Inhibition of insulin-degrading enzyme in human neurons promotes amyloid-ß deposition.

Alzheimer's disease (AD) is characterised by the aggregation and deposition of amyloid-ß (Aß) peptides in the human brain. In age-related late-onset AD, deficient degradation and clearance, rather than enhanced production, of Aß contributes to disease pathology. In the present study, we assessed the contribution of the two key Aß-degrading zinc metalloproteases, insulin-degrading enzyme (IDE) and neprilysin (NEP), to Aß degradation in human induced pluripotent stem cell (iPSC)-derived cortical neurons. Using an Aß fluorescence polarisation assay, inhibition of IDE but not of NEP, blocked the degradation of Aß by human neurons. When the neurons were grown in a 3D extracellular matrix to visualise Aß deposition, inhibition of IDE but not NEP, increased the number of Aß deposits. The resulting Aß deposits were stained with the conformation-dependent, anti-amyloid antibodies A11 and OC that recognise Aß aggregates in the human AD brain. Inhibition of the Aß-forming ß-secretase prevented the formation of the IDE-inhibited Aß deposits. These data indicate that inhibition of IDE in live human neurons grown in a 3D matrix increased the deposition of Aß derived from the proteolytic cleavage of the amyloid precursor protein. This work has implications for strategies aimed at enhancing IDE activity to promote Aß degradation in AD.

Proteolysis of the low-density lipoprotein receptor in hepatocytes is mediated by BMP1 but not by other astacin proteases.

Bone morphogenetic protein 1 (BMP1), a member of the astacin family of zinc-metalloproteases, proteolytically cleaves the low-density lipoprotein receptor (LDLR) within its ligand-binding domain, reducing the binding and cellular uptake of LDL-cholesterol. Here, we aimed to determine whether astacin proteases other than BMP1 may also cleave LDLR. Although human hepatocytes express all six astacin proteases, including the meprins and mammalian tolloid, we found through pharmacological inhibition and genetic knockdown that only BMP1 contributed to the cleavage of LDLR in its ligand-binding domain. We also found that the minimum amino acid change required to render mouse LDLR susceptible to cleavage by BMP1 is mutation at the P1' and P2 positions of the cleavage site. When expressed in cells, the resulting humanised-mouse LDLR internalised LDL-cholesterol. This work provides insight into the biological mechanisms regulating LDLR function.

The role of protein aggregation in the pathogenesis of inclusion body myositis.

Inclusion body myositis (IBM) is characterised by infiltration of CD8+ T-cells and signs of protein aggregation such as rimmed vacuoles and inclusion bodies. Aggregated proteins include those present in neurodegenerative diseases, and also those involved in protein homeostasis. The aim of this review is to discuss the pathological effects of protein aggregates and the process of aggregation following immune attack in IBM. Immune attack is likely to cause protein aggregation by impairing endoplasmic reticulum (ER) and mitochondrial function. Apoptotic and necrotic pathways are activated, possibly leading to nucleo-cytoplasmic coagulation. Overexpression of nuclear and ribosomal proteins in rimmed vacuoles suggests that the vacuoles develop from the collapse of myonuclei and the surrounding ER. Aggregated proteins can activate the NLR family pyrin domain containing 3 (NLRP3) inflammasome or provoke a humoral immune response. Heat shock proteins, ribosomal proteins and protein fragments may provoke interferon-gamma and cytotoxic T-cell responses in a similar manner to Mycobacterium tuberculosis antigens. Persistent provocation can lead to T-cell large granular lymphocytic leukaemia which is resistant to immunosuppression, and would explain the progression from polymyositis to IBM. Protein aggregates may impair the cellular machinery, and proteins may propagate along a myocyte in a prion-like manner. These pathological mechanisms may prevent myocyte regeneration following damage from eccentric muscle contraction, causing weakness and atrophy in a characteristic pattern. Further understanding of the mechanisms of protein aggregation in IBM may lead to additional therapies as well as novel muscle and blood biomarkers. Earlier diagnosis and treatment may result in improved outcomes when effective therapies are available.

3D hydrogel models of the neurovascular unit to investigate blood-brain barrier dysfunction.

The neurovascular unit (NVU), consisting of neurons, glial cells, vascular cells (endothelial cells, pericytes and vascular smooth muscle cells (VSMCs)) together with the surrounding extracellular matrix (ECM), is an important interface between the peripheral blood and the brain parenchyma. Disruption of the NVU impacts on blood-brain barrier (BBB) regulation and underlies the development and pathology of multiple neurological disorders, including stroke and Alzheimer's disease (AD). The ability to differentiate induced pluripotent stem cells (iPSCs) into the different cell types of the NVU and incorporate them into physical models provides a reverse engineering approach to generate human NVU models to study BBB function. To recapitulate the in vivo situation such NVU models must also incorporate the ECM to provide a 3D environment with appropriate mechanical and biochemical cues for the cells of the NVU. In this review, we provide an overview of the cells of the NVU and the surrounding ECM, before discussing the characteristics (stiffness, functionality and porosity) required of hydrogels to mimic the ECM when incorporated into in vitro NVU models. We summarise the approaches available to measure BBB functionality and present the techniques in use to develop robust and translatable models of the NVU, including transwell models, hydrogel models, 3D-bioprinting, microfluidic models and organoids. The incorporation of iPSCs either without or with disease-specific genetic mutations into these NVU models provides a platform in which to study normal and disease mechanisms, test BBB permeability to drugs, screen for new therapeutic targets and drugs or to design cell-based therapies.

Nanoparticle-Enabled Enrichment of Longitudinal Blood Proteomic Fingerprints in Alzheimer's Disease.

Blood-circulating biomarkers have the potential to detect Alzheimer's disease (AD) pathology before clinical symptoms emerge and to improve the outcomes of clinical trials for disease-modifying therapies. Despite recent advances in understanding concomitant systemic abnormalities, there are currently no validated or clinically used blood-based biomarkers for AD. The extremely low concentration of neurodegeneration-associated proteins in blood necessitates the development of analytical platforms to address the "signal-to-noise" issue and to allow an in-depth analysis of the plasma proteome. Here, we aimed to discover and longitudinally track alterations of the blood proteome in a transgenic mouse model of AD, using a nanoparticle-based proteomics enrichment approach. We employed blood-circulating, lipid-based nanoparticles to extract, analyze and monitor AD-specific protein signatures and to systemically uncover molecular pathways associated with AD progression. Our data revealed the existence of multiple proteomic signals in blood, indicative of the asymptomatic stages of AD. Comprehensive analysis of the nanoparticle-recovered blood proteome by label-free liquid chromatography-tandem mass spectrometry resulted in the discovery of AD-monitoring signatures that could discriminate the asymptomatic phase from amyloidopathy and cognitive deterioration. While the majority of differentially abundant plasma proteins were found to be upregulated at the initial asymptomatic stages, the abundance of these molecules was significantly reduced as a result of amyloidosis, suggesting a disease-stage-dependent fluctuation of the AD-specific blood proteome. The potential use of the proposed nano-omics approach to uncover information in the blood that is directly associated with brain neurodegeneration was further exemplified by the recovery of focal adhesion cascade proteins. We herein propose the integration of nanotechnology with already existing proteomic analytical tools in order to enrich the identification of blood-circulating signals of neurodegeneration, reinvigorating the potential clinical utility of the blood proteome at predicting the onset and kinetics of the AD progression trajectory.

Author Correction: Proteolysis of the low density lipoprotein receptor by bone morphogenetic protein-1 regulates cellular cholesterol uptake.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Proteolysis of the low density lipoprotein receptor by bone morphogenetic protein-1 regulates cellular cholesterol uptake.

The development of cardiovascular disease is intimately linked to elevated levels of low-density lipoprotein (LDL) cholesterol in the blood. Hepatic LDL receptor (LDLR) levels regulate the amount of plasma LDL. We identified the secreted zinc metalloproteinase, bone morphogenetic protein 1 (BMP1), as responsible for the cleavage of human LDLR within its extracellular ligand-binding repeats at Gly171↓Asp172. The resulting 120 kDa membrane-bound C-terminal fragment (CTF) of LDLR had reduced capacity to bind LDL and when expressed in LDLR null cells had compromised LDL uptake as compared to the full length receptor. Pharmacological inhibition of BMP1 or siRNA-mediated knockdown prevented the generation of the 120 kDa CTF and resulted in an increase in LDL uptake into cells. The 120 kDa CTF was detected in the livers from humans and mice expressing human LDLR. Collectively, these results identify that BMP1 regulates cellular LDL uptake and may provide a target to modulate plasma LDL cholesterol.

Proteolytic shedding of the prion protein via activation of metallopeptidase ADAM10 reduces cellular binding and toxicity of amyloid-ß oligomers.

The cellular prion protein (PrPC) is a key neuronal receptor for ß-amyloid oligomers (AßO), mediating their neurotoxicity, which contributes to the neurodegeneration in Alzheimer's disease (AD). Similarly to the amyloid precursor protein (APP), PrPC is proteolytically cleaved from the cell surface by a disintegrin and metalloprotease, ADAM10. We hypothesized that ADAM10-modulated PrPC shedding would alter the cellular binding and cytotoxicity of AßO. Here, we found that in human neuroblastoma cells, activation of ADAM10 with the muscarinic agonist carbachol promotes PrPC shedding and reduces the binding of AßO to the cell surface, which could be blocked with an ADAM10 inhibitor. Conversely, siRNA-mediated ADAM10 knockdown reduced PrPC shedding and increased AßO binding, which was blocked by the PrPC-specific antibody 6D11. The retinoic acid receptor analog acitretin, which up-regulates ADAM10, also promoted PrPC shedding and decreased AßO binding in the neuroblastoma cells and in human induced pluripotent stem cell (iPSC)-derived cortical neurons. Pretreatment with acitretin abolished activation of Fyn kinase and prevented an increase in reactive oxygen species caused by AßO binding to PrPC Besides blocking AßO binding and toxicity, acitretin also increased the nonamyloidogenic processing of APP. However, in the iPSC-derived neurons, Aß and other amyloidogenic processing products did not exhibit a reciprocal decrease upon acitretin treatment. These results indicate that by promoting the shedding of PrPC in human neurons, ADAM10 activation prevents the binding and cytotoxicity of AßO, revealing a potential therapeutic benefit of ADAM10 activation in AD.

Quantitative interaction proteomics reveals differences in the interactomes of amyloid precursor protein isoforms.

The generation of the amyloid-ß (Aß) peptides from the amyloid precursor protein (APP) through sequential proteolysis by ß- and γ-secretases is a key pathological event in the initiation and propagation of Alzheimer's disease. Aß and the transcriptionally active APP intracellular domain are generated preferentially from the APP695 isoform compared to the longer APP751 isoform. As the Aß and amyloid precursor protein intracellular domain produced from cleavage of APP695 and APP751 are identical we hypothesised that the two isoforms have differences within their interactomes which mediate the differential processing of the two isoforms. To investigate this, we applied a proteomics-based approach to identify differences in the interactomes of the APP695 and APP751 isoforms. Using stable isotope labelling of amino acids in cell culture and quantitative proteomics, we compared the interactomes of APP695 and APP751 expressed in human SH-SY5Y cells. Through this approach, we identified enrichment of proteins involved in mitochondrial function, the nuclear pore and nuclear transport specifically in the APP695 interactome. Further interrogation of the APP interactome and subsequent experimental validation (co-immunoprecipitation and siRNA knockdown) revealed GAP43 as a specific modulator of APP751 proteolysis, altering Aß generation. Our data indicate that interrogation of the APP interactome can be exploited to identify proteins which influence APP proteolysis and Aß production in an isoform dependent-manner. Cover Image for this issue: doi: 10.1111/jnc.14504.

Modelling Sporadic Alzheimer's Disease Using Induced Pluripotent Stem Cells.

Developing cellular models of sporadic Alzheimer's disease (sAD) is challenging due to the unknown initiator of disease onset and the slow disease progression that takes many years to develop in vivo. The use of human induced pluripotent stem cells (iPSCs) has revolutionised the opportunities to model AD pathology, investigate disease mechanisms and screen potential drugs. The majority of this work has, however, used cells derived from patients with familial AD (fAD) where specific genetic mutations drive disease onset. While these provide excellent models to investigate the downstream pathways involved in neuronal toxicity and ultimately neuronal death that leads to AD, they provide little insight into the causes and mechanisms driving the development of sAD. In this review we compare the data obtained from fAD and sAD iPSC-derived cell lines, identify the inconsistencies that exist in sAD models and highlight the potential role of Aß clearance mechanisms, a relatively under-investigated area in iPSC-derived models, in the study of AD. We discuss the development of more physiologically relevant models using co-culture and three-dimensional culture of iPSC-derived neurons with glial cells. Finally, we evaluate whether we can develop better, more consistent models for sAD research using genetic stratification of iPSCs and identification of genetic and environmental risk factors that could be used to initiate disease onset for modelling sAD. These considerations provide exciting opportunities to develop more relevant iPSC models of sAD which can help drive our understanding of disease mechanisms and identify new therapeutic targets.

Tau Proteolysis in the Pathogenesis of Tauopathies: Neurotoxic Fragments and Novel Biomarkers.

With predictions showing that 131.5 million people worldwide will be living with dementia by 2050, an understanding of the molecular mechanisms underpinning disease is crucial in the hunt for novel therapeutics and for biomarkers to detect disease early and/or monitor disease progression. The metabolism of the microtubule-associated protein tau is altered in different dementias, the so-called tauopathies. Tau detaches from microtubules, aggregates into oligomers and neurofibrillary tangles, which can be secreted from neurons, and spreads through the brain during disease progression. Post-translational modifications exacerbate the production of both oligomeric and soluble forms of tau, with proteolysis by a range of different proteases being a crucial driver. However, the impact of tau proteolysis on disease progression has been overlooked until recently. Studies have highlighted that proteolytic fragments of tau can drive neurodegeneration in a fragment-dependent manner as a result of aggregation and/or transcellular propagation. Proteolytic fragments of tau have been found in the cerebrospinal fluid and plasma of patients with different tauopathies, providing an opportunity to develop these fragments as novel disease progression biomarkers. A range of therapeutic strategies have been proposed to halt the toxicity associated with proteolysis, including reducing protease expression and/or activity, selectively inhibiting protease-substrate interactions, and blocking the action of the resulting fragments. This review highlights the importance of tau proteolysis in the pathogenesis of tauopathies, identifies putative sites during tau fragment-mediated neurodegeneration that could be targeted therapeutically, and discusses the potential use of proteolytic fragments of tau as biomarkers for different tauopathies.

Tau pathology and neurochemical changes associated with memory dysfunction in an optimised murine model of global cerebral ischaemia - A potential model for vascular dementia?

Cerebral ischemia is known to be a major cause of death and the later development of Alzheimer's disease and vascular dementia. However, ischemia induced cellular damage that initiates these diseases remain poorly understood. This is primarily due to lack of clinically relevant models that are highly reproducible. Here, we have optimised a murine model of global cerebral ischaemia with multiple markers to determine brain pathology, neurochemistry and correlated memory deficits in these animals. Cerebral ischaemia in mice was induced by bilateral common carotid artery occlusion. Following reperfusion, the mice were either fixed with 4% paraformaldehyde or decapitated under anaesthesia. Brains were processed for Western blotting or immunohistochemistry for glial (GLT1) and vesicular (VGLUT1, VGLUT2) glutamate transporters and paired helical filament (PHF1) tau. The PHF1 tau is the main component of neurofibrillary tangle, which is the pathological hallmark of Alzheimer's disease and vascular dementia. The novel object recognition behavioural assay was used to investigate the functional cognitive consequences in these mice. The results show consistent and selective neuronal and glial cell changes in the hippocampus and the cortex together with significant reductions in GLT1 (***P < 0.001), VGLUT1 (**P < 0.01) and VGLUT2 (***P < 0.001) expressions in the hippocampus in occluded mice as compared to sham-operated animals. These changes are associated with increased PHF1 (***P < 0.0001) protein and a significant impairment of performance (*p < 0.0006, N = 6/group) in the novel object recognition test. This model represents a useful tool for investigating cellular, biochemical and molecular mechanisms of global cerebral ischaemia and may be an ideal preclinical model for vascular dementia.

Plasma metals as potential biomarkers in dementia: a case-control study in patients with sporadic Alzheimer's disease.

Sporadic Alzheimer's disease (AD) is a neurodegenerative disorder that causes the most prevalent form of age-related dementia but its pathogenesis remains obscure. Altered regulation of metals, particularly pan-cerebral copper deficiency, and more regionally-localized perturbation of other metals, are prominent in AD brain although data on how these CNS perturbations are reflected in the peripheral bloodstream are inconsistent to date. To assess the potential use of metal dysregulation to generate biomarkers in AD, we performed a case-control study of seven essential metals and selenium, measured by inductively coupled plasma mass-spectrometry, in samples from AD and matched control cases. Metals were sodium, potassium, calcium, magnesium, iron, zinc, and copper. In the whole study-group and in female participants, plasma metal levels did not differ between cases and controls. In males by contrast, there was moderate evidence that zinc levels trended towards increase in AD [10.8 (10.2-11.5)] µmol/L, mean (± 95% CI; P = 0.021) compared with controls [10.2 (9.6-10.4)]. Thus alterations in plasma zinc levels differed between genders in AD. In correlational analysis, there was evidence for an increased number of 'strong' metal co-regulations in AD cases and differential co-modulations of metal pairs: copper-sodium (Rcontrol = - 0.03, RAD = 0.65; P = 0.009), and copper-calcium (Rcontrol = - 0.01, RAD = 0.65; P = 0.01) were significant in AD males, potentially consistent with reported evidence for dysregulation of copper in severely damaged brain regions in AD. In conclusion, our data suggest that the measurement of metals co-regulation in plasma may provide a useful representation of those metal perturbations taking place in the AD brain and therefore might be useful as plasma-based biomarkers.

Ablation of Prion Protein in Wild Type Human Amyloid Precursor Protein (APP) Transgenic Mice Does Not Alter The Proteolysis of APP, Levels of Amyloid-ß or Pathologic Phenotype.

The cellular prion protein (PrPC) has been proposed to play an important role in the pathogenesis of Alzheimer's disease. In cellular models PrPC inhibited the action of the ß-secretase BACE1 on wild type amyloid precursor protein resulting in a reduction in amyloid-ß (Aß) peptides. Here we have assessed the effect of genetic ablation of PrPC in transgenic mice expressing human wild type amyloid precursor protein (line I5). Deletion of PrPC had no effect on the α- and ß-secretase proteolysis of the amyloid precursor protein (APP) nor on the amount of Aß38, Aß40 or Aß42 in the brains of the mice. In addition, ablation of PrPC did not alter Aß deposition or histopathology phenotype in this transgenic model. Thus using this transgenic model we could not provide evidence to support the hypothesis that PrPC regulates Aß production.

A Greek Tragedy: The Growing Complexity of Alzheimer Amyloid Precursor Protein Proteolysis.

Proteolysis of the amyloid precursor protein (APP) liberates various fragments including the proposed initiator of Alzheimer disease-associated dysfunctions, amyloid-ß. However, recent evidence suggests that the accepted view of APP proteolysis by the canonical α-, ß-, and γ-secretases is simplistic, with the discovery of a number of novel APP secretases (including δ- and η-secretases, alternative ß-secretases) and additional metabolites, some of which may also cause synaptic dysfunction. Furthermore, various proteins have been identified that interact with APP and modulate its cleavage by the secretases. Here, we give an overview of the increasingly complex picture of APP proteolysis.

Elevation of brain glucose and polyol-pathway intermediates with accompanying brain-copper deficiency in patients with Alzheimer's disease: metabolic basis for dementia.

Impairment of brain-glucose uptake and brain-copper regulation occurs in Alzheimer's disease (AD). Here we sought to further elucidate the processes that cause neurodegeneration in AD by measuring levels of metabolites and metals in brain regions that undergo different degrees of damage. We employed mass spectrometry (MS) to measure metabolites and metals in seven post-mortem brain regions of nine AD patients and nine controls, and plasma-glucose and plasma-copper levels in an ante-mortem case-control study. Glucose, sorbitol and fructose were markedly elevated in all AD brain regions, whereas copper was correspondingly deficient throughout (all P < 0.0001). In the ante-mortem case-control study, by contrast, plasma-glucose and plasma-copper levels did not differ between patients and controls. There were pervasive defects in regulation of glucose and copper in AD brain but no evidence for corresponding systemic abnormalities in plasma. Elevation of brain glucose and deficient brain copper potentially contribute to the pathogenesis of neurodegeneration in AD.

The Role of Tissue Non-specific Alkaline Phosphatase (TNAP) in Neurodegenerative Diseases: Alzheimer's Disease in the Focus.

Tissue non-specific alkaline phosphatase (TNAP) is present on neuronal membranes and induces neuronal toxicity via tau dephosphorylation; a mechanism which could play a role in the neuronal loss seen in Alzheimer's disease (AD). TNAP increases in the plasma following brain injury and cerebrovascular disease. In this chapter we summarise our previous work which looked at changes in TNAP activity in the brain and plasma of AD individuals and discuss whether these changes may be reflective of neuronal loss. Our data demonstrate that TNAP activity is significantly increased in the brain in both the sporadic and familial forms of AD and that TNAP activity is significantly increased in the plasma in AD patients. In addition, we describe a significant inverse correlation between plasma TNAP activity and cognitive function in AD. Using these data we propose a model for TNAP-induced neurodegeneration in AD resulting from tau dephosphorylation following secretion of tau from neuronal cells.

BIN1 is decreased in sporadic but not familial Alzheimer's disease or in aging.

Bridging integrator 1 (BIN1) has been implicated in sporadic Alzheimer's disease (AD) by a number of genome wide association studies (GWAS) in a variety of populations. Here we measured BIN1 in frontal cortex samples from 24 sporadic AD and 24 age-matched non-dementia brains and correlated the expression of this protein with markers of AD. BIN1 was reduced by 87% (p=0.007) in sporadic AD compared to non-dementia controls, but BIN1 in sporadic AD did not correlate with soluble Aß (r(s)=-0.084, p=0.698), insoluble Aß (r(s)=0.237, p=0.269), Aß plaque load (r(s)=0.063, p=0.771) or phospho-tau load (r(s)=-0.160, p=0.489). In contrast to our findings in sporadic AD, BIN1 was unchanged in the hippocampus from 6 cases of familial AD compared to 6 age-matched controls (p=0.488). BIN1 declined with age in a cohort of non-dementia control cases between 25 and 88 years but the correlation was not significant (rs=-0.449, p=0.081). Although BIN1 is known to have a role in endocytosis, and the processing of the amyloid precursor protein (APP) to form amyloid-ß (Aß) peptides is dependent on endocytosis, knockdown of BIN1 by targeted siRNA or the overexpression of BIN1 in a human neuroblastoma cell line (SH-SY5Y) had no effect on APP processing. These data suggest that the alteration in BIN1 is involved in the pathogenesis of sporadic, but not familial AD and is not a consequence of AD neurodegeneration or the ageing process, a finding in keeping with the numerous GWAS that implicate BIN1 in sporadic AD. However, the mechanism of its contribution remains to be established.

Prion protein is decreased in Alzheimer's brain and inversely correlates with BACE1 activity, amyloid-ß levels and Braak stage.

The cellular prion protein (PrP(C)) has been implicated in the development of Alzheimer's disease (AD). PrP(C) decreases amyloid-ß (Aß) production, which is involved in AD pathogenesis, by inhibiting ß-secretase (BACE1) activity. Contactin 5 (CNTN5) has also been implicated in the development of AD by a genome-wide association study. Here we measured PrP(C) and CNTN5 in frontal cortex samples from 24 sporadic AD and 24 age-matched control brains and correlated the expression of these proteins with markers of AD. PrP(C) was decreased in sporadic AD compared to controls (by 49%, p = 0.014) but there was no difference in CNTN5 between sporadic AD and controls (p = 0.217). PrP(C) significantly inversely correlated with BACE1 activity (rs = -0.358, p = 0.006), Aß load (rs = -0.456, p = 0.001), soluble Aß (rs = -0.283, p = 0.026) and insoluble Aß (rs = -0.353, p = 0.007) and PrP(C) also significantly inversely correlated with the stage of disease, as indicated by Braak tangle stage (rs = -0.377, p = 0.007). CNTN5 did not correlate with Aß load (rs = 0.040, p = 0.393), soluble Aß (rs = 0.113, p = 0.223) or insoluble Aß (rs = 0.169, p = 0.125). PrP(C) was also measured in frontal cortex samples from 9 Down's syndrome (DS) and 8 age-matched control brains. In contrast to sporadic AD, there was no difference in PrP(C) in the DS brains compared to controls (p = 0.625). These data are consistent with a role for PrP(C) in regulating Aß production and indicate that brain PrP(C) level may be important in influencing the onset and progression of sporadic AD.

Discovery of biphenylacetamide-derived inhibitors of BACE1 using de novo structure-based molecular design.

ß-Secretase (BACE1), the enzyme responsible for the first and rate-limiting step in the production of amyloid-ß peptides, is an attractive target for the treatment of Alzheimer's disease. In this study, we report the application of the de novo fragment-based molecular design program SPROUT to the discovery of a series of nonpeptide BACE1 inhibitors based upon a biphenylacetamide scaffold. The binding affinity of molecules based upon this designed molecular scaffold was increased from an initial BACE1 IC50 of 323 µM to 27 µM following the synthesis of a library of optimized ligands whose structures were refined using the recently developed SPROUT-HitOpt software. Although a number of inhibitors were found to exhibit cellular toxicity, one compound in the series was found to have useful BACE1 inhibitory activity in a cellular assay with minimal cellular toxicity. This work demonstrates the power of an in silico fragment-based molecular design approach in the discovery of novel BACE1 inhibitors.