Johanna Fälting

A zebrafish model of tauopathy allows in vivo imaging of neuronal cell death and drug evaluation

Our aging society is confronted with a dramatic increase of patients suffering from tauopathies, which include Alzheimer disease and certain frontotemporal dementias. These disorders are characterized by typical neuropathological lesions including hyperphosphorylation and subsequent aggregation of TAU protein and neuronal cell death. Currently, no mechanism-based cures are available. We generated fluorescently labeled TAU transgenic zebrafish, which rapidly recapitulated key pathological features of tauopathies, including phosphorylation and conformational changes of human TAU protein, tangle formation, neuronal and behavioral disturbances, and cell death. Due to their optical transparency and small size, zebrafish larvae are well suited for both in vivo imaging and drug development. TAU-induced neuronal cell death was imaged by time-lapse microscopy in vivo. Furthermore, we used this zebrafish model to identify compounds targeting the TAU kinase glycogen synthase kinase 3beta (GSK3beta). We identified a newly developed highly active GSK3beta inhibitor, AR-534, by rational drug design. AR-534 reduced TAU phosphorylation in TAU transgenic zebrafish. This transgenic zebrafish model may become a valuable tool for further studies of the neuropathology of dementia.

Discovery of AZD3839, a Potent and Selective BACE1 Inhibitor Clinical Candidate for the Treatment of Alzheimer Disease

Background: BACE1 inhibitors target the first step in Aβ formation and are tractable drugs for halting disease progression in Alzheimer disease. Results: AZD3839 is a novel BACE1 inhibitor that effectively reduces brain and CSF Aβ levels in several preclinical species. Conclusion: Based on the preclinical profile, AZD3839 was progressed into Phase I. Significance: AZD3839 may have disease-modifying potential in the treatment of Alzheimer disease. β-Site amyloid precursor protein cleaving enzyme1 (BACE1) is one of the key enzymes involved in the processing of the amyloid precursor protein (APP) and formation of amyloid β peptide (Aβ) species. Because cerebral deposition of Aβ species might be critical for the pathogenesis of Alzheimer disease, BACE1 has emerged as a key target for the treatment of this disease. Here, we report the discovery and comprehensive preclinical characterization of AZD3839, a potent and selective inhibitor of human BACE1. AZD3839 was identified using fragment-based screening and structure-based design. In a concentration-dependent manner, AZD3839 inhibited BACE1 activity in a biochemical fluorescence resonance energy transfer (FRET) assay, Aβ and sAPPβ release from modified and wild-type human SH-SY5Y cells and mouse N2A cells as well as from mouse and guinea pig primary cortical neurons. Selectivity against BACE2 and cathepsin D was 14 and >1000-fold, respectively. AZD3839 exhibited dose- and time-dependent lowering of plasma, brain, and cerebrospinal fluid Aβ levels in mouse, guinea pig, and non-human primate. Pharmacokinetic/pharmacodynamic analyses of mouse and guinea pig data showed a good correlation between the potency of AZD3839 in primary cortical neurons and in vivo brain effects. These results suggest that AZD3839 effectively reduces the levels of Aβ in brain, CSF, and plasma in several preclinical species. It might, therefore, have disease-modifying potential in the treatment of Alzheimer disease and related dementias. Based on the overall pharmacological profile and its drug like properties, AZD3839 has been progressed into Phase 1 clinical trials in man.

Design and synthesis of β-site amyloid precursor protein cleaving enzyme (BACE1) inhibitors with in vivo brain reduction of β-amyloid peptides.

The evaluation of a series of aminoisoindoles as β-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors and the discovery of a clinical candidate drug for Alzheimers disease, (S)-32 (AZD3839), are described. The improvement in permeability properties by the introduction of fluorine adjacent to the amidine moiety, resulting in in vivo brain reduction of Aβ40, is discussed. Due to the basic nature of these compounds, they displayed affinity for the human ether-a-go-go related gene (hERG) ion channel. Different ways to reduce hERG inhibition and increase hERG margins for this series are described, culminating in (S)-16 and (R)-41 showing large in vitro margins with BACE1 cell IC(50) values of 8.6 and 0.16 nM, respectively, and hERG IC(50) values of 16 and 2.8 μM, respectively. Several compounds were advanced into pharmacodynamic studies and demonstrated significant reduction of β-amyloid peptides in mouse brain following oral dosing.

New Aminoimidazoles as β-Secretase (BACE-1) Inhibitors Showing Amyloid-β (Aβ) Lowering in Brain

Amino-2H-imidazoles are described as a new class of BACE-1 inhibitors for the treatment of Alzheimers disease. Synthetic methods, crystal structures, and structure-activity relationships for target activity, permeability, and hERG activity are reported and discussed. Compound (S)-1m was one of the most promising compounds in this report, with high potency in the cellular assay and a good overall profile. When guinea pigs were treated with compound (S)-1m, a concentration and time dependent decrease in Aβ40 and Aβ42 levels in plasma, brain, and CSF was observed. The maximum reduction of brain Aβ was 40-50%, 1.5 h after oral dosing (100 μmol/kg). The results presented highlight the potential of this new class of BACE-1 inhibitors with good target potency and with low effect on hERG, in combination with a fair CNS exposure in vivo.

Core Refinement toward Permeable β-Secretase (BACE-1) Inhibitors with Low hERG Activity

By use of iterative design aided by predictive models for target affinity, brain permeability, and hERG activity, novel and diverse compounds based on cyclic amidine and guanidine cores were synthesized with the goal of finding BACE-1 inhibitors as a treatment for Alzheimers disease. Since synthesis feasibility had low priority in the design of the cores, an extensive synthesis effort was needed to make the relevant compounds. Syntheses of these compounds are reported, together with physicochemical properties and structure-activity relationships based on in vitro data. Four crystal structures of diverse amidines binding in the active site are deposited and discussed. Inhibitors of BACE-1 with 3 μM to 32 nM potencies in cells are shown, together with data on in vivo brain exposure levels for four compounds. The results presented show the importance of the core structure for the profile of the final compounds.

Aminoimidazoles as Bace-1 Inhibitors: The Challenge to Achieve in Vivo Brain Efficacy

The evaluation of a series of bicyclic aminoimidazoles as potent BACE-1 inhibitors is described. The crystal structures of compounds 14 and 23 in complex with BACE-1 reveal hydrogen bond interactions with the protein important for achieving potent inhibition. The optimization of permeability and efflux properties of the compounds is discussed as well as the importance of these properties for attaining in vivo brain efficacy. Compound (R)-25 was selected for evaluation in vivo in wild type mice and 1.5h after oral co-administration of 300μmol/kg (R)-25 and efflux inhibitor GF120918 the brain Aβ40 level was reduced by 17% and the plasma Aβ40 level by 76%.

AZD1080, a novel GSK3 inhibitor, rescues synaptic plasticity deficits in rodent brain and exhibits peripheral target engagement in humans

Abnormal tau phosphorylation resulting in detachment of tau from microtubules and aggregation are critical events in neuronal dysfunction, degeneration, and neurofibrillary pathology seen in Alzheimers disease. Glycogen synthase kinase‐3β (GSK3β) is a key target for drug discovery in the treatment of Alzheimers disease and related tauopathies because of its potential to abnormally phosphorylate proteins and contribute to synaptic degeneration. We report the discovery of AZD1080, a potent and selective GSK3 inhibitor that demonstrates peripheral target engagement in Phase 1 clinical studies. AZD1080 inhibits tau phosphorylation in cells expressing human tau and in intact rat brain. Interestingly, subchronic but not acute administration with AZD1080 reverses MK‐801‐induced deficits, measured by long‐term potentiation in hippocampal slices and in a cognitive test in mice, suggesting that reversal of synaptic plasticity deficits in dysfunctional systems requires longer term modifications of proteins downstream of GSK3β signaling. The inhibitory pattern on tau phosphorylation reveals a prolonged pharmacodynamic effect predicting less frequent dosing in humans. Consistent with the preclinical data, in multiple ascending dose studies in healthy volunteers, a prolonged suppression of glycogen synthase activity was observed in blood mononuclear cells providing evidence of peripheral target engagement with a selective GSK3 inhibitor in humans.

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 Alzheimers 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.

AZD3293: A Novel, Orally Active BACE1 Inhibitor with High Potency and Permeability and Markedly Slow Off-Rate Kinetics

A growing body of pathological, biomarker, genetic, and mechanistic data suggests that amyloid accumulation, as a result of changes in production, processing, and/or clearance of brain amyloid-β peptide (Aβ) concentrations, plays a key role in the pathogenesis of Alzheimer’s disease (AD). Beta-secretase 1 (BACE1) mediates the first step in the processing of amyloid-β protein precursor (AβPP) to Aβ peptides, with the soluble N terminal fragment of AβPP (sAβPPβ) as a direct product, and BACE1 inhibition is an attractive target for therapeutic intervention to reduce the production of Aβ. Here, we report the in vitro and in vivo pharmacological profile of AZD3293, a potent, highly permeable, orally active, blood-brain barrier (BBB) penetrating, BACE1 inhibitor with unique slow off-rate kinetics. The in vitro potency of AZD3293 was demonstrated in several cellular models, including primary cortical neurons. In vivo in mice, guinea pigs, and dogs, AZD3293 displayed significant dose- and time-dependent reductions in plasma, cerebrospinal fluid, and brain concentrations of Aβ40, Aβ42, and sAβPPβ. The in vitro potency of AZD3293 in mouse and guinea pig primary cortical neuronal cells was correlated to the in vivo potency expressed as free AZD3293 concentrations in mouse and guinea pig brains. In mice and dogs, the slow off-rate from BACE1 may have translated into a prolongation of the observed effect beyond the turnover rate of Aβ. The preclinical data strongly support the clinical development of AZD3293, and patients with AD are currently being recruited into a combined Phase 2/3 study to test the disease-modifying properties of AZD3293.

Evidence for dimeric BACE-mediated APP processing.

beta-Secretase (BACE) is an aspartyl protease, which proteolytically processes amyloid precursor protein, making BACE an interesting pharmacological target in Alzheimers disease. To study the enzymatic function of BACE, we mutated either of the two aspartic acid residues in the active site of BACE. This rendered BACE functionally inactive without affecting the degree of glycosylation or endosomal localization. In contrast, substituting both active site aspartic acid residues produced a functionally inactive, endoplasmic reticulum-retained and partially glycosylated BACE. Interestingly, co-expression of the two single active site mutants partially restored beta-site cleavage of amyloid precursor protein, and the restored activity was inhibited with similar dose-dependency and potency as wildtype BACE by a small molecule inhibitor raised against BACE. In sum, our data suggest that two different active site mutants can complement each other in a partially functional BACE dimer and mediate APP processing.