RESUMO
Thrombin acts on the endothelium by activating protease-activated receptors (PARs). The endothelial thrombin-PAR system becomes deregulated during pathological conditions resulting in loss of barrier function and a pro-inflammatory and pro-angiogenic endothelial phenotype. We reported recently that the ion transporter Na(+)/Ca(2+) exchanger (NCX) operating in the Ca(2+)-influx (reverse) mode promoted ERK1/2 activation and angiogenesis in vascular endothelial growth factor-stimulated primary human vascular endothelial cells. Here, we investigated whether Ca(2+) influx through NCX was involved in ERK1/2 activation, angiogenesis, and endothelial barrier dysfunction in response to thrombin. Reverse-mode NCX inhibitors and RNAi-mediated NCX1 knockdown attenuated ERK1/2 phosphorylation in response to thrombin or an agonist of PAR-1, the main endothelial thrombin receptor. Conversely, promoting reverse-mode NCX by suppressing Na(+)-K(+)-ATPase activity enhanced ERK1/2 activation. Reverse-mode NCX inhibitors and NCX1 siRNA suppressed thrombin-induced primary human vascular endothelial cell angiogenesis, quantified as proliferation and tubular differentiation. Reverse-mode NCX inhibitors or NCX1 knockdown preserved barrier integrity upon thrombin stimulation in vitro. Moreover, the reverse-mode NCX inhibitor SEA0400 suppressed Evans' blue albumin extravasation to the lung and kidneys and attenuated edema formation and ERK1/2 activation in the lungs of mice challenged with a peptide activator of PAR-1. Mechanistically, thrombin-induced ERK1/2 activation required NADPH oxidase 2-mediated reactive oxygen species (ROS) production, and reverse-mode NCX inhibitors and NCX1 siRNA suppressed thrombin-induced ROS production. We propose that reverse-mode NCX is a novel mechanism contributing to thrombin-induced angiogenesis and hyperpermeability by mediating ERK1/2 activation in a ROS-dependent manner. Targeting reverse-mode NCX could be beneficial in pathological conditions involving unregulated thrombin signaling.
Assuntos
Cálcio/metabolismo , Glicoproteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Neovascularização Fisiológica/genética , Espécies Reativas de Oxigênio/metabolismo , Trocador de Sódio e Cálcio/genética , Compostos de Anilina/administração & dosagem , Animais , Endotélio/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Sistema de Sinalização das MAP Quinases/genética , Glicoproteínas de Membrana/genética , Camundongos , NADPH Oxidase 2 , NADPH Oxidases/genética , Neovascularização Fisiológica/efeitos dos fármacos , Permeabilidade/efeitos dos fármacos , Éteres Fenílicos/administração & dosagem , RNA Interferente Pequeno/genética , Receptor PAR-1/genética , Receptor PAR-1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Trocador de Sódio e Cálcio/biossíntese , Trocador de Sódio e Cálcio/metabolismo , Trombina/administração & dosagemRESUMO
The metal-dependent phosphatase PPM1D (WIP1) is an important oncogene in cancer, with over-expression of the protein being associated with significantly worse clinical outcomes. In this communication we describe the discovery and optimization of novel 2,4-bisarylthiazoles that phenocopy the knockdown of PPM1D, without inhibiting its phosphatase activity. These compounds cause growth inhibition at nanomolar concentrations, induce apoptosis, activate p53 and display impressive cell-line selectivity. The results demonstrate the potential for targeting phenotypes in drug discovery when tackling challenging targets or unknown mechanisms.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Fosfoproteínas Fosfatases/antagonistas & inibidores , Tiazóis/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Descoberta de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Fenótipo , Fosfoproteínas Fosfatases/metabolismo , Proteína Fosfatase 2C , Relação Estrutura-Atividade , Tiazóis/síntese química , Tiazóis/química , Proteína Supressora de Tumor p53/metabolismoRESUMO
This study describes a novel series of UDP-N-acetylglucosamine acyltransferase (LpxA) inhibitors that was identified through affinity-mediated selection from a DNA-encoded compound library. The original hit was a selective inhibitor of Pseudomonas aeruginosa LpxA with no activity against Escherichia coli LpxA. The biochemical potency of the series was optimized through an X-ray crystallography-supported medicinal chemistry program, resulting in compounds with nanomolar activity against P. aeruginosa LpxA (best half-maximal inhibitory concentration (IC50) <5 nM) and cellular activity against P. aeruginosa (best minimal inhibitory concentration (MIC) of 4 µg/mL). Lack of activity against E. coli was maintained (IC50 > 20 µM and MIC > 128 µg/mL). The mode of action of analogues was confirmed through genetic analyses. As expected, compounds were active against multidrug-resistant isolates. Further optimization of pharmacokinetics is needed before efficacy studies in mouse infection models can be attempted. To our knowledge, this is the first reported LpxA inhibitor series with selective activity against P. aeruginosa.
Assuntos
Aciltransferases/antagonistas & inibidores , Antibacterianos/farmacologia , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Antibacterianos/química , Cristalografia por Raios X , Farmacorresistência Bacteriana/efeitos dos fármacos , Inibidores Enzimáticos/química , Escherichia coli/enzimologia , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Trifluoromethyl group containing oxazolidines (Fox) are conveniently synthesized by condensation of serine esters with trifluoroacetaldehyde hemiacetal or trifluoroacetone. These oxazolidines can undergo N-acylation and amidification reactions and are completely configurationally and hydrolytically stable. Therefore, they can be considered as highly valuable proline surrogates (Tfm-pseudoprolines).
Assuntos
Flúor/química , Metano/química , Oxazóis/síntese química , Prolina/análogos & derivados , Tiazóis/química , Acilação , Ésteres/síntese química , Ésteres/química , Estrutura Molecular , Oxazóis/química , Prolina/químicaRESUMO
The SCAL linker, a safety catch linker, is amongst the most versatile linkers for solid phase synthesis. It was originally described in 1991 by Pátek and Lebl. Yet, its application has been hindered by the low yields of published synthetic routes. Over time, the exceptional versatility of this linker has been demonstrated in several applications of advanced solid phase synthesis of peptides and peptidomimetics. Recently, an updated synthesis of the original linker has also been presented at the 22nd American Peptide Symposium, comprising 10 steps. Herein, the design and synthesis of a next generation SCAL linker, SCAL-2, is reported. SCAL-2 features a simplified molecular architecture, which allows for a more efficient synthesis in 8 steps with superior yields. Both linkers, SCAL and SCAL-2 are compared in terms of their cleavage properties adding valuable information on how to best utilize the versatility of these linkers for solid phase synthesis.
RESUMO
HSP70 is a molecular chaperone and a key component of the heat-shock response. Because of its proposed importance in oncology, this protein has become a popular target for drug discovery, efforts which have as yet brought little success. This study demonstrates that adenosine-derived HSP70 inhibitors potentially bind to the protein with a novel mechanism of action, the stabilization by desolvation of an intramolecular salt-bridge which induces a conformational change in the protein, leading to high affinity ligands. We also demonstrate that through the application of this mechanism, adenosine-derived HSP70 inhibitors can be optimized in a rational manner.
Assuntos
Adenosina/farmacologia , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Adenosina/síntese química , Adenosina/química , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Modelos Moleculares , Conformação Molecular , Relação Estrutura-AtividadeRESUMO
A well-documented obstacle in the synthesis of functionalized rhodamine dyes is the generation of regioisomers which are difficult to separate. These isomers occur due to the use of unsymmetrical anhydride reagents, which possess two potential points of reactivity where condensation with meta-aminophenols can take place. In this report we describe a method which eliminates this problem by using phthalaldehydic acids as anhydride replacements. These reagents provide only one point of reactivity for the aminophenol, thus allowing direct access to single isomer tetramethylrhodamines and avoiding isomer generation altogether. A range of functionalities are shown to be tolerated at the 5- and 6-position of the dye compounds which are prepared in up to gram quantities using our method. The scope of the method is further demonstrated by the preparation of additional rhodamine family members Rhodamine B and X-Rhodamine.
RESUMO
Following the discovery of dibenzo[b,d]thiophen-4-yl)-2-morpholino-4H-chromen-4-one (NU7441) ( Leahy , J. J. J. ; Golding , B. T. ; Griffin , R. J. ; Hardcastle , I. R. ; Richardson , C. ; Rigoreau , L. ; Smith , G. C. M. Bioorg. Med. Chem. Lett. 2004 , 14 , 6083 - 6087) as a potent inhibitor (IC50 = 30 nM) of DNA-dependent protein kinase (DNA-PK), we have investigated analogues in which the chromen-4-one core template has been replaced by aza-heterocyclic systems: 9-substituted 2-morpholin-4-ylpyrido[1,2-a]pyrimidin-4-ones and 8-substituted 2-morpholin-4-yl-1H-quinolin-4-ones. The 8- and 9-substituents were either dibenzothiophen-4-yl or dibenzofuran-4-yl, which were each further substituted at the 1-position with water-solubilizing groups [NHCO(CH2)(n)NR¹R², where n = 1 or 2 and the moiety R¹R²N was derived from a library of primary and secondary amines (e.g., morpholine)]. The inhibitors were synthesized by employing a multiple-parallel approach in which the two heterocyclic components were assembled by Suzuki-Miyaura cross-coupling. Potent DNA-PK inhibitory activity was generally observed across the compound series, with structure-activity studies indicating that optimal potency resided in pyridopyrimidin-4-ones bearing a substituted dibenzothiophen-4-yl group. Several of the newly synthesized compounds (e.g., 2-morpholin-4-yl-N-[4-(2-morpholin-4-yl-4-oxo-4H-pyrido[1,2-a]pyrimidin-9-yl)dibenzothiophen-1-yl]acetamide) combined high potency against the target enzyme (DNA-PK IC50 = 8 nM) with promising activity as potentiators of ionizing radiation-induced cytotoxicity in vitro.
Assuntos
Benzopiranos/química , Proteína Quinase Ativada por DNA/antagonistas & inibidores , Piridinas/síntese química , Pirimidinonas/síntese química , Quinolonas/síntese química , Permeabilidade da Membrana Celular , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Furanos/síntese química , Furanos/química , Furanos/farmacologia , Células HeLa , Humanos , Piridinas/química , Piridinas/farmacologia , Pirimidinonas/química , Pirimidinonas/farmacologia , Quinolonas/química , Quinolonas/farmacologia , Bibliotecas de Moléculas Pequenas , Relação Estrutura-Atividade , Tiofenos/síntese química , Tiofenos/química , Tiofenos/farmacologiaRESUMO
The Wnt signaling pathway is frequently deregulated in cancer due to mutations in genes encoding APC, beta-catenin, and axin. To identify small-molecule inhibitors of Wnt signaling as potential therapeutics, a diverse chemical library was screened using a transcription factor reporter cell line in which the activity of the pathway was induced at the level of Disheveled protein. A series of deconvolution studies was used to focus on three compound series that selectively killed cancer cell lines with constitutive Wnt signaling. Activities of the compounds included the ability to induce degradation of beta-catenin that had been stabilized by a glycogen synthase kinase-3 (GSK-3) inhibitor. This screen illustrates a practical approach to identify small-molecule inhibitors of Wnt signaling that can seed the development of agents suitable to treat patients with Wnt-dependent tumors.
Assuntos
Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Ensaios de Triagem em Larga Escala/métodos , Proteínas Wnt/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Humanos , Células L , Camundongos , Transdução de Sinais , Transcrição Gênica/efeitos dos fármacos , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Xenopus laevis , Peixe-ZebraRESUMO
8-Substituted 2-morpholin-4-yl-quinolin-4-ones and 9-substituted 2-morpholin-4-yl-pyrido[1,2-a]pyrimidin-4-ones with selected aryl and heteroaryl groups as the substituent have been synthesised as potential inhibitors of DNA-dependent protein kinase. A multiple-parallel approach, employing Suzuki cross-coupling methodology, was utilised in the preparation of 8-substituted 2-morpholin-4-yl-quinolin-4-ones. For this purpose 8-bromo-2-morpholin-4-yl-quinolin-4-one was required as an intermediate. This compound was obtained by adapting a literature route in which thermal cyclocondensation of (2-bromoanilino)-morpholin-4-yl-5-methylene-2,2-dimethyl[1,3]dioxane-4,6-dione afforded 8-bromo-2-morpholin-4-yl-quinolin-4-one. A multiple-parallel approach, employing Suzuki cross-coupling methodology, was also utilised to prepare 9-substituted 2-morpholin-4-yl-pyrido[1,2-a]pyrimidin-4-ones using 9-hydroxy-2-morpholin-4-yl-pyrido[1,2-a]pyrimidin-4-one O-trifluoromethanesulfonate as an intermediate. 8-Substituted 2-morpholin-4-yl-quinolin-4-ones and 9-substituted 2-morpholin-4-yl-pyrido[1,2-a]pyrimidin-4-ones were both inhibitors of DNA-dependent protein kinase. When the substituent was dibenzothiophen-4-yl, dibenzofuran-4-yl or biphen-3-yl, IC50 values in the low nanomolar range were observed. Interestingly, the pyridopyrimidinones and quinolinones were essentially equipotent with the corresponding 8-substituted 2-morpholin-4-yl-chromen-4-ones previously reported (I. R. Hardcastle, X. Cockcroft, N. J. Curtin, M. Desage El-Murr, J. J. J. Leahy, M. Stockley, B. T. Golding, L. Rigoreau, C. Richardson, G. C. M. Smith and R. J. Griffin, J. Med. Chem., 2005, 48, 7829-7846).