Small-molecule BACE1 Inhibitors Research Articles

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.

Accelerated long-term forgetting is a BACE1 inhibitor-reversible incipient cognitive phenotype in Alzheimer's disease model mice.

AimAfter the continued failure of β‐secretase (BACE1) inhibitor clinical trials in prodromal as well as mild‐to‐moderate Alzheimer's disease (AD), they are shifting to further earlier or asymptomatic stages. The aim of this study is to explore a cognitive paradigm that allows us to more sensitively detect beneficial effects of BACE1 inhibitors in presymptomatic AD.MethodsGRL‐8234 (33.4 mg/kg, ip), a small‐molecule BACE1 inhibitor, was administered once daily for 28 days to the 5XAFD transgenic mouse model of AD. The contextual fear conditioning was used to evaluate the effects of GRL‐8234 on memory deficits in 5XFAD mice at different ages.ResultsChronic administration of GRL‐8234 to 5XFAD mice rescued their contextual memory deficits, when tested 1 day after training at 6‐8 months but not at 12 months of age. Importantly, 4‐month‐old 5XFAD mice retain the ability to form contextual memory equivalent to wild‐type controls, demonstrating that the standard method of 1‐day memory assessment is not suitable for evaluating BACE1 inhibitor efficacy in ameliorating cognitive declines during earlier disease stages. Despite normal contextual memory formation, young 5XFAD mice showed faster forgetting when a longer delay (28 days) intervened between training and memory testing. Notably, GRL‐8234 administered to 4‐month‐old 5XFAD mice during the 28‐day delay reversed accelerated long‐term forgetting almost completely back to wild‐type control levels.ConclusionThe results provide experimental evidence that accelerated long‐term forgetting represents more sensitive memory testing that can help evaluate BACE1 inhibitor therapy in presymptomatic AD populations.

Highlights in BACE1 Inhibitors for Alzheimer's Disease Treatment.

Alzheimer's disease (AD) is a severe neurodegenerative disorder and the most common type of dementia in the elderly. The clinical symptoms of AD include a progressive loss of memory and impairment of cognitive functions interfering with daily life activities. The main neuropathological features consist in extracellular amyloid-β (Aβ) plaque deposition and intracellular Neurofibrillary tangles (NFTs) of hyperphosphorylated Tau. Understanding the pathophysiological mechanisms that underlie neurodegeneration in AD is essential for rational design of neuroprotective agents able to prevent disease progression. According to the “Amyloid Cascade Hypothesis” the critical molecular event in the pathogenesis of AD is the accumulation of Aβ neurotoxic oligomers. Since the proteolytic processing of Amyloid Precursor Protein (APP) by β-secretase (beta-site APP cleaving enzyme 1, BACE1) is the rate-limiting step in the production of Aβ, this enzyme is considered a major therapeutic target and BACE1 inhibitors have the potential to be disease-modifying drugs for AD treatment. Therefore, intensive efforts to discover and develop inhibitors that can reach the brain and effectively inhibit BACE1 have been pursued by several groups worldwide. The aim of this review is to highlight the progress in the discovery of potent and selective small molecule BACE1 inhibitors over the past decade.

Conformational dynamics of cathepsin D and binding to a small-molecule BACE1 inhibitor.

BACE1 is a major therapeutic target for prevention and treatment of Alzheimer's disease. Developing inhibitors that can selectively target BACE1 in favor of other proteases, especially cathepsin D (CatD), has presented significant challenges. Here, we investigate the conformational dynamics and protonation states of BACE1 and CatD using continuous constant pH molecular dynamics with pH replica-exchange sampling protocol. Despite similar structure, BACE1 and CatD exhibit markedly different active site dynamics. BACE1 displays pH-dependent flap dynamics that controls substrate accessibility, while the CatD flap is relatively rigid and remains open in the pH range 2.5-6. Interestingly, although each protease hydrolyzes peptide bonds, the protonation states of the catalytic dyads are different within the active pH range. The acidic and basic components of the BACE1 catalytic dyad are clear, while either aspartic acid of the CatD catalytic dyad could play the role of acid or base. Finally, we investigate binding of the inhibitor LY2811376 developed by Eli Lilly to BACE1 and CatD. Surprisingly, in the enzyme active pH range, LY2811376 forms a stronger salt bridge with the catalytic dyad in CatD than in BACE1, which might explain the retinal toxicity of the inhibitor related to off-target inhibition of CatD. This work highlights the complexity and challenge in structure-based drug design where receptor-ligand binding induces protonation state change in both the protein and the inhibitor. © 2017 Wiley Periodicals, Inc.

Design, synthesis and in vitro evaluation studies of sulfonyl-amino-acetamides as small molecule BACE-1 inhibitors

The identification of a series of sulfonyl-amino-acetamides as BACE-1 (β-secretase) inhibitors for the treatment of Alzheimer’s disease is reported. The derivatives were designed based on the docking simulation study, synthesized and assessed for BACE-1 inhibition in vitro. The designed ligands revealed desired binding interactions with the catalytic aspartate dyad and occupance of S1 and S2′ active site regions. These in silico results correlated well with in vitro activity. Out of 33 compounds synthesized, 12 compounds showed significant inhibition at 10μM concentration. The most active compound 2.17S had IC50 of 7.90μM against BACE-1, which was concomitant with results of in silico docking study.

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.

Retinal Toxicity Induced by a Novel β-secretase Inhibitor in the Sprague-Dawley Rat.

β-Secretase 1 (BACE1) represents an attractive target for the treatment of Alzheimer's disease. In the course of development of a novel small molecule BACE1 inhibitor (AMG-8718), retinal thinning was observed in a 1-month toxicity study in the rat. To further understand the lesion, an investigational study was conducted whereby rats were treated daily with AMG-8718 for 1 month followed by a 2-month treatment-free phase. The earliest detectable change in the retina was an increase in autofluorescent granules in the retinal pigment epithelium (RPE) on day 5; however, there were no treatment-related light microscopic changes observed in the neuroretina and no changes observed by fundus autofluorescence or routine ophthalmoscopic examination after 28 days of dosing. Following 2 months of recovery, there was significant retinal thinning attributed to loss of photoreceptor nuclei from the outer nuclear layer. Electroretinographic changes were observed as early as day 14, before any microscopic evidence of photoreceptor loss. BACE1 knockout rats were generated and found to have normal retinal morphology indicating that the retinal toxicity induced by AMG-8718 was likely off-target. These results suggest that AMG-8718 impairs phagolysosomal function in the rat RPE, which leads to photoreceptor dysfunction and ultimately loss of photoreceptors.

Design and synthesis of novel 3,5-bis-N-(aryl/heteroaryl) carbamoyl-4-aryl-1,4-dihydropyridines as small molecule BACE-1 inhibitors

Alzheimer disease (AD) is a neuronal dementia for which no treatment has been consolidated yet. Major pathologic hallmark of AD is the aggregated extracellular amyloid-β plaques in the brains of disease sufferers. Aβ-peptide is a major component of amyloid plaques and is produced from amyloid precursor protein (APP) via the proteolysis action. An aspartyl protease known as β-site amyloid precursor protein cleaving enzyme (BACE-1) is responsible for this proteolytic action. Distinctive role of BACE-1 in AD pathogenesis has made it a validated target to develop anti-Alzheimer agents. Our structure-based virtual screening method led to the synthesis of novel 3,5-bis-N-(aryl/heteroaryl) carbamoyl-4-aryl-1,4-dihydropyridine BACE-1 inhibitors (6a-6p; in vitro hits). Molecular docking and DFT-based ab initio studies using B3LYP functional in association with triple-ζ basis set (TZV) proposed binding mode and binding energies of ligands in the active site of the receptor. In vitro BACE-1 inhibitory activities were determined by enzymatic fluorescence resonance energy transfer (FRET) assay. Most of the synthesized dihydropyridine scaffolds were active against BACE-1 while 6d, 6k, 6n and 6a were found to be the most potent molecules with IC50 values of 4.21, 4.27, 4.66 and 6.78 μM, respectively. Superior BACE-1 inhibitory activities were observed for dihydropyridine derivatives containing fused/nonfused thiazole containing groups, possibly attributing to the additional interactions with S2-S3 subpocket residues. Relatively reliable correlation between calculated binding energies and experimental BACE-1 inhibitory activities was achieved (R(2)=0.51). Moreover, compounds 6d, 6k, 6n and 6a exhibited relatively no calcium channel blocking activity with regard to nifedipine suggesting them as appropriate candidates for further modification(s) to BACE-1 inhibitory scaffolds.

Development of a Novel β-Secretase Binding Assay Using the AlphaScreen Platform

Alzheimer’s disease (AD) is a devastating neurodegenerative disease affecting millions of people. β-secretase-1 (BACE1), an enzyme involved in the processing of the amyloid precursor protein (APP) to form Aβ is a validated target for AD. Herein, the authors develop and validate a novel binding assay for BACE1 using the AlphaScreen platform that is amenable for high-throughput screening (HTS). Small-molecule BACE1 inhibitors of the hydroxyethylamine, hydantoin, and sulfamide classes were functionalized by biotin PEG linkers of varying lengths forming probes that were bound to streptavidin donor beads. BACE1 was coupled to nickel-chelate acceptor beads. Upon mixing, probes designed from all three classes registered high signal-to-background values in the AlphaScreen binding assay, where the interaction between probe and BACE1 was completely blocked by free parent compound. A probe from the hydantoin class was chosen for further optimization, where the final assay conditions of 50 nM BACE and 250 nM probe were used and Z′ values >0.75 were commonly observed. IC50 values determined by the AlphaScreen assay format exhibited ~10-fold greater sensitivity when compared with a fluorescence polarization–based activity assay. The assay was miniaturized to a 1536-well format for HTS, in which 525 000 compounds were screened.

AZ-4217: A High Potency BACE Inhibitor Displaying Acute Central Efficacy in Different In Vivo Models and Reduced Amyloid Deposition in Tg2576 Mice

Aβ, the product of APP (amyloid precursor protein), has been implicated in the pathophysiology of Alzheimer's disease (AD). β-Site APP cleaving enzyme1 (BACE1) is the enzyme initiating the processing of the APP to Aβ peptides. Small molecule BACE1 inhibitors are expected to decrease Aβ-peptide generation and thereby reduce amyloid plaque formation in the brain, a neuropathological hallmark of AD. BACE1 inhibition thus addresses a key mechanism in AD and its potential as a therapeutic target is currently being addressed in clinical studies. Here, we report the discovery and the pharmacokinetic and pharmacodynamic properties of BACE1 inhibitor AZ-4217, a high potency compound (IC50 160 pM in human SH-SY5Y cells) with an excellent in vivo efficacy. Central efficacy of BACE1 inhibition was observed after a single dose in C57BL/6 mice, guinea pigs, and in an APP transgenic mouse model of cerebral amyloidosis (Tg2576). Furthermore, we demonstrate that in a 1 month treatment paradigm BACE1 inhibition of Aβ production does lower amyloid deposition in 12-month-old Tg2576 mice. These results strongly support BACE1 inhibition as concretely impacting amyloid deposition and therefore potentially an important approach for therapeutic intervention in AD.

Response surface methodology in docking study of small molecule BACE-1 inhibitors

Computational evaluation of ligand-receptor binding via docking strategy is a well established approach in structure-based drug design. This technique has been applied frequently in developing molecules of biological interest. However, any procedure would require an optimization set up to be more efficient, economic and time-saving. Advantages of modern statistical optimization methods over conventional one-factor-at-a-time studies have been well revealed. The optimization by experimental design provides a combination of factor levels simultaneously satisfying the requirements considered for each of the responses and factors. In this study, response surface method was applied to optimize the prominent factors (number of genetic algorithm runs, population size, maximum number of evaluations, torsion degrees for ligand and number of rotatable bonds in ligand) in AutoDock4.2-based binding study of small molecule β-secretase inhibitors as anti-alzheimer agents. Results revealed that a number of rotatable bonds in ligand and maximum number of docking evaluations were determinant variables affecting docking outputs. The interference between torsion degrees for ligand and number of genetic algorithm runs for docking procedure was found to be the significant interaction term in our model. Optimized docking outputs exhibited a high correlation with experimental fluorescence resonance energy transfer-based IC₅₀s for β-secretase inhibitors (R² = 0.9133).

P2-481: Small molecule BACE-1 inhibitors are potent, selective and orally active

P2-481: Small molecule BACE-1 inhibitors are potent, selective and orally active

O1-04-02: Significant Aβ lowering in CSF and plasma after oral administration of a potent small molecule BACE1 inhibitor in non-human primates

O1-04-02: Significant Aβ lowering in CSF and plasma after oral administration of a potent small molecule BACE1 inhibitor in non-human primates

Acylguanidine inhibitors of β-secretase: Optimization of the pyrrole ring substituents extending into the S1 and S3 substrate binding pockets

Proteolytic cleavage of amyloid precursor protein by beta-secretase (BACE-1) and gamma-secretase leads to formation of beta-amyloid (A beta) a key component of amyloid plaques, which are considered the hallmark of Alzheimer's disease. Small molecule inhibitors of BACE-1 may reduce levels of A beta and thus have therapeutic potential for treating Alzheimer's disease. We recently reported the identification of a novel small molecule BACE-1 inhibitor N-[2-(2,5-diphenyl-pyrrol-1-yl)-acetyl]guanidine (3.a.1). We report here the initial hit-to-lead optimization of this hit and the SAR around the aryl groups occupying the S(1) and S(2') pockets leading to submicromolar BACE-1 inhibitors.

P4-324: Aβ lowering after IV administration of a potent, cell-permeable small molecule BACE1 inhibitor

P4-324: Aβ lowering after IV administration of a potent, cell-permeable small molecule BACE1 inhibitor