BACE : Lead Target for Orchestrated Therapy of Alzheimer's Disease

BACE inhibitors and their use in the treatment of Alzheimer's Disease BACE (β-site of APP cleaving enzyme) is a critical component in Alzheimer's Disease (AD), and the development of BACE inhibitors shows great potential as a therapy for the disease. BACE: Lead Target for Orchestrated Therapy of Alzheimer's Disease covers virtually all aspects of BACE from initial identification, discovery of inhibitors, and challenges in clinical development, while providing a global understanding essential for productive and successful drug discovery. This book details the story of the discovery of BACE and its role in AD and comprehensively discusses: The development of BACE inhibitors as therapeutics for Alzheimer's disease The research that led to the identification of BACE New BACE inhibitors currently being clinically tested ADME (absorption, distribution, metabolism, excretion) and clinical trial design—topics not addressed in current field literature Cutting-edge technology such as high-throughput screening, structure-based drug design, and QSAR in context of BACE inhibitors and Alzheimer's drug discovery Other approaches to BACE inhibition based on interaction with the precursor protein APP By enhancing the reader's understanding of the various aspects of the BACE drug-discovery process, this much-needed reference will serve as a key resource for all scientists involved in Alzheimer's research—and inspire new approaches to treatment of AD.

CHAPTER 1

BACE, APP PROCESSING, AND SIGNAL TRANSDUCTION IN ALZHEIMER'S DISEASE

Dale E. Bredesen and Edward H. Koo

1.1 INTRODUCTION

Alzheimer's disease (AD) is a remarkably, and to date inexplicably, common disease, affecting over five million Americans at a national cost of approximately $150billion annually – a cost that does not begin to address the impact of the disease on families, individuals, and society. With the graying of America, the prediction is for approximately 13million cases by 2050 and, given the late appearance of symptoms in the pathogenic process, many more pre-Alzheimer's cases, including both mild cognitive impairment (MCI) and pre-MCI conditions. Thus, AD is unfolding as one of the most important global health concerns.

Since the first description of the disease just over 100years ago, extensive clinical, pathological, genetic, and biochemical data have been accumulated, implicating the amyloid-beta (Aβ) peptides, especially Aβ1-42, as key mediators in the pathogenesis of this disorder, the so-called amyloid cascade hypothesis. However, the physiological role of these peptides remains unknown, as does the mechanism(s) of their neurodegenerative effect.

The Aβ peptides are derived proteolytically from the β-amyloid precursor protein (APP) by β-site APP cleaving enzyme (BACE) (or β-secretase) cleavage of the extracellular domain, followed by [gamman-secretase cleavage of the transmembrane domain. However, APP is also cleaved at other sites, for example, at the α-site by α-secretase (with ADAM10 being the most likely candidate) and at the cytosolic caspase site by caspases (with caspase-8 and caspase-6 being the most likely candidates, given their P4 preference along with co-immunoprecipitation and kinetic data). With four major cleavage sites, theoretically 14 peptides could be produced, and it is becoming more apparent that other APP-derived peptides beyond Aβ also play critical roles in elements of the Alzheimer's phenotype. Therefore, Aβ may turn out to be one part of a much larger pathogenetic scenario, and thus, BACE may ultimately be one target of a cocktail of drugs that modulates APP processing at more than one site, as well as affecting targets other than APP.

In neurodegenerative diseases such as AD, neurons in various nuclei are lost in disease-specific distributions. However, the neuronal loss is a relatively late event, typically following synaptic dysfunction, synaptic loss, neurite retraction, and the appearance of other abnormalities such as axonal transport defects. This progression argues that cell death programs may play at best only a secondary role in the neurodegenerative process. However, emerging evidence from several laboratories has suggested an alternative possibility: that although cell death itself occurs late in the degenerative process, the pathways involved in cell death signaling do indeed play critical roles in neurodegeneration, both in sub-apoptotic events such as synapse loss and in the ultimate neuronal loss itself.

Although initial comparisons of the intrinsic suicide program in genetically tractable organisms such as the nematode Caenorhabditis elegans failed to disclose obvious relationships to genes associated with familial AD – for example, presenilin-1 and the β-APP do not bear an obvious relationship to any of the major C. elegans cell death genes (ced-3, ced-4, or ced-9) – more recent studies suggest a fundamental relationship between developmental and degenerative processes. For example, Nikolaev et al. recently found that in a culture model of trophic factor withdrawal in developing neurons, one pathway involved in neurite retraction is mediated by a cleavage product of sAPPβ, the latter released after BACE cleavage of APP. A detailed understanding of the interrelationship between fundamental cell death programs and neurodegenerative processes is still evolving, and it promises to offer novel approaches to the treatment of these diseases. In this review, we will discuss BACE cleavage of APP and how it might be involved in certain aspects of Alzheimer's pathology.

1.2 BACE CLEAVAGE OF APP AS A MOLECULAR SWITCHING MECHANISM

Neurons, as well as other cells, depend for their survival on stimulation that is mediated by various receptors and sensors, and programmed cell death may be induced in response to the withdrawal of trophic factors, hormonal support, electrical activity, extracellular matrix support, or other trophic stimuli. For years, it was generally assumed that cells dying as a result of the withdrawal of required stimuli did so because of the loss of a positive survival signal, for example, mediated by receptor tyrosine kinases. While such positive survival signals are clearly critical, data obtained over the past 15 years argue for a complementary effect that is pro-apoptotic, activated by trophic stimulus withdrawal, and mediated by specific receptors dubbed "dependence receptors". Over a dozen such receptors have now been identified, and examples include DCC (deleted in colorectal cancer), Unc5H2 (uncoordinated gene 5 homologue 2), neogenin, rearranged during transfection (RET), Ptc, and APP. These receptors interact in their intracytoplasmic domains with caspases, including apical caspases such as caspase-9, and may therefore serve as sites of induced proximity and activation of these caspases. Caspase activation leads in turn to receptor cleavage, producing pro-apoptotic fragments; however, caspase cleavage site mutation of dependence receptors suppresses the cell death signals mediated by the receptors. A striking example of this effect was obtained in studies of neural tube development: withdrawal of Sonic hedgehog from the developing chick spinal cord led to apoptosis mediated by its receptor, Patched, preventing spinal cord development; however, transfection of a caspase-uncleavable mutant of Patched blocked apoptosis and restored significant development, even in the absence of Sonic hedgehog.

Thus, cellular dependence on specific signals for survival is mediated, at least in part, by specific dependence receptors that induce apoptosis in the absence of the required stimulus – when unoccupied by a trophic ligand, or when bound by a competing, anti-trophic ligand – but block apoptosis following binding to their respective ligands. Expression of these dependence receptors thus creates cellular states of dependence on the associated trophic ligands. These states of dependence are not absolute, since they can be blocked downstream in some cases by the expression of anti-apoptotic genes such as bcl-2 or p35; however, they result in a shift of the apostat toward an increased likelihood of triggering apoptosis. In the aggregate, these receptors may serve as part of a molecular integration system for trophic signals, analogous to the electrical integration system composed of the dendritic arbors within the nervous system.

Although cellular dependence on trophic signals was originally described in the developing nervous system, neurodegeneration may also utilize the same pathways, since APP exhibits several features characteristic of dependence receptors: an intracytoplasmic caspase cleavage site (Asp664), co-immunoprecipitation with an apical caspase (caspase-8), caspase activation, derivative pro-apoptotic peptides (including the A peptide; see below), and suppression of apoptosis induction by mutation of the caspase cleavage site.

These findings raise several questions: first, does BACE cleavage of APP play a role in APP's putative dependence-related pro-apoptotic function? Second, does the caspase cleavage of APP occur in human brain and, if so, is this increased in patients with AD and coordinated with BACE cleavage? Third, if this cleavage is prevented, is the Alzheimer's phenotype affected? These questions are addressed below.

1.3 AD: AN IMBALANCE IN CELLULAR DEPENDENCE?

Although cellular dependence on trophic signals was originally described in molecules and pathways critical for the developing nervous system, degeneration in neurons of the aged organism may also utilize the same pathways. For example, APP, a molecule holding a central position in AD pathogenesis because of its intimate relationship to A peptides, exhibits several features characteristic of dependence receptors: a caspase cleavage site (Asp664), interaction with an apical caspase (caspase-8), derivative pro-apoptotic peptides released after caspase activation, and suppression of apoptosis induction by mutation of the caspase cleavage site. Although APP demonstrates many of the characteristics of a dependence receptor, what is unclear is whether there is a physiological ligand that maintains the balance of APP in favor of survival rather than neuronal death, as has been seen with other trophic factor receptors.

In this context, BACE may hold a surprising central position. This is because cleavage of APP by BACE generates not only the C-terminal fragment of APP that is the direct precursor of Aβ, but this cleavage also releases sAPPβ, which can interact with DR6 to effect neuronal damage (see below). What is unclear is whether BACE cleavage of APP plays a role in APP's putative dependence-related proapoptotic function. And second, whether caspase cleavage of APP occurs in human brain and, if so, is this increased in patients with AD and coordinated with BACE cleavage? And lastly, if this cleavage is prevented, is the Alzheimer's phenotype affected? These questions are addressed below.

Extensive genetic and biochemical data have implicated the Aβ peptide as a central mediator of AD, but the mechanism(s) of action remains controversial: the ability of Aβ to generate a sulfuranyl radical involving methionine 35 has been implicated, and so have its direct effects on postsynaptic structures, the metal-binding property of Aβ, as well as its aggregating property, and its ability to form pore-like structures in membranes, just to list a few of the mechanisms proposed. These proposed mechanisms share a focus on the chemical and physical properties of the A peptide. However, cellular signaling is emerging as a complementary mechanism by which Aβ exerts its critical effects, and multiple candidates have surfaced as key downstream mediators, including APP itself, the insulin receptor, and tau, among others. These cellular signals may also mediate neuronal dependence on trophic support, as described below.

1.4 BACE CLEAVAGE, CASPASE CLEAVAGE, AND NEURONAL TROPHIC DEPENDENCE

Neoepitope antibodies directed against residues 657–664 of human APP disclosed the presence of caspase-cleaved APP fragments in human brain, especially in the hippocampal region, with an approximately fourfold increase in Alzheimer's patients over age-matched controls. However, in brains without Alzheimer's pathology, there was an inverse relationship between age and immunohistochemical detection of APPneo, with a different distribution from AD brains: in the Alzheimer brains, the staining was primarily in neuronal somata and peri-neuronally, whereas in the non-Alzheimer brains, the staining was observed predominantly in the processes. These findings suggest that the caspase cleavage of APP occurs physiologically and is reduced with age, but that this process remains more active and perhaps aberrant in cellular distribution in association with AD.

To test whether preventing the caspase cleavage of APP has any consequences on the Alzheimer's phenotype, a transgenic mouse model of age-associated amyloid pathology was generated in which APP containing the Swedish and Indiana familial AD mutations were combined with a mutation of the caspase site (D664A) was expressed under the control of the neuronal-specific platelet-derived growth factor-B (PDGF-B) promoter. Although the caspase mutation (D664A) had no effect on amyloid production or plaque formation, these animals did not exhibit any overt synapse loss, early p21-activated kinase (PAK) phosphorylation, dentate gyral atrophy, electrophysiological abnormalities (including reductions in excitatory postsynaptic potentials [EPSPs] and long-term potentiation [LTP]), neophobia, or memory deficits that frequently characterize these APP overexpressing mice. These findings indicate that key features of the Alzheimer's associated phenotype in a standard transgenic mouse model depend on the presence of the caspase cleavage site within APP. This finding, when combined with the extensive previous work showing that the Alzheimer's phenotype is critically dependent on Aβ, suggests that the APP caspase site may lie downstream from the Aβ accumulation and that cleavage of this site is one pathway that contributes to neuronal injury. This possibility has received support from studies showing that A interacts directly with APP in the Aβ region itself, leading to APP multimerization, caspase cleavage at Asp664, and cell death signaling.

1.5 BACE CLEAVAGE OF APP, DEPENDENCE RECEPTORS, AND ALZHEIMER PATHOLOGY

The above model of caspase cleavage of APP focuses primarily on the cytosolic region of APP where the caspase site is situated. Experimental evidence suggested that Aβ-induced multimerization and subsequent caspase cleavage of APP can take place with either APP or the BACE-cleaved C99 APP fragment. However, there did not appear to be any preference between these two substrates. How, then, might BACE cleavage of APP relate to the caspase cleavage of APP? Recent work from the Tessier-Lavigne lab provides surprising new insight into this question: following trophic factor withdrawal from developing neurons in culture, BACE was apparently activated, resulting in the shedding of sAPPβ. Following further processing near the amino-terminus of sAPPβ by an unidentified protease, the resulting amino-terminal peptide of the APP ectodomain was able to interact with death receptor 6 (DR6). This binding led to caspase-6 activation and subsequent neurite retraction, which is of interest given previous studies showing that APP is cleavable by caspase-6, and that its caspase site (VEVD) is indeed most compatible with a caspase-6 site. This is a surprising observation because the large sAPP ectodomain (mainly sAPPα) has traditionally been thought to play a neurotrophic rather than toxic role. Nonetheless, increased BACE cleavage of APP should lead to the production of more Aβ peptide, and this in turn will result in more Aβ–C99 interaction and potentially more caspase cleavage of APP. Thus, BACE cleavage of APP results in two downstream pathways that damage neurons: through the sAPPβ fragment interacting with DR6 receptor (via N-APP production) and through Aβ-induced caspase cleavage of APP C99 fragment.

If APP does indeed function as a dependence receptor, AD may be considered a "state of altered dependence." What then is/are the trophic ligand/s for APP? Several candidate APP interactors have been described, such as collagen (types I and IV), heparan sulfate proteoglycan, glypican, laminin, and F-spondin. F-spondin's interaction with APP leads to a reduction in β-secretase activity. Lourenco et al. have recently shown that netrin-1, a multifunctional axon guidance and trophic factor, also binds APP. Furthermore, netrin-1 also interacts with Aβ itself, and Aβ is capable of interfering with netrin-1 binding to APP. The binding of netrin-1 to APP results in enhanced interaction of APP with intracytoplasmic mediators Fe65 and Dab, upregulation of KAI1, and a marked reduction of net Aβ production.

These findings suggest a model in which the Aβ peptide functions as an "anti-trophin," first by blocking netrin's guidance and trophic effects and then through binding to and facilitating APP oligomerization, recruiting and activating caspase-8 (and possibly caspase-6), engendering the processing of APP at Asp664, and inducing neurite retraction, then, ultimately, neuronal cell death. Whether the D664A mutation of APP exerts effects beyond the prevention of caspase cleavage (e.g., an alteration of the intracytoplasmic structure of APP) is not yet known. However, regardless of the mechanism, the results suggest that APP signal transduction may be important in mediating AD, at least in the transgenic mouse model, possibly downstream from Aβ oligomerization and binding of APP. The results also suggest that BACE cleavage and caspase cleavage of APP may work in concert to lead to neurite retraction, and potentially other aspects of the AD phenotype.

The results obtained in the transgenic mouse model of AD also suggest an alternative to the classic models of AD. As noted above, chemical and physical properties of Aβ have been cited as the proximate cause of AD pathophysiology. However, these theories do not explain why Aβ is produced ubiquitously and constitutively, nor do they offer a physiological function for the Aβ peptide, or account for the improvement in AD model mice that occurs with a reduction in tau protein.
(Continues...) Excerpted from BACE by Varghese John. Copyright © 2010 John Wiley & Sons, Inc.. Excerpted by permission of John Wiley & Sons.
All rights reserved. No part of this excerpt may be reproduced or reprinted without permission in writing from the publisher.
Excerpts are provided by Dial-A-Book Inc. solely for the personal use of visitors to this web site. BACE......Page 4 CONTENTS......Page 10 PREFACE......Page 14 ACKNOWLEDGMENTS......Page 16 CONTRIBUTORS......Page 18 1.1 Introduction......Page 20 1.2 BACE Cleavage of APP as a Molecular Switching Mechanism......Page 21 1.3 AD: An Imbalance in Cellular Dependence?......Page 22 1.4 BACE Cleavage, Caspase Cleavage, and Neuronal Trophic Dependence......Page 23 1.5 BACE Cleavage of APP, Dependence Receptors, and Alzheimer Pathology......Page 24 1.6 Key Mutations Proximal of APP Processing to Aβ......Page 28 1.7 Final Remarks......Page 29 2.1 Introduction......Page 34 2.2 The Search for β-Secretase......Page 36 2.3 Validation of the BACE Target......Page 46 2.4 Final Remarks......Page 47 3.1 Introduction......Page 54 3.3 APP Processing......Page 55 3.4 Aspartyl Protease Classification......Page 56 3.5 BACE Structure......Page 57 3.6 Mechanism, Kinetics, Inhibition, and Specificity......Page 58 3.7 Assay Strategies for Inhibitor Finding and Development......Page 64 3.8 Common Assays Used to Identify and Study Inhibitors......Page 67 3.9 BACE Assays......Page 69 3.10 Final Remarks......Page 73 4.2 Elan/Pharmacia (Pfizer)......Page 78 4.3 Oklahoma Medical Research Foundation (OMRF)/Multiple Collaborators......Page 89 4.4 Eli Lilly......Page 91 4.5 Merck......Page 93 4.6 GlaxoSmithKline......Page 99 4.7 Schering Plough......Page 101 4.8 Bristol-Myers Squibb......Page 104 4.9 Novartis......Page 106 4.10 Amgen......Page 107 4.11 Wyeth......Page 109 4.12 Final Remarks......Page 113 5.1 Introduction......Page 126 5.2 Biophysical Methods Applied to BACE Fragment Screens......Page 127 5.3 BACE Inhibitors Identified by Fragment Screening......Page 129 5.4 Final Remarks......Page 138 6.1 Introduction......Page 142 6.2 Preparation of BACE for Structural Studies......Page 145 6.3 Crystallographic Studies of BACE......Page 149 6.4 Structural Studies with BACE Inhibitors: Peptidomimetics and Nonpeptidomimetics......Page 154 6.5 Computational Approaches......Page 164 6.6 Final Remarks......Page 169 7.1 Introduction......Page 178 7.2 BACE1 and Mouse Models of AD......Page 180 7.3 Testing BACE Inhibitors in the Canine Model of Human Aging and AD......Page 182 7.4 BACE Inhibitors and Nonhuman Primates......Page 186 7.5 Final Remarks......Page 187 8.1 Introduction......Page 196 8.2 Development of BACE Inhibitors with Optimized ADME Properties......Page 199 8.3 In Vivo Efficacy of BACE Inhibitors......Page 207 8.4 Toxicology of BACE Inhibitors......Page 211 8.5 Final Remarks......Page 212 9.1 Introduction......Page 216 9.2 Update on Beta-Amyloid Therapies in Clinical Development......Page 217 9.3 Clinical Development of BACE Inhibitors and Other Disease-Modifying Drugs......Page 222 9.4 Final Remarks......Page 231 10.1 Introduction......Page 236 10.2 β-Secretase: Discovery, Function, and Inhibitors......Page 237 10.3 Generation of Aβ Peptides via the Endocytic Pathway......Page 239 10.4 Generation of Anti-APP β-Site Antibodies......Page 240 10.5 Antibody Interference with Aβ Production in Cellular Model......Page 242 10.6 Antibody Interference with Aβ Production in Animal Models......Page 245 10.7 Identification of APP Binding Small Molecules that Block β-Site Cleavage of APP......Page 247 10.8 Final Remarks......Page 249 Introduction......Page 254 Artwork as a Measure of the Progression of AD......Page 255 INDEX......Page 262

BACE inhibitors and their use in the treatment of Alzheimer's Disease

BACE (β-site of APP cleaving enzyme) is a critical component in Alzheimer's Disease (AD), and the development of BACE inhibitors shows great potential as a therapy for the disease. BACE: Lead Target for Orchestrated Therapy of Alzheimer's Disease covers virtually all aspects of BACE from initial identification, discovery of inhibitors, and challenges in clinical development, while providing a global understanding essential for productive and successful drug discovery.

This book details the story of the discovery of BACE and its role in AD and comprehensively discusses:

• The development of BACE inhibitors as therapeutics for Alzheimer's disease

• The research that led to the identification of BACE

• New BACE inhibitors currently being clinically tested

• ADME (absorption, distribution, metabolism, excretion) and clinical trial design—topics not addressed in current field literature

• Cutting-edge technology such as high-throughput screening, structure-based drug design, and QSAR in context of BACE inhibitors and Alzheimer's drug discovery

• Other approaches to BACE inhibition based on interaction with the precursor protein APP

By enhancing the reader's understanding of the various aspects of the BACE drug-discovery process, this much-needed reference will serve as a key resource for all scientists involved in Alzheimer's research—and inspire new approaches to treatment of AD.

Note: CD-ROM/DVD and other supplementary materials are not included as part of eBook file.

BACE : APP processing, and signal transduction in Alzheimer's disease (AD) / Dale E. Bredesen and Edward H. Koo Identification of BACE as an AD target / Robert L. Heinrikson and Sukanto Sinha BACE biological assays / Alfredo G. Tomasselli and Michael J. Bienkowski Peptidic, peptidomimetic, and HTS based BACE inhibitors / James P. Beck and Dustin J. Mergott Fragment-based approaches for identification of BACE inhibitors / Andreas Kuglstatter and Michael Hennig Structure-based drug design of BACE inhibitors : technical and practical aspects of preparation, 3-dimensional structure, and computational analysis / Felix F. Vajdos, Veer Shanmugasundaram, and Alfredo G. Tomasselli Pharmacological models for preclinical testing : from mouse to dog to non-human primates / Jason L. Eriksen, Michael Paul Murphy, and Elizabeth Head Adsorption, distribution, metabolism, excretion (ADME), efficacy & toxicology for BACE inhibitors / Ishrut Hussain and Emmanuel Demont Clinical trials for disease modifying drugs such as BACE inhibitors / Henry Hsu Future strategies for development of novel BACE inhibitors : anti-APP e-site antibody & APP binding small molecule approaches for Alzheimer's disease / Beka Solomon ... [et al.]. BACE inhibitors and their use in the treatment of Alzheimer's Disease. BACE (ß-site of APP cleaving enzyme) is a critical component in Alzheimer's Disease (AD), and the development of BACE inhibitors shows great potential as a therapy for the disease. BACE: Lead Target for Orchestrated Therapy of Alzheimer's Disease covers virtually all aspects of BACE from initial identification, discovery of inhibitors, and challenges in clinical development, while providing a global understanding essential for productive and successful drug discovery. This book details the story of the discovery of BACE and