Monoamine oxidase binding not expected to significantly affect [18F]flortaucipir PET interpretation.

PURPOSE: [18F]-labeled positron emission tomography (PET) radioligands permit in vivo assessment of Alzheimer's disease biomarkers, including aggregated neurofibrillary tau (NFT) with [18F]flortaucipir. Due to structural similarities of flortaucipir with some monoamine oxidase A (MAO-A) inhibitors, this study aimed to evaluate flortaucipir binding to MAO-A and MAO-B and any potential impact on PET interpretation. METHODS: [18F]Flortaucipir autoradiography was performed on frozen human brain tissue slices, and PET imaging was conducted in rats. Dissociation constants were determined by saturation binding, association and dissociation rates were measured by kinetic binding experiments, and IC50 values were determined by competition binding. RESULTS: Under stringent wash conditions, specific [18F]flortaucipir binding was observed on tau NFT-rich Alzheimer's disease tissue and not control tissue. In vivo PET experiments in rats revealed no evidence of [18F]flortaucipir binding to MAO-A; pre-treatment with MAO inhibitor pargyline did not impact uptake or wash-out of [18F]flortaucipir. [18F]Flortaucipir bound with low nanomolar affinity to human MAO-A in a microsomal preparation in vitro but with a fast dissociation rate relative to MAO-A ligand fluoroethyl-harmol, consistent with no observed in vivo binding in rats of [18F]flortaucipir to MAO-A. Direct binding of flortaucipir to human MAO-B was not detected in a microsomal preparation. A high concentration of flortaucipir (IC50 of 1.3 µM) was found to block binding of the MAO-B ligand safinamide to MAO-B on microsomes suggesting that, at micromolar concentrations, flortaucipir weakly binds to MAO-B in vitro. CONCLUSION: These data suggest neither MAO-A nor MAO-B binding will contribute significantly to the PET signal in cortical target areas relevant to the interpretation of [18F]flortaucipir.

Macrocyclic BACE inhibitors: Optimization of a micromolar hit to nanomolar leads.

We have identified macrocyclic inhibitors of the aspartic protease BACE, implicated in the etiology of Alzheimer's disease. An X-ray structure of screening hit 1 in the BACE active site revealed a hairpin conformation suggesting that constrained macrocyclic derivatives may also bind there. Several of the analogs we prepared were >100x more potent than 1, such as 7 (5 nM K(i)).

2-Amino-3,4-dihydroquinazolines as inhibitors of BACE-1 (beta-site APP cleaving enzyme): Use of structure based design to convert a micromolar hit into a nanomolar lead.

A new aspartic protease inhibitory chemotype bearing a 2-amino-3,4-dihydroquinazoline ring was identified by high-throughput screening for the inhibition of BACE-1. X-ray crystallography revealed that the exocyclic amino group participated in a hydrogen bonding array with the two catalytic aspartic acids of BACE-1 (Asp(32), Asp(228)). BACE-1 inhibitory potency was increased (0.9 microM to 11 nM K(i)) by substitution into the unoccupied S(1)' pocket.

A comparative analysis of brain and plasma Abeta levels in eight common non-transgenic mouse strains: validation of a specific immunoassay for total rodent Abeta.

Transgenic mouse models of Alzheimer's disease (AD) are being utilized as models for elucidating AD etiology and potential therapeutic approaches. However, two major drawbacks of these models are: (1) transgenic animals often over-express amyloid beta (Abeta) to high levels compared to that seen in sporadic human AD and (2) the current intellectual property issues surrounding a number of these models make them difficult to utilize in a commercial setting. Our goal was to identify an appropriate non-transgenic mouse strain, devoid of these patent restrictions and test whether amyloid-modulating compounds will lower total brain and plasma Abeta. Plasma and brain samples were collected from eight commonly used mouse strains (C57BL/6, SJL, CF-1, DBA/2, CD-1, 129, FVB and B6D2F1; Charles River Labs) and total Abetalevels were validated and quantified with a rodent-specific monoclonal Abetaantibody. Plasma Abeta in SJL mice was the highest of the eight strains tested (213 pM +/- 21 pM), but was not significantly different than the seven other strains. Total brain Abeta in SJL mice was also the greatest of the mouse strains tested (356 pM +/- 73 pM). SJL, C57BL/6 and CF-1 mice had total brain Abeta levels that were significantly greater than Abeta levels in B6D2F1 mice (242 +/- 20 pM). In vivo efficacy of an Abeta lowering agent was observed in CF-1 mice upon oral administration of the gamma-secretase inhibitors, DAPT and LY-411575. The absolute levels of rodent brain Abeta detected and the efficacy of the gamma-secretase treatment were dependent upon the antibodies used, as well as the extraction methodology. The measurement of total brain Abeta lowering in a common mouse strain could help accelerate drug discovery programs for Alzheimer's disease without relying on costly transgenic animals that overexpress APP in a manner that may not be predictive of the effects of these compounds in human AD.

Interactions among alpha-synuclein, dopamine, and biomembranes: some clues for understanding neurodegeneration in Parkinson's disease.

Parkinson's disease (PD) is a neurologic disorder resulting from the loss of dopaminergic neurons in the brain. Two lines of evidence suggest that the protein alpha-synuclein plays a role in the pathogenesis of PD: Fibrillar alpha-synuclein is a major component of Lewy bodies in diseased neurons, and two mutations in alpha-synuclein are linked to early-onset disease. Accordingly, the fibrillization of alpha-synuclein is proposed to contribute to neurodegeneration in PD. In this report, we provide evidence that oligomeric intermediates of the alpha-synuclein fibrillization pathway, termed protofibrils, might be neurotoxic. Analyses of protofibrillar alpha-synuclein by atomic force microscopy and electron microscopy indicate that the oligomers consist of spheres, chains, and rings. alpha-Synuclein protofibrils permeabilize synthetic vesicles and form pore-like assemblies on the surface of brain-derived vesicles. Dopamine reacts with alpha-synuclein to form a covalent adduct that slows the conversion of protofibrils to fibrils. This finding suggests that cytosolic dopamine in dopaminergic neurons promotes the accumulation of toxic alpha-synuclein protofibrils, which might explain why these neurons are most vulnerable to degeneration in PD. Finally, we note that aggregation of alpha-synuclein likely occurs via different mechanisms in the cell versus the test tube. For example, the binding of alpha-synuclein to cellular membranes might influence its self-assembly. To address this point, we have developed a yeast model that might enable the selection of random alpha-synuclein mutants with different membrane-binding affinities. These variants might be useful to test whether membrane binding by alpha-synuclein is necessary for neurodegeneration in transgenic animal models of PD.

Virus-based expression systems facilitate rapid target in vivo functionality validation and high-throughput screening.

Target validation is one of rate-limiting steps in the modern drug discovery. The authors developed a strategy of combining adenovirus-mediated gene transfer for efficient target functionality validation, both in vivo and in vitro, with baculovirus expression to produce sufficient quantities of protein for high-throughput screening (HTS). The incorporation of green fluorescent protein (GFP) in the adenovirus vectors accelerates recombinant adenovirus plaque purification, whereas the use of epitope and affinity tags facilitates the identification and purification of recombinant protein. In this generalized scheme, the flexible modular design of viral vectors facilitates the transition between target validation and HTS. In the example presented, functional target validation in vivo was achieved by overexpressing the target gene in cell-based models and in the mouse cortex following adenovirus-mediated gene delivery. In this context, target overexpression resulted in the accumulation of a disease-related biomarker both in vitro and in vivo. A baculovirus-based expressional system was then generated to produce enough target protein for HTS. Thus, the use of these viral expression systems represents a generalized method for rapid target functionality validation and HTS assay development, which could be applied to numerous target candidates being elucidated in gene discovery programs.

Emerging beta-amyloid therapies for the treatment of Alzheimer's disease.

Alzheimer's Disease (AD) is a progressive neurodegenerative disorder marked by loss of memory, cognition, and behavioral stability. AD is defined pathologically by extracellular neuritic plaques comprised of fibrillar deposits of beta-amyloid peptide (Abeta) and neurofibrillary tangles comprised of paired helical filaments of hyperphosphorylated tau. Current therapies for AD, such as cholinesterase inhibitors, treat the symptoms but do not modify the progression of the disease. The etiology of AD is unclear. However, data from familial AD mutations (FAD) strongly support the "amyloid cascade hypothesis" of AD, i.e. that neurodegeneration in AD is initiated by the formation of neurotoxic beta-amyloid (Abeta) aggregates; all FAD mutations increase levels of Abeta peptide or density of Abeta deposits. The likely link between Abeta aggregation and AD pathology emphasizes the need for a better understanding of the mechanisms of Abeta production. This review summarizes current therapeutic strategies directed at lowering Abeta levels and decreasing levels of toxic Abeta aggregates through (1) inhibition of the processing of amyloid precursor protein (APP) to Abeta peptide, (2) inhibition, reversal or clearance of Abeta aggregation, (3) cholesterol reduction and (4) Abeta immunization.

Synthesis of (-)-5,8-dihydroxy-3R-methyl-2R-(dipropylamino)-1,2,3,4-tetrahydronaphthalene: an inhibitor of beta-amyloid(1-42) aggregation.

A concise synthesis of the beta-amyloid(1-42 )aggregation inhibitor (-)-5,8-dihydroxy-3R-methyl-2R-(dipropylamino)-1,2,3,4-tetrahydronaphthalene [(-)-2] has been developed. The key step is a regio- and diastereoselective hydroboration-amination sequence to convert alkene into amine. Enantiomeric resolution was achieved by recrystallization of amine as the dibenzoyl-D-tartaric acid salt. Hydroquinone is a potent inhibitor of the fibrillar aggregation of beta-amyloid as determined in two different assay systems.