Restricted Location of PSEN2/γ-Secretase Determines Substrate Specificity and Generates an Intracellular Aß Pool.

γ-Secretases are a family of intramembrane-cleaving proteases involved in various signaling pathways and diseases, including Alzheimer's disease (AD). Cells co-express differing γ-secretase complexes, including two homologous presenilins (PSENs). We examined the significance of this heterogeneity and identified a unique motif in PSEN2 that directs this γ-secretase to late endosomes/lysosomes via a phosphorylation-dependent interaction with the AP-1 adaptor complex. Accordingly, PSEN2 selectively cleaves late endosomal/lysosomal localized substrates and generates the prominent pool of intracellular Aß that contains longer Aß; familial AD (FAD)-associated mutations in PSEN2 increased the levels of longer Aß further. Moreover, a subset of FAD mutants in PSEN1, normally more broadly distributed in the cell, phenocopies PSEN2 and shifts its localization to late endosomes/lysosomes. Thus, localization of γ-secretases determines substrate specificity, while FAD-causing mutations strongly enhance accumulation of aggregation-prone Aß42 in intracellular acidic compartments. The findings reveal potentially important roles for specific intracellular, localized reactions contributing to AD pathogenesis.

Crystal structure reveals conservation of amyloid-ß conformation recognized by 3D6 following humanization to bapineuzumab.

INTRODUCTION: Immunotherapy targeting amyloid-ß peptide is under active clinical investigation for treatment of Alzheimer's disease (AD). Among the hypotheses being investigated for impact on clinical outcome are the preferred epitope or conformation of amyloid-ß to target for treatment, and the mechanism of action underlying immunotherapy. Bapineuzumab (humanized 3D6), a neo-epitope specific antibody recognizing amyloid-ß1-5 with strong preference for an exposed Asp residue at the N-terminus of the peptide, has undergone advanced clinical testing for treatment of AD. METHODS: To gain further insight into the epitope conformation, we interrogated structural details of amino-terminal epitopes in amyloid-ß using x-ray crystallography of 3D6Fab:amyloid-ß complexes. Humanization of 3D6 was carried out using standard procedures integrating recombinant methods, sequence informatics, and homology modeling predictions to identify important mouse framework residues for retention in the finished humanized product. RESULTS: Here we report the crystal structure of a recombinant Fab fragment of 3D6 in complex with amyloid-ß1-7 solved at 2.0 Å resolution. The N-terminus of amyloid-ß is bound to 3D6 as a 310 helix. The amino-terminal Asp residue is buried deepest in the antibody binding pocket, with the Cß atom of residue 6 visible at the entrance to the binding pocket near the surface of the antibody. We further evaluate homology model based predictions used to guide humanization of 3D6 to bapineuzumab, with actual structure of the Fab. The structure of the Fab:amyloid-ß complex validates design of the humanized antibody, and confirms the amyloid-ß epitope recognized by 3D6 as previously mapped by ELISA. CONCLUSIONS: The conformation of amyloid-ß antigen recognized by 3D6 is novel and distinct from other antibodies recognizing N-terminal epitopes. Our result provides the first report demonstrating structural conservation of antigen contact residues, and conformation of antigen recognized, between the parent murine antibody and its humanized version.

Discovery of a novel [3.2.1] benzo fused bicyclic sulfonamide-pyrazoles as potent, selective and efficacious γ-secretase inhibitors.

Structure-activity relationship (SAR) of a novel, potent and metabolically stable series of benzo [3.2.1] bicyclic sulfonamide-pyrazoles as γ-secretase inhibitors are described. Compounds that are efficacious in reducing the cortical Aßx-40 levels in FVB mice via oral dose, as well as those with high selectivity over Notch, are highlighted.

Evidence that enzyme processivity mediates differential Aß production by PS1 and PS2.

The γ-secretase complex cleaves the carboxy-terminal 99 residue (C99) fragment of the amyloid precursor protein (APP) to generate the amyloid-ß (Aß) peptide. The catalytic activity of this complex is mediated either by the presenilin- 1 (PS1) or the presenilin-2 (PS2) subunit. In vitro and in vivo studies have demonstrated that PS1-containing complexes generate more total Aß product than PS2-containing complexes, indicating greater cleavage activity by PS1- containing γ-secretase complexes at the APP γ-site. However, it remains untested whether γ-secretase cleavage at the APP -site, which precedes γ-site cleavage and produces the physiologically active APP intracellular domain (AICD), follows the same rule. Using a novel Swedish APP-GVP substrate to facilitate the parallel detection of Aß and AICD products from PS1-/-/PS2-/- cells co-transfected with either PS1 or PS2, we observed that while PS1 generates more total Aß product than PS2, consistent with published reports, PS1 and PS2 unexpectedly generate equal amounts of AICD product. We also observed that PS1 and PS2 produce equivalent amounts of Notch intracellular domain (NICD), indicating equal cleavage activity at the Notch S3-site (the corollary of the APP -site). Our findings suggest that processivity differences between PS1 and PS2 underlie the differential production of Aß peptide. Taken together these findings offer novel insights into γ- secretase biology and have important implications for therapeutically targeting γ-secretase.

Treatment strategies targeting amyloid ß-protein.

With the advent of the key discovery in the mid-1980s that the amyloid ß-protein (Aß) is the core constituent of the amyloid plaque pathology found in Alzheimer disease (AD), an intensive effort has been underway to attempt to mitigate its role in the hope of treating the disease. This effort fully matured when it was clarified that the Aß is a normal product of cleavage of the amyloid precursor protein, and well-defined proteases for this process were identified. Further therapeutic options have been developed around the concept of anti-Aß aggregation inhibitors and the surprising finding that immunization with Aß itself leads to reduction of pathology in animal models of the disease. Here we review the progress in this field toward the goal of targeting Aß for treatment and prevention of AD and identify some of the major challenges for the future of this area of medicine.

Design, synthesis and structure-activity relationship of novel [3.3.1] bicyclic sulfonamide-pyrazoles as potent γ-secretase inhibitors.

The structure-activity relationship (SAR) of a novel, potent and metabolically stable series of sulfonamide-pyrazoles that attenuate ß-amyloid peptide synthesis via γ-secretase inhibition is detailed herein. Sulfonamide-pyrazoles that are efficacious in reducing the cortical Aßx-40 levels in FVB mice via a single PO dose, as well as sulfonamide-pyrazoles that exhibit selectivity for inhibition of APP versus Notch processing by γ-secretase, are highlighted.

Accelerating drug discovery for Alzheimer's disease: best practices for preclinical animal studies.

Animal models have contributed significantly to our understanding of the underlying biological mechanisms of Alzheimer's disease (AD). As a result, over 300 interventions have been investigated and reported to mitigate pathological phenotypes or improve behavior in AD animal models or both. To date, however, very few of these findings have resulted in target validation in humans or successful translation to disease-modifying therapies. Challenges in translating preclinical studies to clinical trials include the inability of animal models to recapitulate the human disease, variations in breeding and colony maintenance, lack of standards in design, conduct and analysis of animal trials, and publication bias due to under-reporting of negative results in the scientific literature. The quality of animal model research on novel therapeutics can be improved by bringing the rigor of human clinical trials to animal studies. Research communities in several disease areas have developed recommendations for the conduct and reporting of preclinical studies in order to increase their validity, reproducibility, and predictive value. To address these issues in the AD community, the Alzheimer's Drug Discovery Foundation partnered with Charles River Discovery Services (Morrisville, NC, USA) and Cerebricon Ltd. (Kuopio, Finland) to convene an expert advisory panel of academic, industry, and government scientists to make recommendations on best practices for animal studies testing investigational AD therapies. The panel produced recommendations regarding the measurement, analysis, and reporting of relevant AD targets, th choice of animal model, quality control measures for breeding and colony maintenance, and preclinical animal study design. Major considerations to incorporate into preclinical study design include a priori hypotheses, pharmacokinetics-pharmacodynamics studies prior to proof-of-concept testing, biomarker measurements, sample size determination, and power analysis. The panel also recommended distinguishing between pilot 'exploratory' animal studies and more extensive 'therapeutic' studies to guide interpretation. Finally, the panel proposed infrastructure and resource development, such as the establishment of a public data repository in which both positive animal studies and negative ones could be reported. By promoting best practices, these recommendations can improve the methodological quality and predictive value of AD animal studies and make the translation to human clinical trials more efficient and reliable.

Lysine 624 of the amyloid precursor protein (APP) is a critical determinant of amyloid ß peptide length: support for a sequential model of γ-secretase intramembrane proteolysis and regulation by the amyloid ß precursor protein (APP) juxtamembrane region.

γ-Secretase is a multiprotein intramembrane cleaving aspartyl protease (I-CLiP) that catalyzes the final cleavage of the amyloid ß precursor protein (APP) to release the amyloid ß peptide (Aß). Aß is the primary component of senile plaques in Alzheimer's disease (AD), and its mechanism of production has been studied intensely. γ-Secretase executes multiple cleavages within the transmembrane domain of APP, with cleavages producing Aß and the APP intracellular domain (AICD), referred to as γ and ε, respectively. The heterogeneous nature of the γ cleavage that produces various Aß peptides is highly relevant to AD, as increased production of Aß 1-42 is genetically and biochemically linked to the development of AD. We have identified an amino acid in the juxtamembrane region of APP, lysine 624, on the basis of APP695 numbering (position 28 relative to Aß) that plays a critical role in determining the final length of Aß peptides released by γ-secretase. Mutation of this lysine to alanine (K28A) shifts the primary site of γ-secretase cleavage from 1-40 to 1-33 without significant changes to ε cleavage. These results further support a model where ε cleavage occurs first, followed by sequential proteolysis of the remaining transmembrane fragment, but extend these observations by demonstrating that charged residues at the luminal boundary of the APP transmembrane domain limit processivity of γ-secretase.

Amino-caprolactam γ-secretase inhibitors showing potential for the treatment of Alzheimer's disease.

Herein we describe the structure-activity relationship (SAR) of amino-caprolactam analogs derived from amino-caprolactam benzene sulfonamide 1, highlighting affects on the potency of γ-secretase inhibition, selectivity for the inhibition of APP versus Notch processing by γ-secretase and selected pharmakokinetic properties. Amino-caprolactams that are efficacious in reducing the cortical Aß(x-40) levels in FVB mice via a single 100 mpk IP dose are highlighted.

Amyloid precursor protein selective gamma-secretase inhibitors for treatment of Alzheimer's disease.

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.

Discovery of a novel sulfonamide-pyrazolopiperidine series as potent and efficacious gamma-secretase inhibitors (Part II).

Significant improvement in metabolic stability on the pyrazolopiperidine scaffold over the original series were achieved and this stability improvement translated in an improved in vivo efficacy.

Discovery of sulfonamide-pyrazole gamma-secretase inhibitors.

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.

Discovery of a novel sulfonamide-pyrazolopiperidine series as potent and efficacious gamma-secretase inhibitors.

Discovery of a series of pyrazolopiperidine sulfonamide based gamma-secretase inhibitors and its SAR evolution is described. Significant increases in APP potency on the pyrazolopiperidine scaffold over the original N-bicyclic sulfonamide scaffold were achieved and this potency increase translated in an improved in vivo efficacy.

Structural correlates of antibodies associated with acute reversal of amyloid beta-related behavioral deficits in a mouse model of Alzheimer disease.

Immunotherapy targeting of amyloid beta (Abeta) peptide in transgenic mouse models of Alzheimer disease (AD) has been widely demonstrated to resolve amyloid deposition as well as associated neuronal, glial, and inflammatory pathologies. These successes have provided the basis for ongoing clinical trials of immunotherapy for treatment of AD in humans. Acute as well as chronic Abeta-targeted immunotherapy has also been demonstrated to reverse Abeta-related behavioral deficits assessing memory in AD transgenic mouse models. We observe that three antibodies targeting the same linear epitope of Abeta, Abeta(3-7), differ in their ability to reverse contextual fear deficits in Tg2576 mice in an acute testing paradigm. Reversal of contextual fear deficit by the antibodies does not correlate with in vitro recognition of Abeta in a consistent or correlative manner. To better define differences in antigen recognition at the atomic level, we determined crystal structures of Fab fragments in complex with Abeta. The conformation of the Abeta peptide recognized by all three antibodies was highly related and is also remarkably similar to that observed in independently reported Abeta:antibody crystal structures. Sequence and structural differences between the antibodies, particularly in CDR3 of the heavy chain variable region, are proposed to account for differing in vivo properties of the antibodies under study. These findings provide a structural basis for immunotherapeutic strategies targeting Abeta species postulated to underlie cognitive deficits in AD.

N-Bridged bicyclic sulfonamides as inhibitors of gamma-secretase.

The structural modification of a series of [3.3.1] bicyclic sulfonamide based gamma-secretase inhibitors is described. Appropriate substitution on the bicyclic scaffold provides a significant increase in the metabolic stability of the compounds resulting in an improved in vivo metabolic profile.

Design, synthesis, and structure-activity relationship of novel orally efficacious pyrazole/sulfonamide based dihydroquinoline gamma-secretase inhibitors.

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.

Polo-like kinase 2 (PLK2) phosphorylates alpha-synuclein at serine 129 in central nervous system.

Several neurological diseases, including Parkinson disease and dementia with Lewy bodies, are characterized by the accumulation of alpha-synuclein phosphorylated at Ser-129 (p-Ser-129). The kinase or kinases responsible for this phosphorylation have been the subject of intense investigation. Here we submit evidence that polo-like kinase 2 (PLK2, also known as serum-inducible kinase or SNK) is a principle contributor to alpha-synuclein phosphorylation at Ser-129 in neurons. PLK2 directly phosphorylates alpha-synuclein at Ser-129 in an in vitro biochemical assay. Inhibitors of PLK kinases inhibited alpha-synuclein phosphorylation both in primary cortical cell cultures and in mouse brain in vivo. Finally, specific knockdown of PLK2 expression by transduction with short hairpin RNA constructs or by knock-out of the plk2 gene reduced p-Ser-129 levels. These results indicate that PLK2 plays a critical role in alpha-synuclein phosphorylation in central nervous system.

Immunotherapy reduces vascular amyloid-beta in PDAPP mice.

In addition to parenchymal amyloid-beta (Abeta) plaques, Alzheimer's disease (AD) is characterized by Abeta in the cerebral vasculature [cerebral amyloid angiopathy (CAA)] in the majority of patients. Recent studies investigating vascular Abeta (VAbeta) in amyloid precursor protein transgenic mice have suggested that passive immunization with anti-Abeta antibodies may clear parenchymal amyloid but increase VAbeta and the incidence of microhemorrhage. However, the influences of antibody specificity and exposure levels on VAbeta and microhemorrhage rates have not been well established, nor has any clear causal relationship been identified. This report examines the effects of chronic, passive immunization on VAbeta and microhemorrhage in PDAPP mice by comparing antibodies with different Abeta epitopes (3D6, Abeta(1-5); 266, Abeta(16-23)) and performing a 3D6 dose-response study. VAbeta and microhemorrhage were assessed using concomitant Abeta immunohistochemistry and hemosiderin detection. 3D6 prevented or cleared VAbeta in a dose-dependent manner, whereas 266 was without effect. Essentially complete absence of VAbeta was observed at the highest 3D6 dose, whereas altered morphology suggestive of ongoing clearance was seen at lower doses. The incidence of microhemorrhage was increased in the high-dose 3D6 group and limited to focal, perivascular sites. These colocalized with Abeta deposits having altered morphology and apparent clearance in the lower-dose 3D6 group. Our results suggest that passive immunization can reduce VAbeta levels, and modulating antibody dose can significantly mitigate the incidence of microhemorrhage while still preventing or reducing VAbeta. These observations raise the possibility that Abeta immunotherapy can potentially slow or halt the course of CAA development in AD that is implicated in vascular dysfunction.

Antibody capture of soluble Abeta does not reduce cortical Abeta amyloidosis in the PDAPP mouse.

BACKGROUND: In vivo administration of antibodies against the amyloid-beta (Abeta) peptide has been shown to reduce and reverse the progressive amyloidosis that develops in a variety of mouse models of Alzheimer's disease (AD). This work has been extended to clinical trials where subsequent autopsy cases of AD subjects immunized against Abeta showed similar reductions in parenchymal amyloid plaques, suggesting this approach to reduce neuropathology in man is feasible. OBJECTIVE: Multiple hypotheses have been advanced to explain how anti-Abeta antibodies may lower amyloid burden. In this report, we compare approaches utilizing either plaque-binding or peptide-capturing anti-Abeta antibodies for effectiveness in reducing amyloidosis in a mouse model of AD. METHODS: A plaque-binding monoclonal antibody (3D6) and an Abeta peptide-capturing monoclonal antibody (266) were compared in chronic treatment and prevention paradigms using a transgenic mouse model of AD. The effects of antibody therapy on plaque burden and plasma clearance of Abeta were investigated by quantitative imaging and clearance studies of intravenously injected (125)I-Abeta. RESULTS: The plaque-binding antibody 3D6 was highly effective in either treatment or prevention of amyloidosis. In these studies, the peptide-capture antibody 266 showed no reduction in amyloidosis in either paradigm and showed trends towards increasing amyloidosis. Antibody 266 was also found to greatly prolong (>180-fold) the normally rapid peripheral clearance of Abeta, in contrast to that found with 3D6 (>24-fold). CONCLUSION: Reversing and preventing Alzheimer's type amyloidosis is most effectively accomplished with anti-amyloid antibodies that avidly bind plaque.

Identification of gamma-secretase inhibitor potency determinants on presenilin.

Production of amyloid beta peptides (Abeta), followed by their deposition in the brain as amyloid plaques, contributes to the hallmark pathology of Alzheimer disease. The enzymes responsible for production of Abeta, BACE1 and gamma-secretase, are therapeutic targets for treatment of Alzheimer disease. Two presenilin (PS) homologues, referred to as PS1 and PS2, comprise the catalytic core of gamma-secretase. In comparing presenilin selectivity of several classes of gamma-secretase inhibitors, we observed that sulfonamides in general tend to be more selective for inhibition of PS1-comprising gamma-secretase, as exemplified by ELN318463 and BMS299897. We employed a combination of chimeric constructs and point mutants to identify structural determinants for PS1-selective inhibition by ELN318463. Our studies identified amino acid residues Leu(172), Thr(281), and Leu(282) in PS1 as necessary for PS1-selective inhibition by ELN318463. These residues also contributed in part to the PS1-selective inhibition by BMS299897. Alanine scanning mutagenesis of areas flanking Leu(172), Thr(281), and Leu(282) identified additional amino acids that affect inhibitor potency of not only these sulfonamides but also nonsulfonamide inhibitors, without affecting Abeta production and presenilin endoproteolysis. Interestingly, many of these same residues have been identified previously to be important for gamma-secretase function. These findings implicate TM3 and a second region near the carboxyl terminus of PS1 aminoterminal fragment in mediating the activity of gamma-secretase inhibitors. Our observations demonstrate that PS-selective inhibitors of gamma-secretase are feasible, and such inhibitors may allow differential inhibition of Abeta peptide production and Notch signaling.