O-GlcNAcase Inhibitor ASN90 is a Multimodal Drug Candidate for Tau and α-Synuclein Proteinopathies.

Neurodegenerative proteinopathies are characterized by the intracellular formation of insoluble and toxic protein aggregates in the brain that are closely linked to disease progression. In Alzheimer's disease and in rare tauopathies, aggregation of the microtubule-associated tau protein leads to the formation of neurofibrillary tangles (NFT). In Parkinson's disease (PD) and other α-synucleinopathies, intracellular Lewy bodies containing aggregates of α-synuclein constitute the pathological hallmark. Inhibition of the glycoside hydrolase O-GlcNAcase (OGA) prevents the removal of O-linked N-acetyl-d-glucosamine (O-GlcNAc) moieties from intracellular proteins and has emerged as an attractive therapeutic approach to prevent the formation of tau pathology. Like tau, α-synuclein is known to be modified with O-GlcNAc moieties and in vitro these have been shown to prevent its aggregation and toxicity. Here, we report the preclinical discovery and development of a novel small molecule OGA inhibitor, ASN90. Consistent with the substantial exposure of the drug and demonstrating target engagement in the brain, the clinical OGA inhibitor ASN90 promoted the O-GlcNAcylation of tau and α-synuclein in brains of transgenic mice after daily oral dosing. Across human tauopathy mouse models, oral administration of ASN90 prevented the development of tau pathology (NFT formation), functional deficits in motor behavior and breathing, and increased survival. In addition, ASN90 slowed the progression of motor impairment and reduced astrogliosis in a frequently utilized α-synuclein-dependent preclinical rodent model of PD. These findings provide a strong rationale for the development of OGA inhibitors as disease-modifying agents in both tauopathies and α-synucleinopathies. Since tau and α-synuclein pathologies frequently co-exist in neurodegenerative diseases, OGA inhibitors represent unique, multimodal drug candidates for further clinical development.

Substrate determinants in the C99 juxtamembrane domains differentially affect γ-secretase cleavage specificity and modulator pharmacology.

The molecular mechanisms governing γ-secretase cleavage specificity are not fully understood. Herein, we demonstrate that extending the transmembrane domain of the amyloid precursor protein-derived C99 substrate in proximity to the cytosolic face strongly influences γ-secretase cleavage specificity. Sequential insertion of leucines or replacement of membrane-anchoring lysines by leucines elevated the production of Aß42, whilst lowering production of Aß40. A single insertion or replacement was sufficient to produce this phenotype, suggesting that the helical length distal to the ε-site is a critical determinant of γ-secretase cleavage specificity. Replacing the lysine at the luminal membrane border (K28) with glutamic acid (K28E) increased Aß37 and reduced Aß42 production. Maintaining a positive charge with an arginine replacement, however, did not alter cleavage specificity. Using two potent and structurally distinct γ-secretase modulators (GSMs), we elucidated the contribution of K28 to the modulatory mechanism. Surprisingly, whilst lowering the potency of the non-steroidal anti-inflammatory drug-type GSM, the K28E mutation converted a heteroaryl-type GSM to an inverse GSM. This result implies the proximal lysine is critical for the GSM mechanism and pharmacology. This region is likely a major determinant for substrate binding and we speculate that modulation of substrate binding is the fundamental mechanism by which GSMs exert their action.

Development and characterization of a novel membrane assay for full-length BACE-1 at pH 6.0.

ß-Site amyloid precursor protein cleaving enzyme-1 (BACE-1) is a transmembrane aspartic protease that mediates the initial cleavage of the amyloid precursor protein (APP), leading to the generation of amyloid-ß (Aß) peptides that are thought to be causative of Alzheimer's disease (AD). Consequently, inhibition of BACE-1 is an attractive therapeutic approach for the treatment of AD. In general, in vitro biochemical assays to monitor BACE-1 activity have used the extracellular domain of the protein that contains the catalytic active site. This form of BACE-1 is catalytically active at acidic pH and cleaves APP-based peptide substrates at the ß-site. However, this form of BACE-1 does not mimic the natural physiology of BACE-1 and shows minimal activity at pH 6.0, which is more representative of the pH within the intracellular compartments where BACE-1 resides. Moreover, high-throughput screens with recombinant BACE-1 at pH 4.5 have failed to identify tractable leads for drug discovery, and hence, BACE-1 inhibitor development has adopted a rational drug design approach. Here we describe the development and validation of a novel membrane assay comprising full-length BACE-1 with measurable activity at pH 6.0, which could be used for the identification of novel inhibitors of BACE-1.

Reduction of amyloid load and cerebral damage in a transgenic mouse model of Alzheimer's disease by treatment with a beta-sheet breaker peptide.

Genetic, neuropathological, and biochemical studies have provided strong evidence for a central role of amyloid in the pathogenesis of Alzheimer's disease (AD). We have proposed previously that peptides designed as beta-sheet breakers may be useful in preventing the formation of amyloid plaques. In this study, we describe a modified beta-sheet breaker peptide with improved pharmacological properties, a high rate of penetration across the blood-brain barrier, and the ability to induce a dramatic reduction in amyloid deposition in two different transgenic AD models. In addition, we report for the first time a significant increase in neuronal survival and a decrease in brain inflammation associated with the reduction of amyloid plaques. These results demonstrate that the process of amyloid deposition is one of the causes of neurodegeneration in AD. Moreover, our findings indicate that beta-sheet breaker peptides provide a valuable tool for evaluating further the importance of amyloid in the etiology of AD and suggest that these peptides or some of their derivatives might be good candidates for AD treatment.

Amyloid beta40/42 clearance across the blood-brain barrier following intra-ventricular injections in wild-type, apoE knock-out and human apoE3 or E4 expressing transgenic mice.

An important event in the pathogenesis of Alzheimer's disease (AD) is the deposition of the amyloid beta (Abeta)1-40 and 1-42 peptides in a fibrillar form, with Abeta42 typically having a greater propensity to undergo this conformational change. A major risk factor for late-onset AD is the inheritance of the apolipoprotein E (apoE) 4 allele [3,14,31]. We previously proposed that apoE may function as a "pathological chaperone" in the pathogenesis of AD (i.e. modulate the structure of Abeta, promoting or stabilizing a beta-sheet conformation), prior to the discovery of this linkage [7,40,41,42]. Data from apoE knockout / AbetaPP^(V717F) mice, has shown that the presence of apoE is necessary for cerebral amyloid formation [1,2], consistent with our hypothesis. However, in betaPP^(V717F) mice expressing human apoE3 or E4 early Abeta deposition at 9 months is suppressed, but by 15 months both human apoE expressing mice had significant fibrillar Abeta deposits with the apoE4 expressing mice having a 10 fold greater amyloid burden [8,9]. This and other data has suggested that apoE, in addition to having a facilitating role in fibril formation, may also influence clearance of Abeta peptides. In order to address if apoE affects the clearance of Abeta peptides across the blood-brain barrier (BBB) and whether there are differences in the clearance of Abeta40 versus Abeta42, we performed stereotactic, intra-ventricular micro-injections of Abeta40, Abeta42 or control peptides in wild-type, apoE knock-out (KO) or human apoE3 or apoE4 expressing transgenic mice. We found that consistent with other studies [5], Abeta40 is rapidly cleared from the brain across the BBB; however, Abeta42 is cleared much less effectively. This clearance of exogenous Abeta peptides across the BBB does not appear to be affected by apoE expression. This data suggests that Abeta42 production may favor amyloid deposition due to a reduced clearance across the BBB, compared to Abeta40. In addition, our experiments support a role of apoE as a pathological chaperone, and do not suggest an isotype specific role of apoE in exogenous Abeta peptide clearance from the CSF across the BBB.