Fragment-based virtual screening approach and molecular dynamics simulation studies for identification of BACE1 inhibitor leads

Traditional structure-based virtual screening method to identify drug-like small molecules for BACE1 is so far unsuccessful. Location of BACE1, poor Blood Brain Barrier permeability and P-glycoprotein (Pgp) susceptibility of the inhibitors make it even more difficult. Fragment-based drug design method is suitable for efficient optimization of initial hit molecules for target like BACE1. We have developed a fragment-based virtual screening approach to identify/optimize the fragment molecules as a starting point. This method combines the shape, electrostatic, and pharmacophoric features of known fragment molecules, bound to protein conjugate crystal structure, and aims to identify both chemically and energetically feasible small fragment ligands that bind to BACE1 active site. The two top-ranked fragment hits were subjected for a 53 ns MD simulation. Principle component analysis and free energy landscape analysis reveal that the new ligands show the characteristic features of established BACE1 inhibitors. The potent method employed in this study may serve for the development of potential lead molecules for BACE1-directed Alzheimer’s disease therapeutics.     

Inhibition of beta-secretase in vivo via antibody binding to unique loops (D and F) of BACE1

β-Secretase (BACE1) is an attractive drug target for Alzheimer disease. However, the design of clinical useful inhibitors targeting its active site has been extremely challenging. To identify alternative drug targeting sites we have generated a panel of BACE1 monoclonal antibodies (mAbs) that interfere with BACE1 activity in various assays and determined their binding epitopes. mAb 1A11 inhibited BACE1 in vitro using a large APP sequence based substrate (IC(50) ～0.76 nm), in primary neurons (EC(50) ～1.8 nm), and in mouse brain after stereotactic injection. Paradoxically, mAb 1A11 increased BACE1 activity in vitro when a short synthetic peptide was used as substrate, indicating that mAb 1A11 does not occupy the active-site. Epitope mapping revealed that mAb 1A11 binds to adjacent loops D and F, which together with nearby helix A, distinguishes BACE1 from other aspartyl proteases. Interestingly, mutagenesis of loop F and helix A decreased or increased BACE1 activity, identifying them as enzymatic regulatory elements and as potential alternative sites for inhibitor design. In contrast, mAb 5G7 was a potent BACE1 inhibitor in cell-free enzymatic assays (IC(50) ～0.47 nm) but displayed no inhibitory effect in primary neurons. Its epitope, a surface helix 299-312, is inaccessible in membrane-anchored BACE1. Remarkably, mutagenesis of helix 299-312 strongly reduced BACE1 ectodomain shedding, suggesting that this helix plays a role in BACE1 cellular biology. In conclusion, this study generated highly selective and potent BACE1 inhibitory mAbs, which recognize unique structural and functional elements in BACE1, and uncovered interesting alternative sites on BACE1 that could become targets for drug development.     

Unmasking BACE1 in aging and age-related diseases

The beta-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) has long been considered a conventional target for Alzheimer’s disease (AD). Unfortunately, AD clinical trials of most BACE1 inhibitors were discontinued due to ineffective cognitive improvement or safety challenges. Recent studies investigating the involvement of BACE1 in metabolic, vascular, and immune functions have indicated a role in aging, diabetes, hypertension, and cancer. These novel BACE1 functions have helped to identify new ‘druggable’ targets for BACE1 against aging comorbidities. In this review, we discuss BACE1 regulation during aging, and then provide recent insights into its enzymatic and nonenzymatic involvement in aging and age-related diseases. Our study not only proposes the perspective of BACE1’s actions in various systems, but also provides new directions for using BACE1 inhibitors and modulators to delay aging and to treat age-related diseases.     

Structure–activity relationship study of BACE1 inhibitors possessing a chelidonic or 2,6-pyridinedicarboxylic scaffold at the P2 position

We have previously reported potent substrate-based pentapeptidic BACE1 inhibitors possessing a hydroxymethylcarbonyl isostere as a substrate transition-state mimic. While these inhibitors exhibited potent activities in enzymatic and cellular assays (KMI-429 in particular inhibited Aβ production in vivo), these inhibitors contained some natural amino acids that seemed to be required to improve enzymatic stability in vivo and permeability across the blood–brain barrier, so as to be practical drug. Recently, we synthesized non-peptidic and small-sized BACE1 inhibitors possessing a heterocyclic scaffold at the P₂ position. Herein we report the SAR study of BACE1 inhibitors possessing this heterocyclic scaffold, a chelidonic or 2,6-pyridinedicarboxylic moiety.     

Molecular docking based virtual screening of natural compounds as potential BACE1 inhibitors: 3D QSAR pharmacophore mapping and molecular dynamics analysis

Beta-site APP cleaving enzyme1 (BACE1) catalyzes the rate determining step in the generation of Aβ peptide and is widely considered as a potential therapeutic drug target for Alzheimer’s disease (AD). Active site of BACE1 contains catalytic aspartic (Asp) dyad and flap. Asp dyad cleaves the substrate amyloid precursor protein with the help of flap. Currently, there are no marketed drugs available against BACE1 and existing inhibitors are mostly pseudopeptide or synthetic derivatives. There is a need to search for a potent inhibitor with natural scaffold interacting with flap and Asp dyad. This study screens the natural database InterBioScreen, followed by three-dimensional (3D) QSAR pharmacophore modeling, mapping, in silico ADME/T predictions to find the potential BACE1 inhibitors. Further, molecular dynamics of selected inhibitors were performed to observe the dynamic structure of protein after ligand binding. All conformations and the residues of binding region were stable but the flap adopted a closed conformation after binding with the ligand. Bond oligosaccharide interacted with the flap as well as catalytic dyad via hydrogen bond throughout the simulation. This led to stabilize the flap in closed conformation and restricted the entry of substrate. Carbohydrates have been earlier used in the treatment of AD because of their low toxicity, high efficiency, good biocompatibility, and easy permeability through the blood–brain barrier. Our finding will be helpful in identify the potential leads to design novel BACE1 inhibitors for AD therapy.     

A surface plasmon resonance-based biosensor with full-length BACE1 in a reconstituted membrane

A surface plasmon resonance (SPR) biosensor-based assay for membrane-embedded full-length BACE1 (β-site amyloid precursor protein cleaving enzyme 1), a drug target for Alzheimer’s disease, has been developed. It allows the analysis of interactions with the protein in its natural lipid membrane environment. The enzyme was captured via an antibody recognizing a C-terminal His6 tag, after which a lipid membrane was reconstituted on the chip using a brain lipid extract. The interaction between the enzyme and several inhibitors confirmed that the surface was functional. It had slightly different interaction characteristics as compared with a reference surface with immobilized ectodomain BACE1 but had the same inhibitor characteristic pH effect. The possibility of studying interactions with BACE1 under more physiological conditions than assays using truncated enzyme or conditions dictated by high enzyme activity is expected to increase our understanding of the role of BACE1 in Alzheimer’s disease and contribute to the discovery of clinically efficient BACE1 inhibitors. The strategy exploited in the current study can be adapted to other membrane-bound drug targets by selecting suitable capture antibodies and lipid mixtures for membrane reconstitution.     

Triazolyl tryptoline derivatives as β-secretase inhibitors

Tryptoline, a core structure of ochrolifuanine E, which is a hit compound from virtual screening of the Thai herbal database against BACE1 was used as a scaffold for the design of BACE1 inhibitors. The tryptoline was linked with different side chains by 1,2,3-triazole ring readily synthesized by catalytic azide-alkyne cycloaddition reactions. Twenty two triazolyl tryptoline derivatives were synthesized and screened for the inhibitory action against BACE1. JJCA-140 was the most potent inhibitor (IC(50)=1.49 μM) and was 100 times more selective for BACE1 than for Cat-D.     

Computational analysis of BACE1-ligand complex crystal structures and linear discriminant analysis for identification of BACE1 inhibitors with anti P-glycoprotein binding property

More than 100 years of research on Alzheimer’s disease didn’t yield a potential cure for this dreadful disease. Poor Blood Brain Barrier (BBB) permeability and P-glycoprotein binding of BACE1 inhibitors are the major causes for the failure of these molecules during clinical trials. The design of BACE1 inhibitors with a balance of sufficient affinity to the binding site and little or no interaction with P-glycoproteins is indispensable. Identification and understanding of protein–ligand interactions are essential for ligand optimization process. Structure-based drug design (SBDD) efforts led to a steady accumulation of BACE1-ligand crystal complexes in the PDB. This study focuses on analyses of 153 BACE1-ligand complexes for the direct contacts (hydrogen bonds and weak interactions) observed between protein and ligand and indirect contacts (water-mediated hydrogen bonds), observed in BACE1-ligand complex crystal structures. Intraligand hydrogen bonds were analyzed, with focus on ligand P-glycoprotein efflux. The interactions are dissected specific to subsites in the active site and discussed. The observed protein-ligand and intraligand interactions were used to develop the linear discriminant model for the identification of BACE1 inhibitors with less or no P-glycoprotein binding property. Excellent statistical results and model’s ability to correctly predict a new data-set with an accuracy of 92% is achieved. The results are retrospectively analyzed to give input for the design of potential BACE1 inhibitors.     

A novel approach to identifying beta-secretase inhibitors: bis-statine peptide mimetics discovered using structure and spot synthesis

Beta-secretase 1 (BACE1) is an aspartic protease believed to play a critical role in Alzheimer's disease. Inhibitors of this enzyme have been designed by incorporating the non-cleavable hydroxyethylene and statine isosteres into peptides corresponding to BACE1 substrate sequences. We sought to develop new methods to quickly characterize and optimize inhibitors based on the statine core. Minimal sequence requirements for binding were first established using both crystallography and peptide spot synthesis. These shortened peptide inhibitors were then optimized by using spot synthesis to perform iterative cycles of substitution and deletion. The present study resulted in the identification of novel "bis-statine" inhibitors shown by crystallography to have a unique binding mode. Our results demonstrate the application of peptide spot synthesis as an effective method for enhancing peptidomimetic drug discovery.     

Tetrapeptides,as small‐sized peptidic inhibitors; synthesis and their inhibitory activity against BACE1

β‐Site amyloid precursor protein cleaving enzyme 1 (BACE1) is known to be involved in the production of amyloid β‐peptide in Alzheimer's disease and is a major target for current drug design. We previously reported substrate‐based peptidomimetics, KMI‐compounds as potent BACE1 inhibitors. In this study, we designed and synthesized tetrapeptides as low molecular‐sized inhibitors. These exhibited high potency against recombinant BACE1, with the highest IC₅₀ value of 34.6 nM from KMI‐927. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Tripeptidic BACE1 inhibitors devised by in-silico conformational structure-based design

Previously reported pentapeptidic BACE1 inhibitors, designed using a substrate-based approach, were used as lead compounds for the further design of non-peptidic BACE1 inhibitors. Although these peptidic and non-peptidic inhibitors, with a hydroxymethylcarbonyl isostere as a substrate transition-state mimic, exhibited potent BACE1 inhibitory activities, their molecular-sizes appeared a little too big (molecular weight of >600daltons) for developing practical anti-Alzheimer's disease drugs. To develop lower weight BACE1 inhibitors, a series of tripeptidic BACE1 inhibitors were devised using a design approach based on the conformation of a virtual inhibitor bound to the BACE1 active site, also called 'in-silico conformational structure-based design'. Although these tripeptidic BACE1 inhibitors contained some natural amino acid residues, they are expected to be useful as lead compounds for developing the next generation BACE1 inhibitors, due to their low molecular size and unique structural features compared with previously reported inhibitors.     

Identification of inhibitors using a cell-based assay for monitoring Golgi-resident protease activity

Noninvasive real-time quantification of cellular protease activity allows monitoring of enzymatic activity and identification of activity modulators within the protease's natural milieu. We developed a protease activity assay based on differential localization of a recombinant reporter consisting of a Golgi retention signal and a protease cleavage sequence fused to alkaline phosphatase (AP). When expressed in mammalian cells, this protein localizes to Golgi bodies and, on protease-mediated cleavage, AP translocates to the extracellular medium where its activity is measured. We used this system to monitor the Golgi-associated protease furin, a pluripotent enzyme with a key role in tumorigenesis, viral propagation of avian influenza, ebola, and HIV as well as in activation of anthrax, pseudomonas, and diphtheria toxins. This technology was adapted for high-throughput screening of 39,000-compound small molecule libraries, leading to identification of furin inhibitors. Furthermore, this strategy was used to identify inhibitors of another Golgi protease, the beta-site amyloid precursor protein (APP)-cleaving enzyme (BACE). BACE cleavage of the APP leads to formation of the Abeta peptide, a key event that leads to Alzheimer's disease. In conclusion, we describe a customizable noninvasive technology for real-time assessment of Golgi protease activity used to identify inhibitors of furin and BACE.     

BACE: Therapeutic target and potential biomarker for Alzheimer's disease

β-Site APP-cleaving enzyme (BACE) is a membrane-bound aspartyl protease involved in the production of Alzheimer's disease (AD) Aβ amyloid peptides. This enzyme is ubiquitously expressed, with highest levels in the brain and pancreas. Its cellular trafficking is tightly controlled as it recycles between endosomes and trans-Golgi network. BACE expression increases in response to aging and various stress stimuli. It is elevated in the brain cortex of AD sufferers, and increased levels of BACE in the cerebrospinal fluid of patients with mild cognitive impairment may provide an early biomarker of AD. BACE is considered as a rational drug target for AD therapy, and inhibitors are under development. Anomalies in the behaviour and biochemistry of BACE^?/? mice have pointed to the role this enzyme plays in the processing of neuregulin and of voltage-gated sodium channel β-subunit. A full understanding of BACE biology in health and disease is needed to establish a safe AD therapy based on BACE inhibitors.     

Pharmacological applications of a novel neoepitope antibody to a modified amyloid precursor protein-derived beta-secretase product

We have previously described a novel artificial NFEV β-secretase (BACE1) cleavage site, which when introduced into the amyloid-β precursor protein (APP), significantly enhances APP cleavage by BACE1 in in vitro and cellular assays. In this study, we describe the identification and characterization of a single chain fragment of variable region (scFv), specific to the EV neo-epitope derived from BACE1 cleavage of the NFEV-containing peptide, and its conversion to IgG1. Both the scFv displayed on phage and EV-IgG1 show exquisite specificity for binding to the EV neoepitope without cross-reactivity to other NFEV containing peptides or WT-APP KMDA cleavage products. EV-IgG1 can detect as little as 0.3 nmol/L of the EV peptide. EV-IgG1 antibody was purified, conjugated with alkaline phosphatase and utilized in various biological assays. In the BACE1 enzymatic assay using NFEV substrate, a BACE1 inhibitor MRK-3 inhibited cleavage with an IC₅₀ of 2.4 nmol/L with excellent reproducibility. In an APP_NFEV stable SH-SY5Y cellular assay, the EC₅₀ for inhibition of EV-Aβ peptide secretion with MRK-3 was 236 nmol/L, consistent with values derived using an EV polyclonal antibody. In an APP_NFEV knock-in mouse model, both Aβ_EV40 and Aβ_EV42 peptides in brain homogenate showed excellent gene dosage dependence. In conclusion, the EV neoepitope specific monoclonal antibody is a novel reagent for BACE1 inhibitor discovery for both in vitro, cellular screening assays and in vivo biochemical studies. The methods described herein are generally applicable to novel synthetic substrates and enzyme targets to enable robust screening platforms for enzyme inhibitors.     

New evolutions in the BACE1 inhibitor field from 2014 to 2018

β-Site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors offer the potential of disease-modifying treatment for Alzheimer’s disease (AD). Since 2014, major breakthroughs have appeared in the field of BACE1 inhibitors. This review provides an overview of amidine-based BACE1 inhibitors between 2014 and 2018. Herein are summarized i) the structure-activity relationship, ii) the physiological results and iii) the potential risks from a lack of selectivity. This review also summarizes clinical scope, results and outlook of the compounds that have been or are currently under development in clinical trials.     

Significance of interactions of BACE1–Arg235 with its ligands and design of BACE1 inhibitors with P2 pyridine scaffold

Recently, we reported potent substrate-based pentapeptidic BACE1 inhibitors possessing a hydroxymethylcarbonyl isostere as a substrate transition-state mimic. Because these inhibitors contained some natural amino acids, we would need to improve their enzymatic stability in vivo and permeability across the blood–brain barrier, so that they become practically useful. Subsequently, non-peptidic and small-sized BACE1 inhibitors possessing a heterocyclic scaffold, 2,6-pyridenedicarboxylic, chelidamic or chelidonic moiety, at the P₂ position were reported. These inhibitors were designed based on the conformer of docked inhibitor in BACE1. In this study, we discuss the role and significance of interactions between Arg235 of BACE1 and its inhibitor in BACE1 inhibitory mechanism. Moreover, we designed more potent small-sized BACE1 inhibitors with a 2,6-pyridinedicarboxylic scaffold at the P₂ position, that were optimized for the interactions with Arg235 of BACE1.     

Fragment-based discovery and optimization of BACE1 inhibitors

A novel series of 2-aminobenzimidazole inhibitors of BACE1 has been discovered using fragment-based drug discovery (FBDD) techniques. The rapid optimization of these inhibitors using structure-guided medicinal chemistry is discussed.     

BACE1 inhibition induces a specific cerebrospinal fluid β-amyloid pattern that identifies drug effects in the central nervous system

BACE1 is a key enzyme for amyloid-β (Aβ) production, and an attractive therapeutic target in Alzheimer's disease (AD). Here we report that BACE1 inhibitors have distinct effects on neuronal Aβ metabolism, inducing a unique pattern of secreted Aβ peptides, analyzed in cell media from amyloid precursor protein (APP) transfected cells and in cerebrospinal fluid (CSF) from dogs by immunoprecipitation-mass spectrometry, using several different BACE1 inhibitors. Besides the expected reductions in Aβ1-40 and Aβ1-42, treatment also changed the relative levels of several other Aβ isoforms. In particular Aβ1-34 decreased, while Aβ5-40 increased, and these changes were more sensitive to BACE1 inhibition than the changes in Aβ1-40 and Aβ1-42. The effects on Aβ5-40 indicate the presence of a BACE1 independent pathway of APP degradation. The described CSF Aβ pattern may be used as a pharmacodynamic fingerprint to detect biochemical effects of BACE1-therapies in clinical trials, which might accelerate development of novel therapies.     

Discovery of a series of selective and cell permeable beta-secretase (BACE1) inhibitors by fragment linking with the assistance of STD-NMR

Two β-secreatase (BACE1) inhibitors from natural products (cinnamic acid and flavone) were linked to furnish potent, cell permeable BACE1 inhibitors with noncompetitive mode of inhibition, with the assistance of saturated transfer difference (STD)-NMR technique. Some of these conjugates also exhibited selective BACE1 inhibition over other aspartyl proteases such as BACE-2 and renin, as well as poor cytotoxicity. Taken together, conjugates 4 represent a new series of BACE inhibitors warrants further investigation for their potential in Alzheimier’s disease therapy.     

Novel non-peptidic and small-sized BACE1 inhibitors

Recently, we reported substrate-based beta-secretase (BACE1) inhibitors with a hydroxymethylcarbonyl (HMC) isostere as a substrate transition-state mimic. These inhibitors showed potent BACE1 inhibitory activities (approximately 1.2 nM IC(50)). In order to improve in vivo enzymatic stability and permeability across the blood-brain barrier, these penta-peptidic inhibitors would need to be further optimized. On the other hand, non-peptidic inhibitors possessing isophthalic residue at the P(2) position were reported from other research groups. We selected isophthalic-type aromatic residues at the P(2) position and an HMC isostere at the P(1) position as lead compounds. On the basis of the design approach focused on the conformer of docked inhibitor in BACE1, we found novel non-peptidic and small-sized BACE1 inhibitors possessing a 2,6-pyridinedicarboxylic, chelidamic or chelidonic residue at the P(2) position.