RESUMEN
The development of potent gamma-secretase inhibitors having substituted heterocycles attached to a benzobicyclo[4.2.1]nonane core is described. This work led to the identification of [6S,9R,11R]-2',3',4',5,5',6,7,8,9,10-decahydro-2-(5-(4-fluorophenyl)-1-methylpyrazol-3-yl)-5'-(2,2,2-trifluoroethyl)spiro[6,9-methanobenzocyclooctene-11,3'-[1,2,5]thiadiazole] 1',1'-dioxide (16), which has excellent in vitro potency (0.06 nM) and which reduced amyloid-beta in APP-YAC mice with an ED(50) of 1 mg/kg (po). 16 had a good pharmacokinetic profile in three preclinical species.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Péptidos beta-Amiloides/antagonistas & inhibidores , Ciclooctanos/farmacología , Inhibidores de Proteasas/farmacología , Tiadiazoles/farmacología , Administración Oral , Animales , Ciclooctanos/administración & dosificación , Ciclooctanos/síntesis química , Ciclooctanos/farmacocinética , Concentración 50 Inhibidora , Ratones , Inhibidores de Proteasas/administración & dosificación , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/farmacocinética , Tiadiazoles/administración & dosificación , Tiadiazoles/síntesis química , Tiadiazoles/farmacocinéticaRESUMEN
Gamma-secretase is one of the critical enzymes required for the generation of amyloid-beta peptides from the beta-amyloid precursor protein. Because amyloid-beta peptides are generally accepted to play a key role in Alzheimer disease, gamma-secretase inhibition holds the promise for a disease-modifying therapy for this neurodegenerative condition. Although recent progress has enhanced the understanding of the biology and composition of the gamma-secretase enzyme complex, less information is available on the actual interaction of various inhibitor classes with the enzyme. Here we show that the two principal classes of inhibitor described in the scientific and patent literature, aspartyl protease transition state analogue and small molecule non-transition state inhibitors, display fundamental differences in the way they interact with the enzyme. Taking advantage of a gamma-secretase enzyme overexpressing cellular system and different radiolabeled gamma-secretase inhibitors, we observed that the maximal binding of non-transition state gamma-secretase inhibitors accounts only for half the number of catalytic sites of the recombinant enzyme complex. This characteristic stoichiometry can be best accommodated with a model whereby the non-transition state inhibitors bind to a unique site at the interface of a dimeric enzyme. Subsequent competition studies confirm that this site appears to be targeted by the main classes of small molecule gamma-secretase inhibitor. In contrast, the non-steroidal anti-inflammatory drug gamma-secretase modulator sulindac sulfide displayed noncompetitive antagonism for all types of inhibitor. This finding suggests that non-steroidal anti-inflammatory drug-type gamma-secretase modulators target an alternative site on the enzyme, thereby changing the conformation of the binding sites for gamma-secretase inhibitors.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/química , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Antiinflamatorios no Esteroideos/farmacología , Unión Competitiva , Bioquímica/métodos , Dominio Catalítico , Línea Celular , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/farmacología , Humanos , Cinética , Ligandos , Modelos Químicos , Unión Proteica , Sulindac/análogos & derivados , Sulindac/farmacologíaRESUMEN
Previously, chemistry effort on the gem-cyclohexane series of gamma-secretase inhibitors has focused on the 4-position of the cyclohexane ring. Recently chemistry has been directed towards the 3-position and substitution here has also provided compounds with high gamma-secretase activity.
Asunto(s)
Enfermedad de Alzheimer/patología , Ciclohexanos/química , Endopeptidasas/metabolismo , Inhibidores de Proteasas/química , Sulfonas/química , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/enzimología , Secretasas de la Proteína Precursora del Amiloide , Ácido Aspártico Endopeptidasas , Ciclohexanos/farmacología , Humanos , Concentración 50 Inhibidora , Modelos Químicos , Inhibidores de Proteasas/farmacología , Estereoisomerismo , Sulfonas/farmacologíaRESUMEN
The protease gamma-secretase plays a pivotal role in the synthesis of pathogenic amyloid-beta in Alzheimer's disease (AD). Here, we report a further extension to a series of cyclohexyl sulfone-based gamma-secretase inhibitors which has allowed the preparation of highly potent compounds which also demonstrate robust Abeta(40) lowering in vivo (e.g., compound 32, MED 1mg/kg p.o. in APP-YAC mice).
Asunto(s)
Ciclohexanos/administración & dosificación , Ciclohexanos/farmacología , Endopeptidasas/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Sulfonas/administración & dosificación , Sulfonas/farmacología , Administración Oral , Secretasas de la Proteína Precursora del Amiloide , Péptidos beta-Amiloides/efectos de los fármacos , Animales , Ácido Aspártico Endopeptidasas , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Ciclohexanos/química , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Ratones , Estructura Molecular , Fragmentos de Péptidos/efectos de los fármacos , Relación Estructura-Actividad , Sulfonas/química , Factores de TiempoRESUMEN
Presenilins appear to form the active center of gamma-secretase but require the presence of the integral membrane proteins nicastrin, anterior pharynx defective 1, and presenilin enhancer 2 for catalytic function. We have simultaneously overexpressed all of these polypeptides, and we demonstrate functional assembly of the enzyme complex, a substantial increase in enzyme activity, and binding of all components to a transition state analogue gamma-secretase inhibitor. Co-localization of all components can be observed in the Golgi compartment, and further trafficking of the individual constituents seems to be dependent on functional assembly. Apart from its catalytic function, gamma-secretase appears to play a role in the trafficking of the beta-amyloid precursor protein, which was changed upon reconstitution of the enzyme but unaffected by pharmacological inhibition. Because the relative molecular mass and stoichiometry of the active enzyme complex remain elusive, we performed size exclusion chromatography of solubilized gamma-secretase, which yielded evidence of a tetrameric form of the complex, yet almost completely abolished enzyme activity. Gamma-secretase activity was reconstituted upon addition of an independent size exclusion chromatography fraction of lower molecular mass and nonproteinaceous nature, which could be replaced by a brain lipid extract. The same treatment was able to restore enzyme activity after immunoaffinity purification of the gamma-secretase complex, demonstrating that lipids play a key role in preserving the catalytic activity of this protease. Furthermore, these data show that it is important to discriminate between intact, inactive gamma-secretase complexes and the active form of the enzyme, indicating the care that must be taken in the study of gamma-secretase.
Asunto(s)
Endopeptidasas/biosíntesis , Metabolismo de los Lípidos , Glicoproteínas de Membrana/metabolismo , Péptido Hidrolasas/metabolismo , Secretasas de la Proteína Precursora del Amiloide , Precursor de Proteína beta-Amiloide/metabolismo , Ácido Aspártico Endopeptidasas , Sitios de Unión , Western Blotting , Encéfalo/metabolismo , Catálisis , Línea Celular , Membrana Celular/metabolismo , Sistema Libre de Células/metabolismo , Cromatografía , Medios de Cultivo/metabolismo , Medios de Cultivo Condicionados/farmacología , ADN Complementario/metabolismo , Dimerización , Endopeptidasas/metabolismo , Aparato de Golgi/metabolismo , Humanos , Inmunohistoquímica , Péptidos/química , Estructura Terciaria de Proteína , Factores de TiempoRESUMEN
Gamma-secretase performs the final processing step in the generation of amyloid-beta (Abeta) peptides, which are believed to be causative for Alzheimer's disease. Presenilins (PS) are required for gamma-secretase activity and the presence of two essential intramembranous aspartates (D257 and D385) has implicated this region as the putative catalytic centre of an aspartyl protease. The presence of several key hydrogen-bonding residues around the active site of classical aspartyl proteases led us to investigate the role of both the critical aspartates and two nearby conserved hydrogen bond donors in PS1. Generation of cell lines stably overexpressing the D257E, D385E, Y256F and Y389F engineered mutations has enabled us to determine their role in enzyme catalysis and binding of a transition state analogue gamma-secretase inhibitor. Here we report that replacement of either tyrosine residue alters gamma-secretase cleavage specificity, resulting in an increase in the production of the more pathogenic Abeta42 peptide in both cells and membranous enzyme preparations, without affecting inhibitor binding. In contrast, replacement of either of the aspartate residues precludes inhibitor binding in addition to inactivation of the enzyme. Together, these data further incriminate the region around the intramembranous aspartates as the active site of the enzyme, targeted by transition state analogue inhibitors, and highlight the roles of individual residues.
Asunto(s)
Ácido Aspártico/metabolismo , Secuencia Conservada/fisiología , Endopeptidasas/metabolismo , Activación Enzimática/fisiología , Mutagénesis Sitio-Dirigida/fisiología , Secretasas de la Proteína Precursora del Amiloide , Péptidos beta-Amiloides , Precursor de Proteína beta-Amiloide/metabolismo , Ácido Aspártico/química , Ácido Aspártico/genética , Ácido Aspártico Endopeptidasas/farmacología , Sitios de Unión/efectos de los fármacos , Sitios de Unión/fisiología , Western Blotting/métodos , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Sistema Libre de Células , Ácidos Cólicos/farmacología , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/metabolismo , Medios de Cultivo Condicionados/análisis , Detergentes/farmacología , Embrión de Mamíferos , Endopeptidasas/química , Endopeptidasas/genética , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Riñón , Fragmentos de Péptidos , Unión Proteica/fisiología , Pliegue de Proteína , Estructura Terciaria de Proteína , Especificidad por Sustrato , Transfección , Tirosina/química , Tirosina/genética , Tirosina/metabolismoRESUMEN
Gamma-secretase is a multi-component enzyme complex that performs an intramembranous cleavage, releasing amyloid-beta (Abeta) peptides from processing intermediates of the beta-amyloid precursor protein. Because Abeta peptides are thought to be causative for Alzheimer's disease, inhibiting gamma-secretase represents a potential treatment for this neurodegenerative condition. Whereas inhibitors directed at the active center of gamma-secretase inhibit the cleavage of all its substrates, certain non-steroidal antiinflammatory drugs (NSAIDs) have been shown to selectively reduce the production of the more amyloidogenic Abeta(1-42) peptide without inhibiting alternative cleavages. In contrast to the majority of previous studies, however, we demonstrate that in cell-free systems the mode of action of selected NSAIDs and their derivatives, depending on the concentrations used, can either be classified as modulatory or inhibitory. At modulatory concentrations, a selective and, with respect to the substrate, noncompetitive inhibition of Abeta(1-42) production was observed. At inhibitory concentrations, on the other hand, biochemical readouts reminiscent of a nonselective gamma-secretase inhibition were obtained. When these compounds were analyzed for their ability to displace a radiolabeled, transition-state analog inhibitor from solubilized enzyme, noncompetitive antagonism was observed. The allosteric nature of radioligand displacement suggests that NSAID-like inhibitors change the conformation of the gamma-secretase enzyme complex by binding to a novel site, which is discrete from the binding site for transition-state analogs and therefore distinct from the catalytic center. Consequently, drug discovery efforts aimed at this site may identify novel allosteric inhibitors that could benefit from a wider window for inhibition of gamma (42)-cleavage over alternative cleavages in the beta-amyloid precursor protein and, more importantly, alternative substrates.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Endopeptidasas/metabolismo , Regulación Alostérica , Secretasas de la Proteína Precursora del Amiloide , Sitios de Unión , Endopeptidasas/química , Endopeptidasas/efectos de los fármacos , Flurbiprofeno/farmacología , Cinética , Ligandos , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Sulindac/farmacologíaRESUMEN
In this paper, we describe the development of a novel series of high affinity, orally bioavailable 3-amino-1,4 benzodiazepine-based gamma-secretase inhibitors for the potential treatment of Alzheimer's disease. We disclose structure-activity relationships based around the 1, 3 and 5 positions of the benzodiazepine core structure.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Benzodiazepinas/síntesis química , Benzodiazepinas/farmacocinética , Endopeptidasas/metabolismo , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacocinética , Administración Oral , Aminas/síntesis química , Aminas/farmacocinética , Secretasas de la Proteína Precursora del Amiloide , Ácido Aspártico Endopeptidasas , Benzodiazepinas/administración & dosificación , Benzodiazepinas/farmacología , Disponibilidad Biológica , Biotransformación , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/farmacología , Humanos , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Gamma-secretase is the enzyme activity releasing the amyloid-beta peptide from membrane-bound processing intermediates derived from the beta-amyloid precursor protein. Cellular release and subsequent aggregation of the amyloid-beta peptide is thought to be causative for the pathogenesis of Alzheimer's disease. Gamma-secretase performs an unusual intramembranous cleavage and has been closely linked to a macromolecular complex containing presenilins. To generate a molecular probe for gamma-secretase, we have developed a novel biotinylated affinity ligand which is based on a specific inhibitor containing a hydroxyethylene dipeptide isostere, known to serve as a transition state analogue for aspartic proteinases. Using this probe we confirmed the presence of the presenilin heterodimer and mature nicastrin in the active enzyme complex and, furthermore, that substrate binding site(s) and active center(s) are spatially separated. Affinity precipitations suggest that only a discrete fraction of cellular presenilin is present in the active gamma-secretase complex and that both gamma(40)- and gamma(42)-activities are mediated by the same molecular entity. This was also reflected by a co-distribution of both enzyme activities in subcellular fractions enriched for trans-Golgi network membranes.
Asunto(s)
Endopeptidasas/metabolismo , Proteínas de la Membrana/metabolismo , Secretasas de la Proteína Precursora del Amiloide , Ácido Aspártico Endopeptidasas , Western Blotting , Humanos , Técnicas In Vitro , Ligandos , Presenilina-1 , Presenilina-2 , Especificidad por Sustrato , Células Tumorales CultivadasRESUMEN
Novel benzodiazepine-containing gamma-secretase inhibitors for potential use in Alzheimer's disease have been designed that incorporate a substituted hydrocinnamide C-3 side chain. A syn combination of alpha-alkyl or aryl and beta-hydroxy or hydroxymethyl substituents was shown to give highly potent compounds. In particular, (2S,3R)-3-(3,4-difluorophenyl)-2-(4-fluorophenyl)-4-hydroxy-N-((3S)-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)butyramide (34) demonstrated excellent in vitro potency (IC(50) = 0.06 nM). 34 could also be selectively methylated to give [(3)H]-28, which is of use in radioligand binding assays.
Asunto(s)
Benzodiazepinas/síntesis química , Benzodiazepinonas/síntesis química , Endopeptidasas/metabolismo , Inhibidores de Proteasas/síntesis química , Secretasas de la Proteína Precursora del Amiloide , Péptidos beta-Amiloides/antagonistas & inhibidores , Péptidos beta-Amiloides/biosíntesis , Ácido Aspártico Endopeptidasas , Benzodiazepinas/química , Benzodiazepinas/farmacología , Benzodiazepinonas/química , Benzodiazepinonas/farmacología , Diseño de Fármacos , Humanos , Marcaje Isotópico , Ligandos , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
A new series of benzodiazepine-containing gamma-secretase inhibitors with potential use in the treatment of Alzheimer's disease is disclosed. Structure-activity relationships of the pendant hydrocinnamate side-chain which led to the preparation of highly potent inhibitors are described.