RESUMO
The enzyme gamma-secretase has long been considered a potential pharmaceutical target for Alzheimer disease. Presenilin (the catalytic subunit of gamma-secretase) and signal peptide peptidase (SPP) are related transmembrane aspartyl proteases that cleave transmembrane substrates. SPP and gamma-secretase are pharmacologically similar in that they are targeted by many of the same small molecules, including transition state analogs, non-transition state inhibitors, and amyloid beta-peptide modulators. One difference between presenilin and SPP is that the proteolytic activity of presenilin functions only within a multisubunit complex, whereas SPP requires no additional protein cofactors for activity. In this study, gamma-secretase inhibitor radioligands were used to evaluate SPP and gamma-secretase inhibitor binding pharmacology. We found that the SPP enzyme exhibited distinct binding sites for transition state analogs, non-transition state inhibitors, and the nonsteroidal anti-inflammatory drug sulindac sulfide, analogous to those reported previously for gamma-secretase. In the course of this study, cultured cells were found to contain an abundance of SPP binding activity, most likely contributed by several of the SPP family proteins. The number of SPP binding sites was in excess of gamma-secretase binding sites, making it essential to use selective radioligands for evaluation of gamma-secretase binding under these conditions. This study provides further support for the idea that SPP is a useful model of inhibitory mechanisms and structure in the SPP/presenilin protein family.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Anti-Inflamatórios não Esteroides/farmacologia , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Presenilinas/antagonistas & inibidores , Inibidores de Proteases/farmacologia , Sulindaco/análogos & derivados , Doença de Alzheimer/enzimologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Sítios de Ligação , Domínio Catalítico , Linhagem Celular , Humanos , Ligantes , Modelos Moleculares , Presenilinas/metabolismo , Sulindaco/farmacologiaRESUMO
Reduction of brain beta-amyloid peptide (Abeta) synthesis by gamma-secretase inhibitors is a promising approach for the treatment of Alzheimer's disease. However, measurement of central pharmacodynamic effects in the Alzheimer's disease patient will be a challenge. Determination of drug occupancy may facilitate the analysis of efficacy of gamma-secretase inhibitors in a clinical setting. In this study, the relationship of gamma-secretase site occupancy and brain Abeta40 reduction by gamma-secretase inhibitors was examined in Tg2576 mice. [3H](2R,3S)-2-Isobutyl-N1-((S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)-3-propylsuccinamide (IN973) was used as a gamma-secretase radioligand, since it has been shown to bind to gamma-secretase in rat, rhesus, and human brains with high affinity and specificity. We extended these findings by showing that [3H]IN973 bound to gamma-secretase in Tg2576 brains with an affinity, specificity, and regional localization very similar to the other species. To quantify gamma-secretase occupancy by gamma-secretase inhibitors, an ex vivo binding assay was developed using [3H]IN973 and frozen brain sections from drug-treated mice. Gamma-secretase occupancy and brain Abeta40 reduction were found to be highly correlated in animals dosed with either 2-[(1R)-1-[[4-chlorophenyl)-sulfonyl](2,5-difluorophenyl)amino] ethyl]-5-fluoro-benzenepropanoic acid (BMS-299897) or (S)-2-((S)-2-(3,5-difluorophenyl)-2-hydroxyacetamido)-N-((S,Z)-3-methyl-4-oxo-4,5-dihydro-3H-benzo[d][1,2]diazepin-5-yl)propanamide (BMS-433796) over a wide range of doses and times postdose, with the exception of the earliest times postdose. This lag in Abeta40 response to gamma-secretase inhibition is probably related to the delayed clearance of previously produced Abeta40. The excellent correlation between brain Abeta40 and gamma-secretase occupancy suggests that a positron emission tomography ligand for gamma-secretase could be a valuable biomarker to determine whether gamma-secretase inhibitors bind to their target in humans.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Precursor de Proteína beta-Amiloide/biossíntese , Encéfalo/enzimologia , Inibidores Enzimáticos/farmacologia , Precursor de Proteína beta-Amiloide/genética , Animais , Sítios de Ligação , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Ligantes , Camundongos , Camundongos Transgênicos , Estrutura Molecular , Ensaio Radioligante , Fatores de TempoRESUMO
A series of N-alkylbenzenesulfonamides were developed from a high throughput screening hit. Classic and parallel synthesis strategies were employed to produce compounds with good in vitro and in vivo gamma-secretase activity.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Sulfonamidas/química , Sulfonamidas/farmacologia , Peptídeos beta-Amiloides/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Camundongos , Camundongos Transgênicos , Estrutura Molecular , Ligação ProteicaRESUMO
We report on the design of benzodiazepinones as peptidomimetics at the carboxy terminus of hydroxyamides. Structure-activity relationships of diazepinones were investigated and orally active gamma-secretase inhibitors were synthesized. Active metabolites contributing to Abeta reduction were identified by analysis of plasma samples from Tg2576 mice. In particular, (S)-2-((S)-2-(3,5-difluorophenyl)-2-hydroxyacetamido)-N-((S,Z)-3-methyl-4-oxo-4,5-dihydro-3H-benzo[d][1,2]diazepin-5-yl)propanamide (BMS-433796) was identified with an acceptable pharmacodynamic and pharmacokinetic profile. Chronic dosing of BMS-433796 in Tg2576 mice suggested a narrow therapeutic window and Notch-mediated toxicity at higher doses.
Assuntos
Alanina/análogos & derivados , Doença de Alzheimer/enzimologia , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Benzodiazepinonas/farmacologia , Inibidores Enzimáticos/farmacologia , Alanina/farmacologia , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides , Animais , Camundongos , Camundongos Transgênicos , Modelos MolecularesRESUMO
2,3-Benzodiazepin-1,4-diones were designed as peptidomimetics at the carboxy terminus of hydroxyamides. Inhibition of brain Abeta production was improved by one of the compounds containing constrained modification.
Assuntos
Encéfalo/efeitos dos fármacos , Endopeptidases/metabolismo , Inibidores de Proteases/farmacologia , Doença de Alzheimer/enzimologia , Amidas/química , Secretases da Proteína Precursora do Amiloide , Animais , Ácido Aspártico Endopeptidases , Benzodiazepinas/síntese química , Benzodiazepinas/farmacologia , Encéfalo/metabolismo , Linhagem Celular , Desenho de Fármacos , Concentração Inibidora 50 , Cetonas/síntese química , Cetonas/farmacologia , Camundongos , Conformação Molecular , Mimetismo Molecular , Inibidores de Proteases/síntese química , Ligação ProteicaRESUMO
Using a cell-based assay, we have identified optimal residues and key recognition elements necessary for inhibition of gamma-secretase. An (S)-hydroxy group or 3,5-difluorophenylacetyl group at the amino terminus and N-methyltertiary amide moiety at the carboxy terminus provided potent gamma-secretase inhibitors with an IC(50) <10 nM.