Docking studies to explore novel inhibitors against human beta-site APP cleaving enzyme (BACE-1) involved in Alzheimer’s disease

Alzheimer’s disease (AD) is one of the most prominent neurodegenerative disorders, particularly in elder persons over 65 age. It is characterized by progressive cognitive deterioration together with declining activities. Amyloid precursor protein (APP) cleaves at A-beta (Aβ) peptide by rate limiting factor of Beta-site APP cleaving enzyme (BACE-1) in amyloidogenic pathway. Elevated level of BACE-1 leads to the accumulation of an insoluble form of Aβ peptides (Senile Plaques), an important hallmark in the pathogenesis of Alzheimer disease. Five published inhibitors of BACE-1, thiazolidinediones, rosiglitazone, pioglitazone, Sc7 and tartaric acid are available with poor pharmacological properties and intolerable side effects. Therefore, a computational approach was undertaken to design novel inhibitors against human BACE-1. The crystal structure of human BACE-1 was retrieved from the protein data bank and optimized by applying OPLS force field in Maestro v9.2. An ASINEX database (115,000 ligands) was downloaded and compounds were prepared using LigPrep. The optimized ligand dataset was docked into the BACE-1 through sequential application of Glide HTVS, SP and XP methods that penalizes more stringently for minor steric classes subsequently. Finally, seven leads were reported and ranked based on XPGscore with better binding affinity and good pharmacological properties compared with existing inhibitors. Six leads were proposed for human BACE-1. Among the six, lead 1, with XPGscore -8.051Kcal/mol, would be intriguing for rational drug design against Alzheimer’s disease and would be highly encouraging for future Alzheimer’s therapy if tested in animal models

Chronic BACE-1 Inhibitor Administration in TASTPM Mice (APP KM670/671NL and PSEN1 M146V Mutation): An EEG Study

Objective: In this exploratory study, we tested whether electroencephalographic (EEG) rhythms may reflect the effects of a chronic administration (4 weeks) of an anti-amyloid β-site amyloid precursor protein (APP) cleaving enzyme 1 inhibitor (BACE-1; ER-901356; Eisai Co., Ltd., Tokyo, Japan) in TASTPM (double mutation in APP KM670/671NL and PSEN1 M146V) producing Alzheimer's disease (AD) amyloid neuropathology as compared to wild type (WT) mice. Methods: Ongoing EEG rhythms were recorded from a bipolar frontoparietal and two monopolar frontomedial (prelimbic) and hippocampal channels in 11 WT Vehicle, 10 WT BACE-1, 10 TASTPM Vehicle, and 11 TASTPM BACE-1 mice (males; aged 8/9 months old at the beginning of treatment). Normalized EEG power (density) was compared between the first day (Day 0) and after 4 weeks (Week 4) of the BACE-1 inhibitor (10 mg/Kg) or vehicle administration in the 4 mouse groups. Frequency and magnitude of individual EEG delta and theta frequency peaks (IDF and ITF) were considered during animal conditions of behaviorally passive and active wakefulness. Cognitive status was not tested. Results: Compared with the WT group, the TASTPM group generally showed a significantly lower reactivity in frontoparietal ITF power during the active over the passive condition (p < 0.05). Notably, there was no other statistically significant effect (e.g., additional electrodes, recording time, and BACE-1 inhibitor). Conclusions: The above EEG biomarkers reflected differences between the WT and TASTPM groups, but no BACE-1 inhibitor effect. The results suggest an enhanced experimental design with the use of younger mice, longer drug administrations, an effective control drug, and neuropathological amyloid markers

Identification of a sub-micromolar, non-peptide inhibitor of β-secretase with low neural cytotoxicity through in silico screening

Nowadays identification of novel non-peptide β-secretase (BACE-1, hereinafter) inhibitors with low cytotoxicity and good blood–brain barrier (BBB) property holds common interest of drug discovery for Alzheimer’s disease. Twenty SPECS compounds were tested in BACE-1 FRET assays and methylthiazoletetrazolium (MTT) cytotoxicity experiment. Two compounds: 2 and 15 demonstrated IC50 values of 0.53 and 9.4 μM. In addition, 2 showed least toxic effect to the neuroblastoma cells. The results from both in silico and in vitro studies provided new pharmacophoric entities for chemical synthesis and optimization on the current discovered BACE-1 small molecule inhibitors

Unveiling a Novel Transient Druggable Pocket in BACE-1 through Molecular Simulations: Conformational Analysis and Binding Mode of Multisite Inhibitors

The critical role of BACE-1 in the formation of neurotoxic ß-amyloid peptides in the brain makes it an attractive target for an efficacious treatment of Alzheimer's disease. However, the development of clinically useful BACE-1 inhibitors has proven to be extremely challenging. In this study we examine the binding mode of a novel potent inhibitor (compound 1, with IC50 80 nM) designed by synergistic combination of two fragments - huprine and rhein - that individually are endowed with very low activity against BACE-1. Examination of crystal structures reveals no appropriate binding site large enough to accommodate 1. Therefore we have examined the conformational flexibility of BACE-1 through extended molecular dynamics simulations, paying attention to the highly flexible region shaped by loops 8-14, 154-169 and 307-318. The analysis of the protein dynamics, together with studies of pocket druggability, has allowed us to detect the transient formation of a secondary binding site, which contains Arg307 as a key residue for the interaction with small molecules, at the edge of the catalytic cleft. The formation of this druggable 'floppy' pocket would enable the binding of multisite inhibitors targeting both catalytic and secondary sites. Molecular dynamics simulations of BACE-1 bound to huprine-rhein hybrid compounds support the feasibility of this hypothesis. The results provide a basis to explain the high inhibitory potency of the two enantiomeric forms of 1, together with the large dependence on the length of the oligomethylenic linker. Furthermore, the multisite hypothesis has allowed us to rationalize the inhibitory potency of a series of tacrine-chromene hybrid compounds, specifically regarding the apparent lack of sensitivity of the inhibition constant to the chemical modifications introduced in the chromene unit. Overall, these findings pave the way for the exploration of novel functionalities in the design of optimized BACE-1 multisite inhibitors

Unveiling a novel transient druggable pocket in BACE-1 through molecular simulations: conformational analysis and binding mode of multisite inhibitors

The critical role of BACE-1 in the formation of neurotoxic ß-amyloid peptides in the brain makes it an attractive target for an efficacious treatment of Alzheimer’s disease. However, the development of clinically useful BACE-1 inhibitors has proven to be extremely challeng- ing. In this study we examine the binding mode of a novel potent inhibitor (compound 1, with IC50 80 nM) designed by synergistic combination of two fragments—huprine and rhein— that individually are endowed with very low activity against BACE-1. Examination of crystal structures reveals no appropriate binding site large enough to accommodate 1. Therefore we have examined the conformational flexibility of BACE-1 through extended molecular dynamics simulations, paying attention to the highly flexible region shaped by loops 8–14, 154–169 and 307–318. The analysis of the protein dynamics, together with studies of pocket druggability, has allowed us to detect the transient formation of a secondary binding site, which contains Arg307 as a key residue for the interaction with small molecules, at the edge of the catalytic cleft. The formation of this druggable “floppy” pocket would enable the bind- ing of multisite inhibitors targeting both catalytic and secondary sites. Molecular dynamics simulations of BACE-1 bound to huprine-rhein hybrid compounds support the feasibility of this hypothesis. The results provide a basis to explain the high inhibitory potency of the two enantiomeric forms of 1, together with the large dependence on the length of the oligo- methylenic linker. Furthermore, the multisite hypothesis has allowed us to rationalize the inhibitory potency of a series of tacrine-chromene hybrid compounds, specifically regarding the apparent lack of sensitivity of the inhibition constant to the chemical modifications intro- duced in the chromene unit. Overall, these findings pave the way for the exploration of novel functionalities in the design of optimized BACE-1 multisite inhibitors

Alvos terapêuticos na Doença de Alzheimer: relevância da BACE I e o desenvolvimento de inibidores desta secretase

Monografia realizada no âmbito da unidade de Estágio Curricular do Mestrado Integrado em Ciências Farmacêuticas, apresentada à Faculdade de Farmácia da Universidade de CoimbraA Doença de Alzheimer (DA) é uma doença neurodegenerativa progressiva e irreversível que apresenta duas lesões características: depósitos β-amilóide (Aβ) e tranças neurofibrilares (NFTs). Clinicamente, a DA é caracterizada por perda progressiva e gradual de memória e de outras funções cognitivas. A hipótese globalmente aceite para a patogénese da DA baseia-se na teoria da cascata amilóide e subsequente patologia de tau, uma vez que a toxicidade amilóide induz a hiperfosforilação da proteína tau. A descoberta recente de biomarcadores pré-clínicos é importante para o diagnóstico da DA em fases precoces da doença. Ao longo dos anos as investigações para a cura da DA têm sido incessantes, contudo ainda não existem terapêuticas capazes de impedir a progressão da doença. A BACE 1 (β-secretase) é a enzima que inicia a produção de Aβ, e é apontada como um alvo terapêutico primordial no tratamento da DA. A inibição da BACE 1 apresenta consequências benéficas na diminuição das concentrações Aβ. Durante a última década o foco tem sido o desenvolvimento de inibidores com boas propriedades farmacológicas que inibam eficazmente a BACE 1. Alguns inibidores mostraram-se eficazes nos estudos pré-clínicos e entraram recentemente em ensaios clínicos, sendo o MK-8931 o que se encontra em fase mais avançada. O potencial terapêutico da inibição de BACE.I pode ser a chave para um tratamento eficaz da DA no futuro.Alzheimer’s Disease (AD) is a progressive neurodegenerative disease and its hallmarks consist in amyloid-β deposits (Aβ) and neurofibrillary tangles (NFTs). Clinically, AD is characterized by progressive memory loss and other cognitive deficits. The globally accepted hypothesis to explain DA is based on the amyloid-β cascade theory and subsequent tau pathology, since amyloid toxicity induces tau protein hyper-phosphorylation. The recent discovery of pre-clinical biomarkers can be used to diagnose AD at an early stage. Over the years, the research for AD cure has been relentless, however there are still no effective therapies available to prevent the advance of the disease. BACE 1 (β-secretase), the enzyme that leads to the production of Aβ in the brain, is considered to be a major therapeutic target in treating Alzheimer ’s Disease. Inhibition of BACE 1 shows beneficial effects in lowering cerebral Aβ concentrations. During the last decade the focus has been in the development of inhibitors with drug-like properties that can effectively inhibit BACE 1. Some BACE 1 inhibitors have shown safety and efficacy in preclinical studies, and have recently entered human clinical trials. Thus far, MK-8931 has advanced the farthest and is in phase 3. The therapeutic potential of inhibiting BACE 1 may be the key to an effective treatment of AD in the future

From virtual screening hits targeting a cryptic pocket in BACE-1 to a nontoxic brain permeable multitarget anti-Alzheimer lead with disease-modifying and cognition-enhancing effects

Starting from six potential hits identified in a virtual screening campaign directed to a cryptic pocket of BACE-1, at the edge of the catalytic cleft, we have synthesized and evaluated six hybrid compounds, designed to simultaneously reach BACE-1 secondary and catalytic sites and to exert additional activities of interest for Alzheimer's disease (AD). We have identified a lead compound with potent in vitro activity towards human BACE-1 and cholinesterases, moderate Ab42 and tau antiaggregating activity, and brain permeability, which is nontoxic in neuronal cells and zebrafish embryos at concentrations above those required for the in vitro activities. This compound completely restored short- and long-term memory in a mouse model of AD (SAMP8) relative to healthy control strain SAMR1, shifted APP processing towards the non-amyloidogenic pathway, reduced tau phosphorylation, and increased the levels of synaptic proteins PSD95 and synaptophysin, thereby emerging as a promising disease-modifying, cognitionenhancing anti-AD lead

Binding of Beta-site Amyloid Precursor Protein Cleaving Enzyme 1 (BACE1) Inhibitor Aminoquinoline (68K) for Possible Treatment of Alzheimer\u27s Disease

Alzheimer’s Disease (AD), affecting approximately 24 million people worldwide, is characterized by the formation of amyloid-β plaques within the brain. Alzheimer’s research has been focused on limiting amyloid-β production through developing inhibitors for the enzymes needed within the amyloid cascade. This project focuses on the aminoquinoline class of inhibitors, of which 68K (PDB: 5i3Y) is the most effective because of its strong Kd and IC50 values. The students of the Honors Protein Modeling class at Nova Southeastern University modeled the interaction between Beta-site Amyloid Precursor Protein Cleaving Enzyme 1 (BACE-1) and 68K. Using Jmol a model was developed, and 3D printed to show how the inhibitor (68K) fit into the enzyme’s active site. This model highlights important aspects of the interactions between the ligand and the BACE-1 enzyme. 68K has strong interactions with 32 amino acid residues in BACE1, some of which are intertwined with one another. For example, BACE-1’s residues Val69, Pro70, and Tyr71 are known collectively as “the flap”. “The flap” is a β-hairpin loop structure that is positioned directly over BACE-1’s catalytic dyad, a group of amino acids within the active site of the enzyme. “The flap” is also responsible for regulating access to the enzyme’s catalytic dyad (Asp 32 and Asp 228) by a given substrate (or inhibitor). Researchers found the inhibitor 68K to have interactions with the flap which maximizes the strength of the interaction with BACE-1 residues, thus minimizing the distance between the inhibitor’s various functional groups and accommodating their specific polarities. Being able to visualize the protein structure using a 3D model aids in the understanding of how the ligand inhibits this enzyme leading to the progression of AD.https://nsuworks.nova.edu/protein_modeling_reports/1011/thumbnail.jp

Analyse der BACE-1-Regulation in der Alzheimer-Krankheit und im Down-Syndrom

Die Alzheimer-Krankheit (AD), die häufigste Form der Demenz, manifestiert sich klinisch meist ab dem 65. Lebensjahr mit langsam progredienten Gedächtnis-, Orientierungs- und Aufmerksamkeitsstörungen. Neuropathologische Korrelate sind die Amyloid-Plaques, die hauptsächlich aus extrazellulären Aggregaten des β-Amyloid-Proteins (Aβ) zusammengesetzt sind, und intrazelluläre neurofibrilläre Bündel, die aus Aggregaten des mikrotubulus-assoziierten Proteins Tau bestehen. Der „Auslöser“ der Alzheimer-Krankheit ist das β-Amyloid-Protein (Amyloid-Kaskaden-Hypothese), welches durch proteolytische Prozessierung von βAPP (β-Amyloid-Vorläufer-Protein) entsteht. Dies geschieht, indem zuerst BACE-1 (β-site APP cleaving enzyme 1) und anschließend der γ-Sekretase-Komplex βAPP schneiden. Menschen mit Down-Syndrom (DS), welches die häufigste autosomale Chromosomenaberration darstellt, entwickeln bereits ab einem Alter von 40 Jahren die typischen neuropathologischen Kennzeichen der Alzheimer-Krankheit und zeigen mit einem Durchschnittsalter von 56 Jahren die klinischen Symptome der Demenz. Die Gründe für das frühe Auftreten der Amyloid-Plaques sind noch nicht vollständig geklärt. Das Gen für βAPP befindet sich auf dem Chromosom 21, welches im DS dreifach vorhanden ist, und wird daher mit der frühen Plaque-Pathologie im Down-Syndrom in Zusammenhang gebracht. BACE-1 konnte durch Überexpressions- und Knock-out-Experimente eindeutig als alleinige β-Sekretase identifiziert werden. In Gehirnen von Alzheimer-Demenz-Patienten konnten im Vergleich zu Kontrollgehirnen erhöhte BACE-1-Proteinmengen und BACE-1-Aktivitäten nachgewiesen werden. Es war deshalb interessant zu untersuchen, ob auch im Gehirn von Down-Syndrom-Patienten die BACE-1-Proteinexpression erhöht ist. Durch eine Hochregulation von BACE-1 im Down-Syndrom könnte vermehrt βAPP prozessiert und somit Aβ gebildet werden. Daher war es das Ziel der vorliegenden Arbeit die Expression von BACE-1 in Gehirnen (Temporal- und Frontallappen) von DS-Patienten im Vergleich zu Kontrollgehirnen zu analysieren. In der Western-Blot-Analyse der Gewebeproben konnte gezeigt werden, dass die BACE-1-Proteinmengen im Down-Syndrom im Vergleich zu den Kontrollen (K) 1,4-fach erhöht waren. Die Proteinexpressionen von βAPP zeigten sich im DS im Vergleich zu den Kontrollen 1,4-fach erhöht. Diese Ergebnisse erzielten keine Signifikanz, zeigten aber deutliche Trends im Expressionsverhalten. Dies könnte auf die Anzahl der untersuchten Gehirne (Temporal- und Frontallappen je 3 DS, 4 AD und 5 K), Qualitätsmängel der Gehirnproben oder einer ungleichen Verteilung der Proteinexpression im Gewebe zurückzuführen sein. Es ist daher notwendig mehr Gehirne zu untersuchen. Zudem wäre es interessant in Gehirnschnitten das Verteilungsmuster der BACE-1-Expression im DS genauer zu studieren. Die Ergebnisse deuten jedoch auf eine Hochregulation von BACE-1 im DS-Gehirn und somit auf eine Beteiligung der Protease an der Plaquepathologie des Down-Syndroms hin. BACE-1 scheint also sowohl in der Pathogenese der Alzheimer-Demenz als auch in der des Down-Syndroms eine zentrale Rolle einzunehmen. Daher ist es sehr interessant die Regulation von BACE-1 weiter zu analysieren. Da in p25-überexprimierenden Mäusen erhöhte BACE-1-Proteinexpressionen gezeigt werden konnten, vermutete man eine Beteiligung von p25 an der Regulation der β-Sekretase. p25, das im Gehirn der AD-Patienten vermehrt gebildet wird und aus der Proteolyse von p35 entsteht, bindet und aktiviert die Kinase cdk5. cdk5 phosphoryliert unter anderem das Tau-Protein und wird daher mit der Bildung der neurofibrillären Bündel in Zusammenhang gebracht. Durch die Hochregulation von BACE-1 könnte p25 in der Pathogenese der Alzheimer-Erkrankung eine neue Bedeutung zugeschrieben werden. Zur Analyse der p25 induzierten Veränderungen in den Neuronen wurden humane Neuroblastomazellen mit einem induzierbaren p25-Expressionsvektor, Sp25-Zellen, verwendet. In diesen p25-überexprimierenden Zellen konnten sowohl in der Western-Blot-Analyse als auch in der BACE-1-Aktivitätsmessung erhöhte BACE-1-Proteinexpressionen bzw. BACE-1-Aktivitäten gezeigt werden. Die Northern-Blot-Analyse der Sp25-Zellen ergab erhöhte BACE-1-mRNA-Spiegel, die sich jedoch in einer für endogene BACE-1-mRNA untypische Größe detektieren ließen. p35, das membrangebundene Vorläufer-Protein von p25, war indes nicht in der Lage die BACE-1-Proteinexpression in humanen Neuroblastomazellen zu erhöhen. Die Ergebnisse der Sp25-Zellen konnten in p25-überexprimierenden murinen Neuroblastomazellen, Np25-Zellen, nicht reproduziert werden. Daher ist es notwendig, den p25-induzierten BACE-1-Regulationsmechanismus auf seine Reproduzierbarkeit, z.B. in weiteren In-vivo-Modellen, zu überprüfen