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1.
BMC Neurosci ; 17: 16, 2016 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-27103572

RESUMO

BACKGROUND: Regeneration of new myelin is impaired in persistent multiple sclerosis (MS) lesions, leaving neurons unable to function properly and subject to further degeneration. Current MS therapies attempt to ameliorate autoimmune-mediated demyelination, but none directly promote the regeneration of lost and damaged myelin of the central nervous system (CNS). Development of new drugs that stimulate remyelination has been hampered by the inability to evaluate axonal myelination in a rapid CNS culture system. RESULTS: We established a high throughput cell-based assay to identify compounds that promote myelination. Culture methods were developed for initiating myelination in vitro using primary embryonic rat cortical cells. We developed an immunofluorescent phenotypic image analysis method to quantify the morphological alignment of myelin characteristic of the initiation of myelination. Using γ-secretase inhibitors as promoters of myelination, the optimal growth, time course and compound treatment conditions were established in a 96 well plate format. We have characterized the cortical myelination assay by evaluating the cellular composition of the cultures and expression of markers of differentiation over the time course of the assay. We have validated the assay scalability and consistency by screening the NIH clinical collection library of 727 compounds and identified ten compounds that promote myelination. Half maximal effective concentration (EC50) values for these compounds were determined to rank them according to potency. CONCLUSIONS: We have designed the first high capacity in vitro assay that assesses myelination of live axons. This assay will be ideal for screening large compound libraries to identify new drugs that stimulate myelination. Identification of agents capable of promoting the myelination of axons will likely lead to the development of new therapeutics for MS patients.


Assuntos
Axônios/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Esclerose Múltipla/tratamento farmacológico , Bainha de Mielina/efeitos dos fármacos , Regeneração Nervosa/efeitos dos fármacos , Secretases da Proteína Precursora do Amiloide/farmacologia , Animais , Axônios/fisiologia , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Córtex Cerebral/fisiologia , Meios de Cultivo Condicionados/farmacologia , Imunofluorescência/métodos , Esclerose Múltipla/fisiopatologia , Bainha de Mielina/fisiologia , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/fisiologia , Ratos
2.
Bioorg Med Chem Lett ; 23(16): 4674-9, 2013 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-23856050

RESUMO

The structure activity relationship of the prime region of conformationally restricted hydroxyethylamine (HEA) BACE inhibitors is described. Variation of the P1' region provided selectivity over Cat-D with a series of 2,2-dioxo-isothiochromanes and optimization of the P2' substituent of chromane-HEA(s) with polar substituents provided improvements in the compound's in vitro permeability. Significant potency gains were observed with small aliphatic substituents such as methyl, n-propyl, and cyclopropyl when placed at the C-2 position of the chromane.


Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Cromanos/química , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Sítios de Ligação , Células Cultivadas , Etilaminas/síntese química , Etilaminas/química , Etilaminas/farmacologia , Concentração Inibidora 50 , Modelos Moleculares , Relação Estrutura-Atividade
3.
Bioorg Med Chem Lett ; 20(20): 6034-9, 2010 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-20822903
4.
Bioorg Med Chem Lett ; 20(16): 4789-94, 2010 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-20634069
5.
Bioorg Med Chem Lett ; 19(22): 6386-91, 2009 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-19811916

RESUMO

Using structure-guided design, hydroxyethylamine BACE-1 inhibitors were optimized to nanomolar Abeta cellular inhibition with selectivity against cathepsin-D. X-ray crystallography illuminated the S1' residues critical to this effort, which culminated in compounds 56 and 57 that exhibited potency and selectivity but poor permeability and high P-gp efflux.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Desenho de Fármacos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Secretases da Proteína Precursora do Amiloide/química , Secretases da Proteína Precursora do Amiloide/genética , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/genética , Humanos , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Especificidade por Substrato
6.
J Med Chem ; 50(21): 5161-7, 2007 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-17880055

RESUMO

The B1 receptor is an attractive target for the treatment of pain and inflammation. A series of 3-carboxamido-5-phenacylamino pyrazole B1 receptor antagonists are described that exhibit good potency against B1 and high selectivity over B2. Initially, N-unsubstituted pyrazoles were studied, but these compounds suffered from extensive glucuronidation in primates. This difficulty could be surmounted by the use of N-substituted pyrazoles. Optimization efforts culminated in compound 41, which has high receptor potency and metabolic stability.


Assuntos
Benzamidas/síntese química , Antagonistas de Receptor B1 da Bradicinina , Pirazóis/síntese química , Benzamidas/química , Benzamidas/farmacologia , Cristalografia por Raios X , Fibroblastos/metabolismo , Humanos , Técnicas In Vitro , Pulmão/citologia , Estrutura Molecular , Pirazóis/química , Pirazóis/farmacologia , Ensaio Radioligante , Relação Estrutura-Atividade
7.
BMC Res Notes ; 9(1): 444, 2016 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-27629829

RESUMO

BACKGROUND: Newly proliferated oligodendrocyte precursor cells (OPCs) migrate and surround lesions of patients with multiple sclerosis (MS) and other demyelinating diseases, but fail to differentiate into oligodendrocytes (OLs) and remyelinate remaining viable axons. The abundance of secreted inflammatory factors within and surrounding these lesions likely plays a major inhibitory role, promoting cell death and preventing OL differentiation and axon remyelination. To identify clinical candidate compounds that may protect existing and differentiating OLs in patients, we have developed a high throughput screening (HTS) assay that utilizes purified rat OPCs. RESULTS: Using a fluorescent indicator of cell viability coupled with image quantification, we developed an assay to allow the identification of compounds that promote OL viability and differentiation in the presence of the synergistic inflammatory cytokines, tumor necrosis factor α and interferon-γ. We have utilized this assay to screen the NIH clinical collection library and identify compounds that protect OLs and promote OL differentiation in the presence of these inflammatory cytokines. CONCLUSION: This primary OL-based cytokine protection assay is adaptable for HTS and may be easily modified for profiling of compounds in the presence of other potentially inhibitory molecules found in MS lesions. This assay should be of use to those interested in identifying drugs for the treatment of MS and other demyelinating diseases.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Oligodendroglia/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Inflamação , Interferon gama/metabolismo , Masculino , Esclerose Múltipla/patologia , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Ratos , Fator de Necrose Tumoral alfa/metabolismo
8.
BMC Res Notes ; 9(1): 419, 2016 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-27592856

RESUMO

BACKGROUND: Multiple sclerosis is caused by an autoimmune response resulting in demyelination and neural degeneration. The adult central nervous system has the capacity to remyelinate axons in part through the generation of new oligodendrocytes (OLs). To identify clinical candidate compounds that may promote remyelination, we have developed a high throughput screening (HTS) assay to identify compounds that promote the differentiation of oligodendrocyte precursor cells (OPCs) into OLs. RESULTS: Using acutely dissociated and purified rat OPCs coupled with immunofluorescent image quantification, we have developed an OL differentiation assay. We have validated this assay with a known promoter of differentiation, thyroid hormone, and subsequently used the assay to screen the NIH clinical collection library. We have identified twenty-seven hit compounds which were validated by dose response analysis and the generation of half maximal effective concentration (EC50) values allowed for the ranking of efficacy. The assay identified novel promoters of OL differentiation which we attribute to (1) the incorporation of an OL toxicity pre-screen to allow lowering the concentrations of toxic compounds and (2) the utilization of freshly purified, non-passaged OPCs. These features set our assay apart from other OL differentiation assays used for drug discovery efforts. CONCLUSIONS: This acute primary OL-based differentiation assay should be of use to those interested in screening large compound libraries for the identification of drugs for the treatment of MS and other demyelinating diseases.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Oligodendroglia/citologia , Oligodendroglia/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Animais , Técnicas In Vitro , Esclerose Múltipla/tratamento farmacológico , Ratos , Células-Tronco/citologia
9.
J Med Chem ; 45(2): 259-62, 2002 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-11784130

RESUMO

By use of the effectively cleaved beta-secretase (BACE) substrate (1), incorporation of a statine in P(1) resulted in a weak inhibitor 13 of the enzyme. Further substitution of P(1)'-Asp by P(1)'-Val in 13 results in a potent inhibitor 22 of BACE. Removal of the P(10)-P(5) residues on the N-terminal part of inhibitor 22 resulted in no loss of potency (23). C-terminal truncations of inhibitor 22 generally led to significant loss of potency.


Assuntos
Ácido Aspártico Endopeptidases/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Oligopeptídeos/síntese química , Secretases da Proteína Precursora do Amiloide , Encéfalo/enzimologia , Endopeptidases , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Oligopeptídeos/química , Relação Estrutura-Atividade , Especificidade por Substrato
10.
J Med Chem ; 47(1): 158-64, 2004 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-14695829

RESUMO

The hydroxyethylene (HE) transition state isostere was developed as a scaffold to provide potent, small molecule inhibitors of human beta-secretase (BACE). The previous work on the statine series proved critical to the discovery of HE structure-activity relationships. Compound 20 with the N-terminal isophthalamide proved to be the most potent HE inhibitor (IC(50) = 30 nM) toward BACE. Unlike the statine series, we identified HE inhibitors without carboxylic acids on the C terminus, leading to enhanced cell penetration and making them attractive candidates for further drug development in Alzheimer's disease.


Assuntos
Amidas/síntese química , Ácido Aspártico Endopeptidases/química , Dipeptídeos/química , Etilenos/síntese química , Ácidos Ftálicos/síntese química , Inibidores de Proteases/síntese química , Amidas/química , Secretases da Proteína Precursora do Amiloide , Desenho de Fármacos , Endopeptidases , Etilenos/química , Humanos , Modelos Moleculares , Mimetismo Molecular , Ácidos Ftálicos/química , Inibidores de Proteases/química , Relação Estrutura-Atividade
11.
J Med Chem ; 46(10): 1799-802, 2003 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-12723942

RESUMO

We describe the development of statine-based peptidomimetic inhibitors of human beta-secretase (BACE). The conversion of the peptide inhibitor 1 into cell-permeable peptidomimetic inhibitors of BACE was achieved through an iterative strategy of conceptually subdividing 1 into three regions: an N-terminal portion, a central statine-containing core, and a C-terminus. Replacement of the amino acid residues of 1 with moieties with less peptidic character was done with retention of BACE enzyme inhibitory activity. This approach led to the identification of the cell-permeable BACE inhibitor 38 that demonstrated BACE-mechanism-selective inhibition of Abeta secretion in human embryonic kidney cells.


Assuntos
Aminoácidos/síntese química , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Oligopeptídeos/química , Aminoácidos/química , Aminoácidos/farmacologia , Secretases da Proteína Precursora do Amiloide , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/biossíntese , Linhagem Celular , Endopeptidases , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Mimetismo Molecular , Relação Estrutura-Atividade
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