ABSTRACT
Herein, we describe our strategy to design metabolically stable γ-secretase inhibitors which are selective for inhibition of Aß generation over Notch. We highlight our synthetic strategy to incorporate diversity and chirality. Compounds 30 (ELND006) and 34 (ELND007) both entered human clinical trials. The in vitro and in vivo characteristics for these two compounds are described. A comparison of inhibition of Aß generation in vivo between 30, 34, Semagacestat 41, Begacestat 42, and Avagacestat 43 in mice is made. 30 lowered Aß in the CSF of healthy human volunteers.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid beta-Peptides/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Heterocyclic Compounds, 3-Ring/pharmacology , Pyrazoles/pharmacology , Quinolines/pharmacology , Receptors, Notch/antagonists & inhibitors , Sulfonamides/pharmacology , Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Peptides/cerebrospinal fluid , Amyloid beta-Peptides/metabolism , Animals , Area Under Curve , Basic Helix-Loop-Helix Transcription Factors/genetics , Dogs , Dose-Response Relationship, Drug , Drug Design , Drug Stability , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacokinetics , Gene Expression/drug effects , Heterocyclic Compounds, 3-Ring/chemistry , Homeodomain Proteins/genetics , Humans , Male , Mice , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Models, Chemical , Molecular Structure , Pyrazoles/chemical synthesis , Pyrazoles/pharmacokinetics , Quinolines/chemical synthesis , Quinolines/pharmacokinetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Notch/metabolism , Structure-Activity Relationship , Sulfonamides/chemistry , Time Factors , Transcription Factor HES-1ABSTRACT
INTRODUCTION: Inhibition of gamma-secretase presents a direct target for lowering Aß production in the brain as a therapy for Alzheimer's disease (AD). However, gamma-secretase is known to process multiple substrates in addition to amyloid precursor protein (APP), most notably Notch, which has limited clinical development of inhibitors targeting this enzyme. It has been postulated that APP substrate selective inhibitors of gamma-secretase would be preferable to non-selective inhibitors from a safety perspective for AD therapy. METHODS: In vitro assays monitoring inhibitor potencies at APP γ-site cleavage (equivalent to Aß40), and Notch ε-site cleavage, in conjunction with a single cell assay to simultaneously monitor selectivity for inhibition of Aß production vs. Notch signaling were developed to discover APP selective gamma-secretase inhibitors. In vivo efficacy for acute reduction of brain Aß was determined in the PDAPP transgene model of AD, as well as in wild-type FVB strain mice. In vivo selectivity was determined following seven days x twice per day (b.i.d.) treatment with 15 mg/kg/dose to 1,000 mg/kg/dose ELN475516, and monitoring brain Aß reduction vs. Notch signaling endpoints in periphery. RESULTS: The APP selective gamma-secretase inhibitors ELN318463 and ELN475516 reported here behave as classic gamma-secretase inhibitors, demonstrate 75- to 120-fold selectivity for inhibiting Aß production compared with Notch signaling in cells, and displace an active site directed inhibitor at very high concentrations only in the presence of substrate. ELN318463 demonstrated discordant efficacy for reduction of brain Aß in the PDAPP compared with wild-type FVB, not observed with ELN475516. Improved in vivo safety of ELN475516 was demonstrated in the 7d repeat dose study in wild-type mice, where a 33% reduction of brain Aß was observed in mice terminated three hours post last dose at the lowest dose of inhibitor tested. No overt in-life or post-mortem indications of systemic toxicity, nor RNA and histological end-points indicative of toxicity attributable to inhibition of Notch signaling were observed at any dose tested. CONCLUSIONS: The discordant in vivo activity of ELN318463 suggests that the potency of gamma-secretase inhibitors in AD transgenic mice should be corroborated in wild-type mice. The discovery of ELN475516 demonstrates that it is possible to develop APP selective gamma-secretase inhibitors with potential for treatment for AD.
ABSTRACT
Utilizing a pharmacophore hypothesis, previously described gamma-secretase inhibiting HTS hits were evolved into novel tricyclic sulfonamide-pyrazoles, with high in vitro potency, good brain penetration, low metabolic stability, and high clearance.
Subject(s)
Alzheimer Disease/drug therapy , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid Precursor Protein Secretases/metabolism , Pyrazoles/pharmacology , Sulfonamides/pharmacology , Animals , Crystallography, X-Ray , Humans , Inhibitory Concentration 50 , Models, Molecular , Pyrazoles/chemistry , Pyrazoles/pharmacokinetics , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship , Sulfonamides/chemistry , Sulfonamides/pharmacokineticsABSTRACT
In this Letter, we report our strategy to design potent and metabolically stable gamma-secretase inhibitors that are efficacious in reducing the cortical Abetax-40 levels in FVB mice via a single PO dose.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Protease Inhibitors/chemical synthesis , Pyrazoles/chemistry , Quinolines/chemistry , Sulfonamides/chemistry , Administration, Oral , Amyloid Precursor Protein Secretases/metabolism , Animals , Drug Design , Mice , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Quinolines/chemical synthesis , Quinolines/pharmacology , Structure-Activity RelationshipABSTRACT
A comprehensive, unbiased inventory of synuclein forms present in Lewy bodies from patients with dementia with Lewy bodies was carried out using two-dimensional immunoblot analysis, novel sandwich enzyme-linked immunosorbent assays with modification-specific synuclein antibodies, and mass spectroscopy. The predominant modification of alpha-synuclein in Lewy bodies is a single phosphorylation at Ser-129. In addition, there is a set of characteristic modifications that are present to a lesser extent, including ubiquitination at Lys residues 12, 21, and 23 and specific truncations at Asp-115, Asp-119, Asn-122, Tyr-133, and Asp-135. No other modifications are detectable by tandem mass spectrometry mapping, except for a ubiquitous N-terminal acetylation. Small amounts of Ser-129 phosphorylated and Asp-119-truncated alpha-synuclein are present in the soluble fraction of both normal and disease brains, suggesting that these Lewy body-associated forms are produced during normal metabolism of alpha-synuclein. In contrast, ubiquitination is only detected in Lewy bodies and is primarily present on phosphorylated synuclein; it therefore likely occurs after phosphorylated synuclein has deposited into Lewy bodies. This invariant pattern of specific phosphorylation, truncation, and ubiquitination is also present in the detergent-insoluble fraction of brain from patients with familial Parkinson's disease (synuclein A53T mutation) as well as multiple system atrophy, suggesting a common pathogenic pathway for both genetic and sporadic Lewy body diseases. These observations are most consistent with a model in which preferential accumulation of normally produced Ser-129 phosphorylated alpha-synuclein is the key event responsible for the formation of Lewy bodies in various Lewy body diseases.
