RESUMEN
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.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Oligodendroglía/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Femenino , Inflamación , Interferón gamma/metabolismo , Masculino , Esclerosis Múltiple/patología , Oligodendroglía/citología , Oligodendroglía/metabolismo , Ratas , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
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.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos/métodos , Oligodendroglía/citología , Oligodendroglía/efectos de los fármacos , Células Madre/efectos de los fármacos , Animales , Técnicas In Vitro , Esclerosis Múltiple/tratamiento farmacológico , Ratas , Células Madre/citologíaRESUMEN
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.
Asunto(s)
Axones/efectos de los fármacos , Corteza Cerebral/efectos de los fármacos , Evaluación Preclínica de Medicamentos/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Esclerosis Múltiple/tratamiento farmacológico , Vaina de Mielina/efectos de los fármacos , Regeneración Nerviosa/efectos de los fármacos , Secretasas de la Proteína Precursora del Amiloide/farmacología , Animales , Axones/fisiología , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Corteza Cerebral/fisiología , Medios de Cultivo Condicionados/farmacología , Técnica del Anticuerpo Fluorescente/métodos , Esclerosis Múltiple/fisiopatología , Vaina de Mielina/fisiología , Oligodendroglía/efectos de los fármacos , Oligodendroglía/fisiología , RatasRESUMEN
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.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Cromanos/química , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Sitios de Unión , Células Cultivadas , Etilaminas/síntesis química , Etilaminas/química , Etilaminas/farmacología , Concentración 50 Inhibidora , Modelos Moleculares , Relación Estructura-ActividadRESUMEN
The structure-activity relationship of the prime region of hydroxyethylamine BACE inhibitors is described. Variation in the aryl linker region with 5- and 6-membered heterocycles provided compounds such as 33 with improved permeability and reduced P-gp liability compared to benzyl amine analog 1.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/metabolismo , Etilaminas/química , Etilaminas/farmacología , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Secretasas de la Proteína Precursora del Amiloide/química , Ácido Aspártico Endopeptidasas/química , Cristalografía por Rayos X , Etilaminas/síntesis química , Humanos , Modelos Moleculares , Inhibidores de Proteasas/síntesis química , Unión Proteica , Relación Estructura-ActividadRESUMEN
Herein we describe further evolution of hydroxyethylamine inhibitors of BACE-1 with enhanced permeability characteristics necessary for CNS penetration. Variation at the P2' position of the inhibitor with more polar substituents led to compounds 19 and 32, which retained the potency of more lipophilic analog 1 but with much higher observed passive permeability in MDCK cellular assay.
Asunto(s)
Acetamidas/química , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Butanoles/química , Ciclohexilaminas/química , Inhibidores de Proteasas/química , Acetamidas/síntesis química , Acetamidas/farmacocinética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Sitios de Unión , Butanoles/síntesis química , Butanoles/farmacocinética , Permeabilidad de la Membrana Celular/efectos de los fármacos , Cristalografía por Rayos X , Ciclohexilaminas/síntesis química , Ciclohexilaminas/farmacocinética , Humanos , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/farmacocinética , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/química , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Diseño de Fármacos , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Secretasas de la Proteína Precursora del Amiloide/química , Secretasas de la Proteína Precursora del Amiloide/genética , Ácido Aspártico Endopeptidasas/química , Ácido Aspártico Endopeptidasas/genética , Humanos , Modelos Moleculares , Estructura Molecular , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Benzamidas/síntesis química , Antagonistas del Receptor de Bradiquinina B1 , Pirazoles/síntesis química , Benzamidas/química , Benzamidas/farmacología , Cristalografía por Rayos X , Fibroblastos/metabolismo , Humanos , Técnicas In Vitro , Pulmón/citología , Estructura Molecular , Pirazoles/química , Pirazoles/farmacología , Ensayo de Unión Radioligante , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Amidas/síntesis química , Ácido Aspártico Endopeptidasas/química , Dipéptidos/química , Etilenos/síntesis química , Ácidos Ftálicos/síntesis química , Inhibidores de Proteasas/síntesis química , Amidas/química , Secretasas de la Proteína Precursora del Amiloide , Diseño de Fármacos , Endopeptidasas , Etilenos/química , Humanos , Modelos Moleculares , Imitación Molecular , Ácidos Ftálicos/química , Inhibidores de Proteasas/química , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Aminoácidos/síntesis química , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Inhibidores Enzimáticos/síntesis química , Oligopéptidos/química , Aminoácidos/química , Aminoácidos/farmacología , Secretasas de la Proteína Precursora del Amiloide , Péptidos beta-Amiloides/antagonistas & inhibidores , Péptidos beta-Amiloides/biosíntesis , Línea Celular , Endopeptidasas , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Imitación Molecular , Relación Estructura-ActividadRESUMEN
Potent, small molecule A beta inhibitors have been prepared that incorporate an alanine core bracketed by an N-terminal arylacetyl group and various C-terminal amino alcohols. The compounds exhibit stereospecific inhibition as demonstrated in an in vitro assay.
Asunto(s)
Amino Alcoholes/síntesis química , Amino Alcoholes/farmacología , Péptidos beta-Amiloides/antagonistas & inhibidores , Dipéptidos/síntesis química , Dipéptidos/farmacología , Secretasas de la Proteína Precursora del Amiloide , Péptidos beta-Amiloides/genética , Ácido Aspártico Endopeptidasas , Línea Celular , Supervivencia Celular/efectos de los fármacos , Endopeptidasas/metabolismo , Humanos , Mutación , Relación Estructura-Actividad , TransfecciónRESUMEN
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.