ABSTRACT
A series of (N-benzyl-N-phenylsulfonamido)alkyl amides were developed from classic and parallel synthesis strategies. Compounds with good in vitro and in vivo γ-secretase activity were identified and described.
Subject(s)
Amides/chemistry , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Protease Inhibitors/chemistry , Sulfonamides/chemistry , Amides/chemical synthesis , Amides/metabolism , Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Peptides/metabolism , Peptide Fragments/metabolism , Protease Inhibitors/chemical synthesis , Protease Inhibitors/metabolism , Protein Binding , Structure-Activity RelationshipABSTRACT
The amyloid-beta (Abeta) peptide, which likely plays a key role in Alzheimer disease, is derived from the amyloid-beta precursor protein (APP) through consecutive proteolytic cleavages by beta-site APP-cleaving enzyme and gamma-secretase. Unexpectedly gamma-secretase inhibitors can increase the secretion of Abeta peptides under some circumstances. This "Abeta rise" phenomenon, the same inhibitor causing an increase in Abeta at low concentrations but inhibition at higher concentrations, has been widely observed. Here we show that the Abeta rise depends on the beta-secretase-derived C-terminal fragment of APP (betaCTF) or C99 levels with low levels causing rises. In contrast, the N-terminally truncated form of Abeta, known as "p3," formed by alpha-secretase cleavage, did not exhibit a rise. In addition to the Abeta rise, low betaCTF or C99 expression decreased gamma-secretase inhibitor potency. This "potency shift" may be explained by the relatively high enzyme to substrate ratio under conditions of low substrate because increased concentrations of inhibitor would be necessary to affect substrate turnover. Consistent with this hypothesis, gamma-secretase inhibitor radioligand occupancy studies showed that a high level of occupancy was correlated with inhibition of Abeta under conditions of low substrate expression. The Abeta rise was also observed in rat brain after dosing with the gamma-secretase inhibitor BMS-299897. The Abeta rise and potency shift are therefore relevant factors in the development of gamma-secretase inhibitors and can be evaluated using appropriate choices of animal and cell culture models. Hypothetical mechanisms for the Abeta rise, including the "incomplete processing" and endocytic models, are discussed.
Subject(s)
Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Peptides/metabolism , Gene Expression Regulation, Enzymologic , Animals , Brain/metabolism , Butyrates/pharmacology , Cell Line , Enzyme Inhibitors/pharmacology , Female , Humans , Hydrocarbons, Halogenated/pharmacology , Mice , Protein Binding , Protein Structure, Tertiary , Rats , Substrate SpecificityABSTRACT
Alzheimer's disease (AD) is a progressive neurodegenerative disease. Amyloid beta (Abeta) peptides are hypothesized to cause the initiation and progression of AD based on pathologic data from AD patients, genetic analysis of mutations that cause early onset forms of AD, and preclinical studies. Based on this hypothesis, beta-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) inhibitors are an attractive therapeutic approach for AD because cleavage of the APP by BACE1 is required to form Abeta. In this study, three potent BACE1 inhibitors are characterized. All three inhibitors decrease Abeta formation in cultured cells with IC(50) values less than 10 nM. Analysis of APP C-terminal fragments by immunoblotting and Abeta peptides by mass spectrometry showed that these inhibitors decreased Abeta by inhibiting BACE1. An assay for Abeta1-40 in mice was developed and used to show that these BACE1 inhibitors decreased plasma Abeta1-40, but not brain Abeta1-40, in wild-type mice. Because these BACE1 inhibitors were substrates for P-glycoprotein (P-gp), a member of the ATP-binding cassette superfamily of efflux transporters, these inhibitors were administered to P-gp knockout (KO) mice. These studies showed that all three BACE1 inhibitors decreased brain Abeta1-40 in P-gp KO mice, demonstrating that P-gp is a major limitation for development of BACE1 inhibitors to test the amyloid hypothesis. A comparison of plasma Abeta1-40 and brain Abeta1-40 dose responses for these three compounds revealed differences in relative ED(50) values, indicating that factors other than P-gp can also contribute to poor brain activity by BACE1 inhibitors.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid beta-Peptides/metabolism , Aspartic Acid Endopeptidases/antagonists & inhibitors , Brain , Enzyme Inhibitors/pharmacology , Peptide Fragments/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Amyloid Precursor Protein Secretases/physiology , Amyloid beta-Peptides/blood , Amyloid beta-Protein Precursor/genetics , Amyloid beta-Protein Precursor/metabolism , Animals , Aspartic Acid Endopeptidases/physiology , Blotting, Western , Brain/drug effects , Brain/enzymology , Brain/metabolism , Cell Line , Cell Membrane Permeability , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacokinetics , Enzyme-Linked Immunosorbent Assay , Humans , Mice , Mice, Knockout , Molecular Structure , Peptide Fragments/blood , Protein Binding , Substrate SpecificityABSTRACT
The synthesis and gamma-secretase inhibition data for a series of carbamate-appended N-alkylsulfonamides are described. Carbamate 54 was found to significantly reduce brain Abeta in transgenic mice. 54 was also found to possess markedly improved brain levels in transgenic mice compared to previously disclosed 1 and 2.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Carbamates/chemistry , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Sulfonamides/chemistry , Sulfonamides/pharmacology , Alzheimer Disease/metabolism , Amyloid beta-Peptides/antagonists & inhibitors , Amyloid beta-Peptides/metabolism , Animals , Brain/drug effects , Brain/metabolism , Mice , Mice, Transgenic , Structure-Activity RelationshipABSTRACT
The synthesis and gamma-secretase inhibition data for a series of nitrogen-appended N-alkylsulfonamides (11-47) are described. Inhibition of brain Abeta in transgenic mice was demonstrated by two of these compounds (23 and 44).
Subject(s)
Amines/chemistry , Amines/pharmacology , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Sulfonamides/chemistry , Sulfonamides/pharmacology , Alkylation , Amines/chemical synthesis , Amyloid beta-Peptides/genetics , Amyloid beta-Peptides/metabolism , Animals , Azoles/chemical synthesis , Azoles/chemistry , Azoles/pharmacology , Brain/drug effects , Brain/enzymology , Brain/metabolism , Hydrophobic and Hydrophilic Interactions , Inhibitory Concentration 50 , Mice , Mice, Transgenic , Peptide Fragments/metabolism , Protease Inhibitors/chemical synthesis , Structure-Activity Relationship , Sulfonamides/chemical synthesisABSTRACT
A series of amino-caprolactam sulfonamides were developed from a screening hit. Compounds with good in vitro and in vivo gamma-secretase activity are reported.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Caprolactam/pharmacology , Enzyme Inhibitors/pharmacology , Caprolactam/chemistry , Enzyme Inhibitors/chemistryABSTRACT
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.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid beta-Protein Precursor/biosynthesis , Brain/enzymology , Enzyme Inhibitors/pharmacology , Amyloid beta-Protein Precursor/genetics , Animals , Binding Sites , Brain/drug effects , Brain/metabolism , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemistry , Ligands , Mice , Mice, Transgenic , Molecular Structure , Radioligand Assay , Time FactorsABSTRACT
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.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Sulfonamides/chemistry , Sulfonamides/pharmacology , Amyloid beta-Peptides/metabolism , Animals , Brain/drug effects , Brain/metabolism , Mice , Mice, Transgenic , Molecular Structure , Protein BindingABSTRACT
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.
Subject(s)
Alanine/analogs & derivatives , Alzheimer Disease/enzymology , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Benzodiazepinones/pharmacology , Enzyme Inhibitors/pharmacology , Alanine/pharmacology , Alzheimer Disease/metabolism , Amyloid beta-Peptides , Animals , Mice , Mice, Transgenic , Models, MolecularABSTRACT
RATIONALE: Alzheimer's dementia (AD) patients have profound deficits in cognitive and social functions, mediated in part by a decline in cholinergic function. Acetylcholinesterase inhibitors (AChEI) are the most commonly prescribed treatment for the cognitive deficits in AD patients, but their therapeutic effects are small, and it is still not clear if they primarily affect attention, memory, or some other cognitive/behavioral functions. OBJECTIVES: The objective of the present experiments was to explore the effects of donepezil (Aricepttrade mark), an AChEI, on behavioral deficits related exclusively to cholinergic dysfunction. MATERIALS AND METHODS: The effects of donepezil were assessed in Sprague-Dawley rats with scopolamine-induced deficits in a battery of cognitive/behavioral tests. RESULTS: Scopolamine produced deficits in contextual and cued fear conditioning, the 5-choice serial reaction time test, delayed nonmatching to position, the radial arm maze, and the Morris water maze. Analyses of the pattern and size of the effects revealed that donepezil produced very large effects on scopolamine-induced deficits in psychomotor function (approximately 20-50% of the variance), moderate-sized effects on scopolamine-induced deficits in simple conditioning and attention (approximately 3-10% of the variance), but only small effects on scopolamine-induced deficits in higher cognitive functions of working memory and spatial mapping (approximately 1% of the variance). CONCLUSIONS: These results are consistent with the limited efficacy of donepezil on higher cognitive function in AD patients, and suggest that preclinical behavioral models could be used not only to determine if novel treatments have some therapeutic potential, but also to predict more precisely what the pattern and size of the effects might be.
Subject(s)
Cholinesterase Inhibitors/pharmacology , Cognition Disorders/drug therapy , Indans/pharmacology , Piperidines/pharmacology , Psychomotor Disorders/drug therapy , Animals , Attention/drug effects , Cognition Disorders/chemically induced , Conditioning, Psychological/drug effects , Donepezil , Male , Maze Learning/drug effects , Memory/drug effects , Muscarinic Antagonists , Psychomotor Disorders/chemically induced , Rats , Rats, Sprague-Dawley , ScopolamineABSTRACT
Recent findings suggest that Alzheimer's dementia may be mediated by soluble beta amyloid (Abeta) more than the deposits of aggregated, insoluble Abeta, and vulnerability to cognitive deficits after scopolamine challenge may help identify AD even in patients that are still pre-symptomatic. The objectives of the present experiments were to determine if vulnerability to cognitive deficits after scopolamine challenge is related to levels of soluble Abeta, and if levels of soluble Abeta are more closely related to cognitive deficits than levels of insoluble Abeta, even in aged, transgenic mice, after they have developed very high levels of insoluble Abeta. Aged F-344 rats and young mice over-expressing the Swedish mutation in the human amyloid precursor protein (APPsw; Tg2576+) had elevated levels of soluble Abeta, and were more vulnerable to scopolamine challenge in the Morris water maze (MWM), relative to young rats and Tg2576- mice; but, among individual animals, higher levels of soluble Abeta were not correlated with vulnerability to scopolamine. On the other hand, in aged Tg2576+ mice, cognitive deficits were related to levels of soluble Abeta, not insoluble Abeta, despite the fact that the levels of insoluble Abeta were thousands of times higher than the levels of soluble Abeta. The results of the present experiments suggest that vulnerability to cognitive deficits after scopolamine challenge is not related to elevated levels of soluble Abeta, but that high levels of soluble Abeta are more closely correlated with cognitive deficits than the amount insoluble Abeta, even after large amounts of aggregated, insoluble Abeta have been deposited.
Subject(s)
Aging/metabolism , Amyloid beta-Peptides/metabolism , Brain/metabolism , Maze Learning/physiology , Acoustic Stimulation , Amyloid beta-Peptides/chemistry , Amyloid beta-Protein Precursor/genetics , Analysis of Variance , Animals , Conditioning, Classical/physiology , Fear , Male , Maze Learning/drug effects , Mice , Mice, Transgenic , Muscarinic Antagonists/pharmacology , Polymers/chemistry , Rats , Rats, Inbred F344 , Reflex, Startle/physiology , Scopolamine/pharmacology , SolubilityABSTRACT
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.
Subject(s)
Brain/drug effects , Endopeptidases/metabolism , Protease Inhibitors/pharmacology , Alzheimer Disease/enzymology , Amides/chemistry , Amyloid Precursor Protein Secretases , Animals , Aspartic Acid Endopeptidases , Benzodiazepines/chemical synthesis , Benzodiazepines/pharmacology , Brain/metabolism , Cell Line , Drug Design , Inhibitory Concentration 50 , Ketones/chemical synthesis , Ketones/pharmacology , Mice , Molecular Conformation , Molecular Mimicry , Protease Inhibitors/chemical synthesis , Protein BindingABSTRACT
Antagonists of serotonin 6 (5-HT6) receptors have been reported to enhance cognition in animal models of learning, although this finding has not been universal. We have assessed the therapeutic potential of the specific 5-HT6 receptor antagonists 4-amino-N-(2,6-bis-methylamino-pyrimidin-4-yl)-benzenesulfonamide (Ro 04-6790) and 5-chloro-N-(4-methoxy-3-piperazin-1-yl-phenyl)-3-methyl-2-benzothiophenesulfonamide (SB-271046) in rodent models of cognitive function. Although mice express the 5-HT6 receptor and the function of this receptor has been investigated in mice, all reports of activity with 5-HT6 receptor antagonists have used rat models. In the present study, receptor binding revealed that the pharmacological properties of the mouse receptor are different from the rat and human receptor: Ro 04-6790 does not bind to the mouse 5-HT6 receptor, so all in vivo testing included in the present report was conducted in rats. We replicated previous reports that 5-HT6 receptor antagonists produce a stretching syndrome previously shown to be mediated through cholinergic mechanisms, but Ro 04-6790 and SB-271046 failed to attenuate scopolamine-induced deficits in a test of contextual fear conditioning. We also failed to replicate the significant effects reported previously in both an autoshaping task and in a version of the Morris water maze. The results of our experiments are not consistent with previous reports that suggested that 5-HT6 antagonists might have therapeutic potential for cognitive disorders.
