Selective BACE1 Inhibitors Research Articles

BACE1 inhibitors: A promising therapeutic approach for the management of Alzheimer’s disease

Alzheimer’s disease is a neurological disorder marked by the accumulation of amyloid beta (Aβ) aggregates, resulting from mutations in the amyloid precursor protein. The enzyme β-secretase, also known as β-site amyloid precursor protein cleaving enzyme 1 (BACE1), plays a crucial role in generating Aβ peptides. With no targeted therapy available for Alzheimer’s disease, inhibiting BACE1 aspartic protease has emerged as a primary treatment target. Since 1999, compounds demonstrating potential binding to the BACE1 receptor have advanced to human trials. Structural optimization of synthetically derived compounds, coupled with computational approaches, has offered valuable insights for developing highly selective leads with drug-like properties. This review highlights pivotal studies on the design and development of BACE1 inhibitors as anti-Alzheimer’s disease agents. It summarizes computational methods employed in facilitating drug discovery for potential BACE1 inhibitors and provides an update on their clinical status, indicating future directions for novel BACE1 inhibitors. The promising clinical results of Elenbecestat (E-2609) catalyze the development of effective, selective BACE1 inhibitors in the future.

Rational design of novel diaryl ether-linked benzimidazole derivatives as potent and selective BACE1 inhibitors

Due to the large size and high flexibility of the catalytic active site of BACE1 enzyme, the development of nonpeptide inhibitors with optimal pharmacological properties is still highly demanding. In this work, we have discovered 2-aminobenzimidazole-containg ether scaffolds having potent and selective inhibitory potentials against BACE1 enzyme. We have synthesized novel 29 compounds and optimization of aryl linker region resulted in highly potent BACE1 inhibitory activities with EC50 values of 0.05–2.71 μM. The aryloxy-phenyl analogs 20j showed the EC50 value as low as 0.07 μM in the enzyme assay, whereas, the benzyloxyphenyl dervative 24b was comparatively less effective in the enzyme assay. But interestingly the latter was more effective in the cell assay (EC50 value 1.2 μM). While comparing synthesized derivatives in the cell assay using PC12-APPSW cell, compound 27f appeared as the most potent BACE1 inhibitor having EC50 value 0.7 μM. This scaffold also showed high selectivity over BACE2 enzyme and cathepsin D. Furthermore, the research findings were bolstered through the incorporation of molecular docking, molecular dynamics, and DFT studies. We firmly believe that these discoveries will pave the way for the development of a novel class of small-molecule selective BACE1 inhibitors.

Combination strategy employing BACE1 inhibitor and memantine to boost cognitive benefits in Alzheimer's disease therapy.

The β-secretase BACE1 initiates amyloid-β (Aβ) generation and represents a long-standing prime therapeutic target for the treatment of Alzheimer's disease (AD). However, BACE1 inhibitors tested to date in clinical trials have yielded no beneficial outcomes. In fact, prior BACE1 inhibitor trials targeted at ~ 50-90% Aβ reductions in symptomatic or prodromal AD stages have ended in the discontinuation due to futility and/or side effects, including cognitive worsening rather than expected improvement at the highest dose. We tested whether a combination strategy with the selective BACE1 inhibitor GRL-8234 and the FDA-approved symptomatic drug memantine may provide synergistic cognitive benefits within their safe dose range. The drug effects were evaluated in the advanced symptomatic stage of 5XFAD mice that developed extensive cerebral Aβ deposition. Chronic combination treatment with 33.4-mg/kg GRL-8234 and 10-mg/kg memantine, but not either drug alone, rescued cognitive deficits in 5XFAD mice at 12months of age (the endpoint after 60-day drug treatment), as assessed by the contextual fear conditioning, spontaneous alternation Y-maze and nest building tasks. Intact baseline performances of wild-type control mice on three cognitive paradigms demonstrated that combination treatment did not augment potential cognitive side effects of individual drugs. Biochemical and immunohistochemical examination showed that combination treatment did not synergistically reduce the β-amyloidogenic processing of amyloid precursor protein or Aβ levels in 5XFAD mouse brains. A combination strategy with BACE1 inhibitors and memantine may be able to increase the effectiveness of individual drugs within their safe dose range in AD therapy.

Novel Bace 1 selective inhibitor does not affect dendritic spine plasticity

AbstractBackgroundAll clinical BACE‐inhibitor trials for the treatment of Alzheimer´s Disease have failed due to insufficient efficacy or side effects like worsening of cognitive symptoms. The latter could have been the result of the inhibition of BACE at the synapse where it is expressed in high amounts. We have previously shown that prolonged inhibition of BACE interferes with structural and functional synaptic plasticity (Filser et al. Biol. Psych 2015, Blume et al. Front. Aging Neurosci. 2018), most likely due to the diminished processing of the physiological BACE substrate Seizure protein 6 (Sez6, Zhu et al. Biol. Psych. 2018), which is exclusively processed by BACE1 and it is required for dendritic spine plasticity. The aim of the current study was to elucidate the effects of selective BACE1 inhibitors on dendritic spine plasticity at a dose that affects Sez6 cleavage to various degrees but not completely.MethodBy using longitudinal in vivo two‐photon microscopy, we investigated the effect on dendritic spine dynamics of pyramidal layer V neurons in the somatosensory cortex in mice treated with 2 novel highly selective BACE1 inhibitors developed at Shionogi in comparison to Elenbecestat which reduce soluble Sez6 to 27% (Elenbecastat), 17% (Shionogi Compound 1) and 39% (Shionogi Compound2) of baseline levels in untreated animals. For the detection of the soluble Sez6 ectodomain, the BACE cleavage product from full‐length Sez6, Western blot analysis was performed using a highly specific Sez6 antibody generated in the lab of Stefan Lichtenthaler (Pigoni et al. Mol. Neurodeg. 2016).ResultWe observed that BACE1 selective inhibitors do not alter dendritic spine density and plasticity in cortical neurons if at least 27% of soluble Sez6 remains. In detail, the density of gained and lost spines does not change under the treatment with Shionogi Compound 2 or Elenbecastat. Also, no differences in the stability of dendritic spines were found during treatment with these two compounds. With Compound 1 however, a significant spine loss occurs after 21 days of treatment.ConclusionSelective BACE1 inhibitors do not alter dendritic spine plasticity in mice if dosing reduces synaptic BACE1 cleavage of Sez6 not below 27% of baseline soluble Sez6 levels. BACE1 selective inhibitors may be potential novel candidates for clinical trials treating AD if dosing is carefully adjusted to the amount of Sez6 cleavage, which can be easily monitored during the first weeks of treatment.

New Highly Selective BACE1 Inhibitors and Their Effects on Dendritic Spine Density In Vivo.

β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) is considered a therapeutic target to combat Alzheimer's disease by reducing β-amyloid in the brain. To date, all clinical trials involving the inhibition of BACE1 have been discontinued due to a lack of efficacy or undesirable side effects such as cognitive worsening. The latter could have been the result of the inhibition of BACE at the synapse where it is expressed in high amounts. We have previously shown that prolonged inhibition of BACE interferes with structural synaptic plasticity, most likely due to the diminished processing of the physiological BACE substrate Seizure protein 6 (Sez6) which is exclusively processed by BACE1 and is required for dendritic spine plasticity. Given that BACE1 has significant amino acid similarity with its homolog BACE2, the inhibition of BACE2 may cause some of the side effects, as most BACE inhibitors do not discriminate between the two. In this study, we used newly developed BACE inhibitors that have a different chemotype from previously developed inhibitors and a high selectivity for BACE1 over BACE2. By using longitudinal in vivo two-photon microscopy, we investigated the effect on dendritic spine dynamics of pyramidal layer V neurons in the somatosensory cortex in mice treated with highly selective BACE1 inhibitors. Treatment with those inhibitors showed a reduction in soluble Sez6 (sSez6) levels to 27% (elenbecestat, Biogen, Eisai Co., Ltd., Tokyo, Japan), 17% (Shionogi compound 1) and 39% (Shionogi compound 2), compared to animals fed with vehicle pellets. We observed a significant decrease in the number of dendritic spines with Shionogi compound 1 after 21 days of treatment but not with Shionogi compound 2 or with elenbecestat, which did not show cognitive worsening in clinical trials. In conclusion, highly selective BACE1 inhibitors do alter dendritic spine density similar to non-selective inhibitors if soluble (sSez6) levels drop too much. Low-dose BACE1 inhibition might be reasonable if dosing is carefully adjusted to the amount of Sez6 cleavage, which can be easily monitored during the first week of treatment.

Selected Class of Enamides Bearing Nitro Functionality as Dual-Acting with Highly Selective Monoamine Oxidase-B and BACE1 Inhibitors.

A small series of nitro group-bearing enamides was designed, synthesized (NEA1–NEA5), and evaluated for their inhibitory profiles of monoamine oxidases (MAOs) and β-site amyloid precursor protein cleaving enzyme 1 (β-secretase, BACE1). Compounds NEA3 and NEA1 exhibited a more potent MAO-B inhibition (IC50 value = 0.0092 and 0.016 µM, respectively) than the standards (IC50 value = 0.11 and 0.14 µM, respectively, for lazabemide and pargyline). Moreover, NEA3 and NEA1 showed greater selectivity index (SI) values toward MAO-B over MAO-A (SI of >1652.2 and >2500.0, respectively). The inhibition and kinetics studies suggested that NEA3 and NEA1 are reversible and competitive inhibitors with Ki values of 0.013 ± 0.005 and 0.0049 ± 0.0002 µM, respectively, for MAO-B. In addition, both NEA3 and NEA1 showed efficient BACE1 inhibitions with IC50 values of 8.02 ± 0.13 and 8.21 ± 0.03 µM better than the standard quercetin value (13.40 ± 0.04 µM). The parallel artificial membrane permeability assay (PAMPA) method demonstrated that all the synthesized derivatives can cross the blood–brain barrier (BBB) successfully. Docking analyses were performed by employing an induced-fit docking approach in the GLIDE module of Schrodinger, and the results were in agreement with their in vitro inhibitory activities. The present study resulted in the discovery of potent dual inhibitors toward MAO-B and BACE1, and these lead compounds can be fruitfully explored for the generation of newer, clinically active agents for the treatment of neurodegenerative disorders.

JNJ-67569762, A 2-Aminotetrahydropyridine-Based Selective BACE1 Inhibitor Targeting the S3 Pocket: From Discovery to Clinical Candidate.

The discovery of a novel 2-aminotetrahydropyridine class of BACE1 inhibitors is described. Their pKa and lipophilicity were modulated by a pending sulfonyl group, while good permeability and brain penetration were achieved via intramolecular hydrogen bonding. BACE1 selectivity over BACE2 was achieved in the S3 pocket by a novel bicyclic ring system. An optimization addressing reactive metabolite formation, cardiovascular safety, and CNS toxicity is described, leading to the clinical candidate JNJ-67569762 (12), which gave robust dose-dependent BACE1-mediated amyloid β lowering without showing BACE2-dependent hair depigmentation in preclinical models. We show that 12 has a favorable projected human dose and PK and hence presented us with an opportunity to test a highly selective BACE1 inhibitor in humans. However, 12 was found to have a QT effect upon repeat dosing in dogs and its development was halted in favor of other selective leads, which will be reported in the future.

Structure-Based Approaches to Improving Selectivity through Utilizing Explicit Water Molecules: Discovery of Selective β-Secretase (BACE1) Inhibitors over BACE2.

BACE1 is an attractive target for disease-modifying treatment of Alzheimer's disease. BACE2, having high homology around the catalytic site, poses a critical challenge to identifying selective BACE1 inhibitors. Recent evidence indicated that BACE2 has various roles in peripheral tissues and the brain, and therefore, the chronic use of nonselective inhibitors may cause side effects derived from BACE2 inhibition. Crystallographic analysis of the nonselective inhibitor verubecestat identified explicit water molecules with different levels of free energy in the S2' pocket. Structure-based design targeting them enabled the identification of propynyl oxazine 3 with improved selectivity. Further optimization efforts led to the discovery of compound 6 with high selectivity. The cocrystal structures of 7, a close analogue of 6, bound to BACE1 and BACE2 confirmed that one of the explicit water molecules is displaced by the propynyl group, suggesting that the difference in the relative water displacement cost may contribute to the improved selectivity.

Prediction of Drug Potencies of BACE1 Inhibitors: A Molecular Dynamics Simulation and MM_GB(PB)SA Scoring

Alzheimer’s disease (AD) is a progressive neurodegenerative brain disorder. One of the important therapeutic approaches of AD is the inhibition of β-site APP cleaving enzyme-1 (BACE1). This enzyme plays a central role in the synthesis of the pathogenic β-amyloid peptides (Aβ) in Alzheimer’s disease. A group of potent BACE1 inhibitors with known X-ray structures (PDB ID 5i3X, 5i3Y, 5iE1, 5i3V, 5i3W, 4LC7, 3TPP) were studied by molecular dynamics simulation and binding energy calculation employing MM_GB(PB)SA. The calculated binding energies gave Kd values of 0.139 µM, 1.39 nM, 4.39 mM, 24.3 nM, 1.39 mM, 29.13 mM, and 193.07 nM, respectively. These inhibitors showed potent inhibitory activities in enzymatic and cell assays. The Kd values are compared with experimental values and the structures are discussed in view of the energy contributions to binding. Drug likeness of these inhibitors is also discussed. Accommodation of ligands in the catalytic site of BACE1 is discussed depending on the type of fragment involved in each structure. Molecular dynamics (MD) simulations and energy studies were used to explore the recognition of the selected BACE1 inhibitors by Asp32, Asp228, and the hydrophobic flap. The results show that selective BACE1 inhibition may be due to the formation of strong electrostatic interactions with Asp32 and Asp228 and a large number of hydrogen bonds, in addition to π–π and van der Waals interactions with the amino acid residues located inside the catalytic cavity. Interactions with the ligands show a similar binding mode with BACE1. These results help to rationalize the design of selective BACE1 inhibitors.

A series of molecular modeling techniques to reveal selective mechanisms of inhibitors to β-Site amyloid precursor protein cleaving enzyme 1 (BACE1) and β-site amyloid precursor protein cleaving enzyme 2 (BACE2)

Inhibition of β-Site amyloid precursor protein cleaving enzyme 1 (BACE1) has been shown to be an effective treatment for Alzheimer’s disease. A wealth of research has focused on finding highly selective small-molecule inhibitors targeting the BACE1 over its close homologue BACE2 to avoid potential side effects. However, given the highly structural similarities of BACE1 and BACE2, designing highly selective BACE1 inhibitors remains a huge challenge. Recently, it has been reported that a potential BACE1 inhibitor named C28 (∼52-fold selectivity) exhibited greater selectivity to BACE1 over BACE2 than the previously reported inhibitors AZD3293 and AZD3839 (∼1.5-fold and 14-fold selectivity). However, few computational studies have been performed to reveal its underlying mechanisms. In this study, a series of molecular modeling techniques were performed to reveal the selective mechanisms. Classical molecular dynamics (cMD) simulations indicated that the major variations appeared to be controlled by overall protein dynamics. Free energy calculations further suggested that the binding affinities of AZD3293 to BACE1 and BACE2 are similar, but the binding affinity of AZD3839 and C28 to BACE1 is much higher than to BACE2, and that the major variations are electrostatic interactions. The protein dynamics and energy differences were further observed in accelerated molecular dynamics (aMD) simulations. In addition, the umbrella sampling simulations revealed the inhibitors’ different patterns of dissociation from the binding pockets of BACE1 and BACE2, and that different energy barriers were responsible for the selectivity. The physical principles revealed by this study may facilitate the rational design of more potent BACE1 selective inhibitors. Communicated by Ramaswamy H. Sarma

Structure-Based Design of Selective β-Site Amyloid Precursor Protein Cleaving Enzyme 1 (BACE1) Inhibitors: Targeting the Flap to Gain Selectivity over BACE2.

BACE1 inhibitors hold potential as agents in disease-modifying treatment for Alzheimer's disease. BACE2 cleaves the melanocyte protein PMEL in pigment cells of the skin and eye, generating melanin pigments. This role of BACE2 implies that nonselective and chronic inhibition of BACE1 may cause side effects derived from BACE2. Herein, we describe the discovery of potent and selective BACE1 inhibitors using structure-based drug design. We targeted the flap region, where the shape and flexibility differ between these enzymes. Analysis of the cocrystal structures of an initial lead 8 prompted us to incorporate spirocycles followed by its fine-tuning, culminating in highly selective compounds 21 and 22. The structures of 22 bound to BACE1 and BACE2 revealed that a relatively high energetic penalty in the flap of the 22-bound BACE2 structure may cause a loss in BACE2 potency, thereby leading to its high selectivity. These findings and insights should contribute to responding to the challenges in exploring selective BACE1 inhibitors.

Toward β-Secretase-1 Inhibitors with Improved Isoform Selectivity.

BACE1 is responsible for the first step in APP proteolysis, leading to toxic Aβ production, and has been indicated to play a key role in the pathogenesis of Alzheimer's disease. The related isoform BACE2 is thought to be involved in processing of the pigment cell-specific melanocyte protein. To avoid potential effects on pigmentation, we investigated the feasibility for developing isoform-selective BACE1 inhibitors. Cocrystal structures of 47 compounds were analyzed and clustered according to their selectivity profiles. Selective BACE1 inhibitors were found to exhibit two distinct conformational features proximal to the flap and the S3 subpocket. Several new molecules were designed and tested to make use of this observation. The combination of a pyrimidinyl C-ring and a methylcyclohexyl element resulted in lead molecule 28, which exhibited ∼50-fold selectivity. Compared to a nonselective BACE1/2 inhibitor, 28 showed significantly less inhibition of PMEL processing in human melanocytes, indicating good functional selectivity of this inhibitor class.

Asp32 and Asp228 determine the selective inhibition of BACE1 as shown by docking and molecular dynamics simulations

Inhibition of β-site amyloid–β-protein precursor cleaving enzyme 1 (BACE1) represents a promising approach for the treatment of Alzheimer's disease (AD). However, the development of a selective BACE1 inhibitor is difficult due to its highly flexible catalytic site and homology to other aspartic proteases, including BACE2 and Cathepsin D (CTSD). Aiming to better understand the structural factors responsible for selective BACE1 inhibition, we performed alignment studies, molecular dynamics (MD) simulations and docking studies to explore the recognition of four selective BACE1 inhibitors by aspartyl proteases. The results show that selective BACE1 inhibition may be due to the formation of strong electrostatic interactions with Asp32 and Asp228 and a large number of hydrogen bonds, π-π and Van der Waals interactions with the amino acid residues located inside the catalytic cavity, which has different volume and shape compared to BACE2 and CTSD. Hindrance effects avoid the accommodation of ligands in the small catalytic site of BACE2, resulting in a lower affinity and the high cavity of CTSD results in the formation of a small number of interactions with the ligands, although they show a similar binding mode with BACE1. These results might help to rationalize the design of selective BACE1 inhibitors.

Discovery of polymethoxyflavones from black ginger (Kaempferia parviflora) as potential β-secretase (BACE1) inhibitors

Inhibition of β-site amyloid precursor protein cleaving enzyme 1 (BACE1) has emerged as a prime target in Alzheimer's disease research because BACE1 is the rate-limiting enzyme involved in the abnormal production of β-amyloid peptides. In the course of exploring natural BACE1 inhibitors, the present study focused on the major polymethoxyflavones from Kaempferia parviflora: 5,7-dimethoxyflavone (DMF), 5,7,4′-trimethoxyflavone (TMF), and 3,5,7,3′,4′-pentamethoxyflavone (PMF). All polymethoxyflavones exhibited strong BACE1 inhibitory potency, with no significant suppression of α-secretase or other serine protease activities, indicating that they were relatively specific and selective BACE1 inhibitors. Dixon and Lineweaver–Burk plots indicated that they produced non-competitive inhibition of substrate cleavage. An in silico docking study of human BACE1 with polymethoxyflavones demonstrated the binding energy and the interacting residues. Our findings indicated that Kaempferia parviflora could provide a novel therapy for the prevention of Alzheimer's disease and provide a scaffold for exploration of more potent natural BACE1 inhibitors.

Beneficial effects of the β-secretase inhibitor GRL-8234 in 5XFAD Alzheimer's transgenic mice lessen during disease progression.

The β-secretase enzyme BACE1, which initiates the cleavage of amyloid precursor protein (APP) into the amyloid-β (Aβ) peptide, is a prime therapeutic target for Alzheimer's disease (AD). However, recent investigations using genetic animal models raise concern that therapeutic BACE1 inhibition may encounter the dramatic reduction of efficacy in ameliorating AD-like pathology and memory deficits during disease progression. Here, we compared the effects of the potent and selective small-molecule BACE1 inhibitor GRL-8234 in different pathological stages of AD mouse model. Specifically, we administered GRL-8234 (33.4 mg/kg, i.p.) once daily for 2 months to 5XFAD transgenic mice, which showed modest (4 months) and massive (10 months of age) Aβ plaque deposition at starting points. Chronic treatments with GRL-8234 reversed memory impairments, as tested by the spontaneous alternation Y-maze task, in the younger 5XFAD group concomitant with significant reductions in cerebral Aβ42 levels. In contrast, only marginal reductions of Aβ42 were observed in 12-month-old 5XFAD mice treated with GRL-8234 and their memory function remained impaired. We found that not only BACE1 but also full-length APP expression was significantly elevated with progressive Aβ accumulation in 5XFAD mice, while GRL-8234 failed to affect these detrimental mechanisms that further accelerate plaque growth in brains of older 5XFAD mice. Therefore, our results provide important insights into the mechanisms by which Aβ accumulation and related memory impairments become less responsive to rescue by BACE1 inhibition during the course of AD development.

Combining an amyloid-beta (Aβ) cleaving enzyme inhibitor with a γ-secretase modulator results in an additive reduction of Aβ production.

A major hallmark of Alzheimer's disease (AD) is the deposition of amyloid-β (Aβ) peptides in amyloid plaques. Aβ peptides are produced by sequential cleavage of the amyloid precursor protein by the β amyloid cleaving enzyme (BACE) and the γ-secretase (γ-sec) complex. Pharmacological treatments that decrease brain levels of in particular the toxic Aβ42 peptide are thought to be promising approaches for AD disease modification. Potent and selective BACE1 inhibitors as well as γ-sec modulators (GSMs) have been designed. Pharmacological intervention of secretase function is not without risks of either on- or off-target adverse effects. One way of improving the therapeutic window could be to combine treatment on multiple targets, using smaller individual doses and thereby minimizing adverse effect liability. We show that combined treatment of primary cortical neurons with a BACE1 inhibitor and a GSM gives an additive effect on Aβ42 level change compared with the individual treatments. We extend this finding to C57BL/6 mice, where the combined treatment results in reduction of brain Aβ42 levels reflecting the sum of the individual treatment efficacies. These results show that pharmacological targeting of two amyloid precursor protein processing steps is feasible without negatively interfering with the mechanism of action on individual targets. We conclude that targeting Aβ production by combining a BACE inhibitor and a GSM could be a viable approach for therapeutic intervention in AD modification.

The selective BACE1 inhibitor VIa reduces Aβ production in a mouse model of Alzheimer’s disease (846.4)

We are pursuing potentβ‐site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors in an effort to identify suitable Alzheimer’s disease (AD) drug candidates. Employing a homogeneous, time‐resolved fluorescence technique, we determined the selectivity of compound VIa. We then conducted a cell‐based assay using the huAPPswe/huBACE1 transgenic CHO cell to investigate the effect on the β‐cleavage of APP. The effect of VIa anti‐AD was valued using both acute and subchronic dosing paradigms in PrPhAPPswe/PS1ΔE9 transgenic mice. Our results have shown that the novel compound VIa exhibits potent inhibitory effects with and IC50=5.9 nM and displays 30.8‐fold, 7500‐fold and 17533‐fold selectivity against the other aspartic proteases BACE2, cathepsin D and renin, respectively. In cellular assays, VIa moderately reduces Aβ production: Aβ1‐40 with an IC50 =143 nM and 1 nM VIa reduced Aβ1‐42 by 40.17%. Concomitant with VIa inhibiting the β‐cleavage of amyloid precursor protein (APP), VIa increases the production of sAPPα with an approximate EC50 of 16.5 nM. In testing this compound’s efficacy in vivo, the oral administration of VIa resulted in a significant decrease in Aβ1‐40 and Aβ1‐42 in the blood of a mouse model of AD by 17.5‐72.44% and 14.5‐80.32%, respectively. This result indicates that the novel compound VIa is a small, potent, selective, and nonpeptidic BACE1 inhibitor.Grant Funding Source: the National Science and Technology Major Project (2012ZX09301003‐002‐001, 2013ZX09508104)

Discovery of pyrazole as C-terminus of selective BACE1 inhibitors

We recently discovered and reported dual inhibitor 5 of AChE and BACE1 with N-benzylpiperidine ethyl as C-terminus. Compound 5 showed potent inhibitory activities for BACE1, and could reduce endogenous Aβ1–40 production in APP transgenic mice. In present work, we rapidly identified substituted triazole as the C-terminus of compound 5 by replacing the benzylpiperidine ethyl group with click chemistry and tested these synthesized compounds by in situ screening assay. As revealed by the crystal structures of BACE1 in complex with our triazole compound 12, we found that Pro70 and Thr72 located in the flap region were the critical components for binding with these inhibitors. With the aid of the crystal structure, a new series of five-membered heterocyclic compounds was prepared in order to explore the structure–activity relationship (SAR) of this class of molecules. From these efforts, pyrazole was discovered as a novel C-terminus of BACE1 inhibitors. After further modification of pyrazole with variable substituents, compound 37 exhibited good potency in enzyme inhibition assay (IC50 = 0.025 μM) and compound 33 showed moderate inhibition effects on Aβ production of APP transfected HEK293 cells. Moreover, these pyrazole derivatives demonstrated good selectivity versus cathepsin D. Our results indicated that the vicinity of Pro70 and Thr72 might be utilized as a subsite, and the discovered pyrazole derivatives might provide useful hints for developing novel BACE1 inhibitors as anti-AD drugs.

Design and synthesis of thiophene dihydroisoquinolines as novel BACE1 inhibitors

Utilizing a structure based design approach, combined with extensive medicinal chemistry execution, highly selective, potent and novel BACE1 inhibitor 8 (BACE1 Alpha assay IC50=8nM) was made from a weak μM potency hit in an extremely efficient way. The detailed SAR and general design approaches will be discussed.

Loss of Bace2 in zebrafish affects melanocyte migration and is distinct from Bace1 knock out phenotypes

Alzheimer's disease is the most frequent dementia. Pathologically, Alzheimer's disease is characterized by the accumulation of senile plaques composed of amyloid β-peptide (Aβ). Two proteases, β- and γ-secretase proteolytically generate Aβ from its precursor, the ß-amyloid precursor protein (APP). Inhibition of β-secretase, also referred to as beta-site APP cleaving enzyme (BACE1) or γ-secretase is therefore of prime interest for the development of amyloid-lowering drugs. To assess the in vivo function of zebrafish Bace1 (zBace1), we generated zBace1 knock out fish by zinc finger nuclease-mediated genome editing. bace1 mutants (bace1-/-) are hypomyelinated in the PNS while the CNS is not affected. Moreover, the number of mechanosensory neuromasts is elevated in bace1-/-. Mutations in zebrafish Bace2 (zBace2) revealed a distinct melanocyte migration phenotype, which is not observed in bace1-/-. Double homozygous bace1-/-; bace2-/- fish do not enhance the single mutant phenotypes indicating non-redundant distinct physiological functions. Single homozygous bace1 mutants as well as double homozygous bace1 and bace2 mutants are viable and fertile suggesting that Bace1 is a promising drug target without major side effects. The identification of a specific bace2 -/- associated phenotype further allows improving selective Bace1 inhibitors and to distinguish between Bace 1 and Bace 2 inhibition in vivo. Inhibition of BACE1 protease activity has therapeutic importance for Alzheimer's disease. Analysis of BACE1 and BACE2 knock-out zebrafish revealed that they exhibit distinct phenotypes. bace1 mutants display hypomyelination in the PNS and supernumerary neuromasts while in bace2 mutants the shape and migration of melanocytes is affected. These phenotypes are not further enhanced in the viable double mutants. Our data suggest that blocking BACE1 activity is a safe therapeutic approach.