patologia , Receptores Nucleares Órfãos/metabolismo , Fosfolipídeos/metabolismo , 1-Acilglicerofosfocolina O-Aciltransferase/metabolismo , Animais , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Ácidos Graxos/farmacologia , Humanos , Inflamação/metabolismo , Mediadores da Inflamação/metabolismo , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/patologia , Receptores X do Fígado , Masculino , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Camundongos , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Transdução de Sinais/efeitos dos fármacos
11.
Proc Natl Acad Sci U S A ; 110(50): E4904-12, 2013 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-24191014

RESUMO

Pathogenic microorganisms and toxins have evolved a variety of mechanisms to gain access to the host-cell cytosol and thereby exert virulent effects upon the host. One common mechanism of cellular entry requires trafficking to an acidified endosome, which promotes translocation across the host membrane. To identify small-molecule inhibitors that block this process, a library of 30,000 small molecules was screened for inhibitors of anthrax lethal toxin. Here we report that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone, the most active compound identified in the screen, inhibits intoxication by lethal toxin and blocks the entry of multiple other acid-dependent bacterial toxins and viruses into mammalian cells. This compound, which we named EGA, also delays lysosomal targeting and degradation of the EGF receptor, indicating that it targets host-membrane trafficking. In contrast, EGA does not block endosomal recycling of transferrin, retrograde trafficking of ricin, phagolysosomal trafficking, or phagosome permeabilization by Franciscella tularensis. Furthermore, EGA does not neutralize acidic organelles, demonstrating that its mechanism of action is distinct from pH-raising agents such as ammonium chloride and bafilomycin A1. EGA is a powerful tool for the study of membrane trafficking and represents a class of host-targeted compounds for therapeutic development to treat infectious disease.


Assuntos
Toxinas Bacterianas/antagonistas & inibidores , Endossomos/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Semicarbazonas/farmacologia , Internalização do Vírus/efeitos dos fármacos , Aminas , Animais , Transporte Biológico/fisiologia , Caspase 1/metabolismo , Cromatografia Líquida , Endossomos/fisiologia , Citometria de Fluxo , Células HeLa , Humanos , Macrófagos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Estrutura Molecular , Fagocitose/efeitos dos fármacos , Fagocitose/fisiologia , Semicarbazonas/química , Bibliotecas de Moléculas Pequenas , Relação Estrutura-Atividade
12.
Cancer Res ; 73(9): 2850-62, 2013 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-23440422

RESUMO

The sterol regulatory element-binding proteins (SREBP) are key transcriptional regulators of lipid metabolism and cellular growth. It has been proposed that SREBP signaling regulates cellular growth through its ability to drive lipid biosynthesis. Unexpectedly, we find that loss of SREBP activity inhibits cancer cell growth and viability by uncoupling fatty acid synthesis from desaturation. Integrated lipid profiling and metabolic flux analysis revealed that cancer cells with attenuated SREBP activity maintain long-chain saturated fatty acid synthesis, while losing fatty acid desaturation capacity. We traced this defect to the uncoupling of fatty acid synthase activity from stearoyl-CoA desaturase 1 (SCD1)-mediated desaturation. This deficiency in desaturation drives an imbalance between the saturated and monounsaturated fatty acid pools resulting in severe lipotoxicity. Importantly, replenishing the monounsaturated fatty acid pool restored growth to SREBP-inhibited cells. These studies highlight the importance of fatty acid desaturation in cancer growth and provide a novel mechanistic explanation for the role of SREBPs in cancer metabolism.


Assuntos
Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Neoplasias/metabolismo , Animais , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Ácido Graxo Sintases/metabolismo , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos NOD , Modelos Estatísticos , Transplante de Neoplasias , Transdução de Sinais , Estearoil-CoA Dessaturase/metabolismo , Esteróis/metabolismo
13.
Chembiochem ; 13(4): 528-30, 2012 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-22315190

RESUMO

The influence of water: crystallization of (R/S)-α,ß-CHF-dATP with the preorganized pol ß-DNA complex shows that (S)-α,ß-CHF-dATP is preferentially bound to the active site with the C=F fluorine proximal to a structural water bound to Asp276.


Assuntos
DNA Polimerase beta/química , Nucleotídeos de Desoxiadenina/química , Cristalografia por Raios X , DNA Polimerase beta/metabolismo , Nucleotídeos de Desoxiadenina/metabolismo , Modelos Moleculares , Estrutura Molecular , Estereoisomerismo
14.
Phosphorus Sulfur Silicon Relat Elem ; 186(4): 966-967, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21572560

RESUMO

A series of novel ß,γ-methylene-, monofluoromethylene-, and difluoromethylene-bisphosphonophosphate alkyl monoesters was synthesized. The compounds were conveniently detected during preparative HPLC using post-column derivatization with a phosphate-specific chemosensor.

15.
J Org Chem ; 76(12): 5132-6, 2011 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-21462930

RESUMO

The first examples of α-azido bisphosphonates [(RO)(2)P(O)](2)CXN(3) (1, R = i-Pr, X = Me; 2, R = i-Pr, X = H; 3, R = H, X = Me; 4, R = H, X = H) and corresponding ß,γ-CXN(3) dGTP (5-6) and α,ß-CXN(3) dATP (7-8) analogues are described. The individual diastereomers of 7 (7a/b) were obtained by HPLC separation of the dADP synthetic precursor (14a/b).


Assuntos
Azidas/química , Difosfonatos/síntese química , Nucleotídeos/química , Estrutura Molecular , Estereoisomerismo
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