RESUMO
There is a substantial body of evidence indicating that beta-amyloid peptides (Abeta) are critical factors in the onset and development of Alzheimer's disease (AD). One strategy for combating AD is to reduce or eliminate the production of Abeta through inhibition of the gamma-secretase enzyme, which cleaves Abeta from the amyloid precursor protein (APP). We demonstrate here that chronic treatment for 3 months with 3 mg/kg of the potent, orally bioavailable and brain-penetrant gamma-secretase inhibitor N-[cis-4-[(4-chlorophenyl)-sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]-1,1,1-trifluoromethanesulfonamide (MRK-560) attenuates the appearance of amyloid plaques in the Tg2576 mouse. These reductions in plaques were also accompanied by a decrease in the level of reactive gliosis. The morphometric and histological measures agreed with biochemical analysis of Abeta(40) and Abeta(42) in the cortex. Interestingly, the volume of the plaques across treatment groups did not change, indicating that reducing Abeta levels does not significantly alter deposit growth once initiated. Furthermore, we demonstrate that these beneficial effects can be achieved without causing histopathological changes in the ileum, spleen, or thymus as a consequence of blockade of the processing of alternative substrates, such as the Notch family of receptors. This indicates that in vivo a therapeutic window between these substrates seems possible--a key concern in the development of this approach to AD. An understanding of the mechanisms whereby MRK-560 shows differentiation between the APP and Notch proteolytic pathway of gamma-secretase should provide the basis for the next generation of gamma-secretase inhibitors.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Peptídeos beta-Amiloides/metabolismo , Inibidores de Proteases/farmacologia , Sulfonamidas/farmacologia , Sulfonas/farmacologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/antagonistas & inibidores , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Feminino , Masculino , Camundongos , Receptores Notch/antagonistas & inibidores , Receptores Notch/metabolismoRESUMO
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.
Assuntos
Secretases da Proteína Precursora do Amiloide/química , Secretases da Proteína Precursora do Amiloide/metabolismo , Anti-Inflamatórios não Esteroides/farmacologia , Ligação Competitiva , Bioquímica/métodos , Domínio Catalítico , Linhagem Celular , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Humanos , Cinética , Ligantes , Modelos Químicos , Ligação Proteica , Sulindaco/análogos & derivados , Sulindaco/farmacologiaRESUMO
c-Jun N-terminal kinase (JNK) activation is linked to the aberrant cell death in several neurodegenerative disorders, including Parkinson's and Alzheimer's disease. The sequence similarity among the JNK isoforms and fellow MAP kinase family member p38 has rendered the challenge of producing JNK3-specific inhibitors difficult. Using the crystal structure of JNK3 complexed with JNK inhibitors, potential compound-interacting amino acid residues were mutated to the corresponding residues in p38. The effects of these mutations on the kinetic parameters with three compounds were examined: a JNK3- (vs. p38-) selective inhibitor (SP 600125); a p38-selective inhibitor (Merck Z); and a potent combined JNK3 and p38 inhibitor (Merck Y). The data confirm the role of the JNK3 residues Ile-70 and Val-196 in both inhibitor and ATP-binding. Remarkably, the Ile-70-Val and Val-196-Ala mutations caused an increase and decrease, respectively, in the binding affinity of the p38-specific compound, Merck Z, of 10-fold. The Ile-70-Val effect may be due to the increased capacity of the active site to accommodate Merck Z, whereas the Val-196-Ala mutant may induce an unfavourable conformational change. Conservative mutations of the Asn-152 and Gln-155 residues inactivated the JNK3 enzyme, possibly interfering with protein folding in a critical hinge region of the protein.
Assuntos
Trifosfato de Adenosina/química , Proteína Quinase 10 Ativada por Mitógeno/química , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Alanina/química , Aminoácidos/química , Animais , Antracenos/farmacologia , Sítios de Ligação , Biotinilação , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Transferência Ressonante de Energia de Fluorescência , Glutationa/metabolismo , Humanos , Imidazóis/farmacologia , Concentração Inibidora 50 , Isoleucina/química , Cinética , Sistema de Sinalização das MAP Quinases , Modelos Biológicos , Modelos Químicos , Modelos Moleculares , Mutação , Fosforilação , Mutação Puntual , Ligação Proteica , Dobramento de Proteína , Pirimidinas/farmacologia , Valina/químicaRESUMO
The efficacy of gamma-secretase inhibitors in vivo has, to date, been generally assessed in transgenic mouse models expressing increased levels of amyloid-beta (Abeta) peptide thereby allowing the detection of changes in Abeta production. However, it is not clear whether the in vivo potency of gamma-secretase inhibitors is independent of the level of amyloid precursor protein expression. In other words, does a gamma-secretase inhibitor have the same effect in nontransgenic physiological animals versus transgenic overexpressing animals? In the present study, an immunoassay has been developed which can detect Abeta(40) in the rat brain, where concentrations are much lower than those seen in transgenic mice such as Tg2576 (c. 0.7 and 25 nM, respectively) and in cerebrospinal fluid (CSF, c. 0.3 nM). Using this immunoassay, the effects of the gamma-secretase inhibitor LY-411575 [N(2)-[(2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl]-N(1)-[(7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl]-L-alaninamide] were assessed and robust dose-dependent reductions in rat brain and CSF Abeta(40) levels were observed with ID(50) values of 1.3 mg/kg for both brain and CSF. These values were comparable with those calculated for LY-411575 in transgenic mice. Time course experiments using LY-411575 demonstrated comparable temporal reductions in rat brain and CSF Abeta(40), further suggesting these two pools of Abeta are related. Accordingly, when all the data for the dose-response curve and time course were correlated, a strong association was observed between the brain and CSF Abeta(40) levels. These data demonstrate the utility of the rat as a novel approach for assessing the effects of gamma-secretase inhibitors on central nervous system Abeta(40) levels in vivo.
Assuntos
Alanina/análogos & derivados , Alanina/farmacologia , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Azepinas/farmacologia , Encéfalo/efeitos dos fármacos , Endopeptidases/metabolismo , Fragmentos de Peptídeos/líquido cefalorraquidiano , Inibidores de Proteases/farmacologia , Secretases da Proteína Precursora do Amiloide , Peptídeos beta-Amiloides/metabolismo , Animais , Ácido Aspártico Endopeptidases , Encéfalo/enzimologia , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Fragmentos de Peptídeos/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
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.