Malcolm A. Leissring, Ph.D. - US grants

DESCRIPTION (provided by applicant): Steady-state levels of the amyloid beta-protein (ABeta), which accumulates abnormally in Alzheimer's disease (AD), reflect the balance between the key processes of production, via the beta- and gamma- secretases, and proteolytic degradation, by one or more proteases. While therapies based on pharmacological blockade of the secretases have been extensively studied, very little work has examined the complimentary approach of activating or disinhibiting ABeta degrading proteases. Recent evidence shows that molecules that bind tightly to ABeta in the periphery (e.g., antibodies, gelsolin) can decrease levels of ABeta in the brain in a reaction akin to dialysis, indicating that central and peripheral pools of ABeta exist in rapid equilibrium. By extension, we hypothesize that enhancing degradation of ABeta in the periphery will also lower brain ABeta levels, an idea we call the "peripheral degradation hypothesis." To test this new hypothesis, and to identify novel drug targets and small molecule pharmacophores acting on peripheral ABeta degradation, we propose the following Aims. First, to directly test the peripheral degradation hypothesis, ABeta-degrading proteases will be overexpressed exclusively in the periphery of APP transgenic mice by using adenovirus with liver-specific promoters. Second, to identify the principal ABeta-degrading proteases in the periphery in vivo, the rate of clearance of intravenously administered AB3eta will be compared in wildtype mice and in mice lacking specific ABeta-degrading proteases. In addition, the major ABeta-degrading proteases in human serum will be identified using protease-specific inhibitors. Third, in collaboration with the Laboratory for Drug Discovery in Neurodegeneration, a high throughput ABeta-degradation assay will be performed on approximately 60,000 small molecule pharmacophores using human serum or recombinant proteases as the source of ABeta degrading activity. Collectively, the studies outlined in this proposal will evaluate--and potentially enhance--the potential of peripheral ABeta-degrading proteases as novel therapeutic targets in AD.

[unreadable] DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is characterized by abnormal accumulation of the amyloid ?-protein (A?) in brain regions subserving memory and cognition. In recent years, proteases that degrade A??have been identified as potent and rate-limiting regulators of cerebral A??levels and amyloidogenesis in vivo. Significantly, orally bioavailable compounds that activate A?-degrading proteases have already been identified that are effective in reducing AD-type pathology in animal models and are currently entering clinical trials. Converging lines of evidence strongly implicate insulin-degrading enzyme (IDE) as a particularly important A?-degrading protease. Nonetheless, there is a surprising lack of pharmacological tools targeting IDE, or indeed any member of the unusual zinc-metalloprotease superfamily to which it belongs. Importantly, accumulating evidence shows how IDE activity might be augmented pharmacologically by any of several mechanisms, including the displacement of endogenous inhibitors or modulation of its expression and/or secretion into the extracellular space. Moreover, new crystal structures of IDE show that this protease possesses unorthodox structural features that can be targeted to directly activate the protease as much as 4000 percent. The purpose of this proposal is to utilize our well-characterized fluorescence polarization-based A?-degradation assay (Leissring et al., JBC 2003, Appendix) to search for chemical modulators of IDE within the Small Molecule Library of the MLSCN. We propose a series of secondary assays to confirm discovered hits, to establish their specificity for IDE, and to identify cell-penetrant compounds. Probes suitable for use in cultured cells or in vivo will be used in downstream experiments to resolve several outstanding questions about the role of IDE in AD pathogenesis that can only be addressed via a chemical biology approach. In addition, chemical activators of IDE with suitable properties could serve as pharmacophores for the development of novel AD therapies. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Proteases that degrade the amyloid [unreadable]-protein (A[unreadable]), which accumulates abnormally in Alzheimer's disease (AD), have emerged as critical regulators of amyloidogenesis in vivo, yet have only begun to be explored for their therapeutic potential. Accumulating experimental, genetic and animal modeling studies implicate insulin-degrading enzyme (IDE), as a particularly important A[unreadable]-degrading protease. Importantly, recent evidence shows how IDE activity might be increased by any of several mechanisms, including the displacement of endogenous inhibitors and modulation of its secretion into the extracellular space. Moreover, new crystal structures of IDE show that this protease possesses unorthodox enzymological properties that can be exploited to directly activate the protease as much as 40-fold. Here we propose to conduct ultra high- throughput screening (uHTS) on a chemically diverse library of ~550,000 compounds using a cell-based assay optimized for the detection of IDE activators. The development and implementation of the primary assay will largely be performed by Scripps Florida's highly experienced uHTS Core for a modest cost, permitting greater attention to be focused on critical secondary assays essential for identifying bone fide IDE activators and characterizing their mechanism(s) of action. Our long-term goal is to identify pharmacophores suitable for use in cultured cells and in vivo, which may lead to the development of novel therapies to treat this devastating disease. PUBLIC HEALTH RELELVANCE: The goal of this proposal is to test a large collection of molecules for their potential to affect fundamental biological processes known to regulate Alzheimer's disease, specifically relating to insulin-degrading enzyme. Discovered molecules will be evaluated for their possible therapeutic potential, and potentially developed into novel drugs through future funding proposals. [unreadable] [unreadable]

PROJECT SUMMARY/ABSTRACT Insulin-degrading enzyme (IDE) is an evolutionarily and structurally distinctive zinc-metallopeptidase that is the best characterized mediator of insulin catabolism in vivo. IDE is strongly implicated in the pathogenesis and potential treatment of type 2 diabetes mellitus (T2DM) as well as other highly prevalent diseases. For more than 60 years, pharmacological inhibition of IDE has been viewed as an attractive way to promote endogenous insulin signaling and thereby treat T2DM, and the feasibility of this fundamental idea was recently confirmed in vivo (Maianti et al., Nature 2014). Despite the considerable promise of IDE inhibitors as experimental probes and as potential pharmacophores, only a limited number of inhibitors have emerged. Existing inhibitors do not possess the properties needed to address a large number of outstanding questions about the fundamental biology of IDE, such as the differing roles of intracellular and extracellular pools of IDE. Moreover, current IDE inhibitors lack appropriate medicinal properties, such as oral bioavailability and long-term stability in vivo, needed to be developed into drugs for clinical use. The objective of this proposal is to use fragment-based drug design (FBDD) to develop a diverse set of potent, selective, small-molecule, zinc-targeting IDE inhibitors suitable for use as experimental probes and/or as lead compounds for future drug development. Specifically, in Aim 1, we will screen several custom libraries of metal-binding pharmacophores (MBPs), synthesized in-house, to identify novel chemical moieties targeting the catalytic zinc atom within the active site of IDE. In Aim 2, we will synthesize sublibraries of compounds containing these zinc-targeting moieties; test them for potency, selectivity and other properties; then repeat this process until IDE inhibitors with the desired set of features are obtained. In Aim 3, we will characterize key properties of developed inhibitors, using a battery of in vitro, cell-based, and in vivo assays. An innovative goal of this project is to develop compounds that are, to the greatest extent possible, selective for different substrates of interest, a novel feature of IDE that results from its unusual tertiary structure and atypical mechanism of substrate binding. Accordingly, throughout all phases of the project, we will test all compounds against multiple substrates in parallel. Using this approach, we have so far identified >25 structurally diverse MBPs that inhibit IDE with remarkable potency. Moreover, through the optimization of just a subset of identified MBPs, we have already succeeded in developing drug-like compounds with nanomolar potency. Notably, these novel compounds selectively inhibit the degradation of insulin and also show strong specificity for IDE versus numerous other proteases. The successful completion of this project will result in novel IDE inhibitors suitable for diverse experimental and medicinal applications.

PROJECT SUMMARY/ABSTRACT The specific pathogenic mechanisms causing Alzheimer disease (AD) are known with certainty only for extremely rare forms of the disease attributable to genetic mutations, known as famial AD (fAD). However, for the vast majority of cases, known as sporadic AD (sAD), the specific cause(s) remain obscure. fAD-linked mutations are known to perturb the processing and/or aggregation of the amyloid ß-peptide (Aß), thus strongly implicating Aß in the pathogenesis fAD, but there is considerable uncertainty about the role of Aß in the etiology of sAD. A major impediment to the resolution of this question has been the lack of animal models that faithfully recapitulate the physiological milieu from which sAD emerges. Most existing AD mouse models express superphysiological levels of the amyloid precursor protein (APP) harboring fAD-linked mutations and under the control of heterologous promoters. Although these models successfully reproduce amyloid deposits and other associated features of the disease, they are not useful for uncovering the specific mechanisms that trigger amyloidosis in the absence of fAD mutations or hyperphysiological levels of Aß (and other APP metabolites), mechanisms that by definition must be operative in sAD. To address these limitations, we have developed a novel mouse model wherein gene targeting was used to ?humanize? the Aß portion of murine APP. The resulting animals, dubbed APPKI-hAßwt mice, express wild-type human Aß at physiological levels under the control of the endogenous murine App promoter. As is true for normal humans, these animals develop diffuse deposits of human Aß in an age-dependent manner, but do not form the dense-core amyloid plaques characterizing AD. Accordingly, these mice are ideal for investigating the pathophysiological mechanisms responsible for transforming ?normal? Aß deposition to the pathological variety that occurs in AD. Multiple forms of acute brain injury, such as head trauma and ischemia/hypoperfusion, are established risk factors for sAD, and these are known to result in transient elevations in Aß. However, it remains to be established whether transient increases in human Aß per se are responsible for triggering AD-type pathology, rather than myriad other sequelae resulting from brain trauma, in the absence of fAD mutations or overexpression of Aß/APP. Accordingly, the objective of the present proposal is to use the APPKI-hAßwt line to investigate whether a single transient elevation in Aß early in life is sufficient to produce AD-type pathology later in life. To investigate this question cleanly, we will employ a novel pharmacological approach wherein we increase Aß levels using highly selective inhibitors of two Aß-degrading proteases?neprilysin and insulin- degrading enzyme, as well as a broad-spectrum metalloprotease inhibitor. The successful completion of this project is expected to yield new insights into the specific molecular mechanisms underlying the initiation of sAD, which could facilitate the development of acute interventions to mitigate AD risk following brain injury.

PROJECT SUMMARY/ABSTRACT Rare forms of familial Alzheimer disease (fAD) are known to be caused by life-long, genetically determined perturbations in the production of the amyloid ß-protein (Aß), but the cause(s) of the vast majority of so-called sporadic AD (sAD) cases remains remarkably poorly defined. This proposal will use state-of-the-art mouse models of sAD together with several highly innovative approaches to address key temporal and spatial aspects of sAD pathogenesis for the first time, with critical implications for the development of effective therapies. Whereas fAD is attributable to chronic perturbations in the production of Aß, we hypothesize that sAD is triggered by impairments in the clearance of Aß?specifically by transient impairments in Aß clearance. This hypothesis is consistent with evidence showing that several established risk factors for sAD, such as brain trauma, stress, or poor sleep, lead to short-lived or episodic increases in cerebral Aß levels due to reduced Aß clearance. To model this novel mechanistic hypothesis, we employ innovative methods to inhibit Aß clearance transiently and reversibly by blocking either blood-brain barrier transport of Aß or its proteolytic degradation. Because we aim to define the triggers for sAD, we require an animal model that does not develop AD-type pathology on its own, as most AD mouse models do. To this end, we will use an innovative new sAD mouse model, the APPNL-F/hAß mice, which expresses wild-type human Aß only, under the control of the endogenous murine App promoter, with the minimal genetic mutations needed to model sAD. As is true for normal humans, this sAD mouse model develops diffuse deposits of human Aß in an age-dependent manner, and forms very minimal dense-core plaques only at very advanced ages. Accordingly, these mice are ideal for investigating the pathophysiological mechanisms responsible for triggering the conversion of ?normal? Aß deposition to the pathological type in sAD. We hypothesize further that the Aß-dependent pathological mechanisms most relevant to sAD occur much earlier than the ages studied in clinical trials, with clinical symptoms emerging only much later, specifically in the context of aging. Accordingly, we will define the temporal window most relevant to the emergence of AD by increasing Aß levels in APPNL-F/hAß mice transiently at various ages, then evaluating the consequences for the development of AD-type pathology longitudinally, up to and including old age. Finally, we will test the novel hypothesize that spatially distinct pools of Aß (e.g., extra- vs. intracellular) impact the pathogenesis of AD in qualitatively different ways. Specifically, we postulate that intracellular Aß is more relevant than extracellular Aß to the neurodegeneration and memory loss that characterize AD. To test this, we will selectively increase extra- vs. intracellular pools of Aß by reversibly downregulating Aß-degrading proteases that, as our preliminary results show, selectively regulate these distinct pools of Aß. Collectively, these experiments will allow us to investigate, cleanly and for the first time, many critical temporal and spatial aspects of AD pathogenesis, yielding novel insights that will inform improved approaches to the treatment of sAD.