Gareth Wayne

Oral administration of a potent and selective non-peptidic BACE-1 inhibitor decreases β-cleavage of amyloid precursor protein and amyloid-β production in vivo

Generation and deposition of the amyloid β (Aβ) peptide following proteolytic processing of the amyloid precursor protein (APP) by BACE‐1 and γ‐secretase is central to the aetiology of Alzheimers disease. Consequently, inhibition of BACE‐1, a rate‐limiting enzyme in the production of Aβ, is an attractive therapeutic approach for the treatment of Alzheimers disease. We have designed a selective non‐peptidic BACE‐1 inhibitor, GSK188909, that potently inhibits β‐cleavage of APP and reduces levels of secreted and intracellular Aβ in SHSY5Y cells expressing APP. In addition, we demonstrate that this compound can effectively lower brain Aβin vivo. In APP transgenic mice, acute oral administration of GSK188909 in the presence of a p‐glycoprotein inhibitor to markedly enhance the exposure of GSK188909 in the brain decreases β‐cleavage of APP and results in a significant reduction in the level of Aβ40 and Aβ42 in the brain. Encouragingly, subchronic dosing of GSK188909 in the absence of a p‐glycoprotein inhibitor also lowers brain Aβ. This pivotal first report of central Aβ lowering, following oral administration of a BACE‐1 inhibitor, supports the development of BACE‐1 inhibitors for the treatment of Alzheimers disease.

Heparan sulfate regulates amyloid precursor protein processing by BACE1, the Alzheimer's β-secretase

Cleavage of amyloid precursor protein (APP) by the Alzheimers β-secretase (BACE1) is a key step in generating amyloid β-peptide, the main component of amyloid plaques. Here we report evidence that heparan sulfate (HS) interacts with β-site APP-cleaving enzyme (BACE) 1 and regulates its cleavage of APP. We show that HS and heparin interact directly with BACE1 and inhibit in vitro processing of peptide and APP substrates. Inhibitory activity is dependent on saccharide size and specific structural characteristics, and the mechanism of action involves blocking access of substrate to the active site. In cellular assays, HS specifically inhibits BACE1 cleavage of APP but not alternative cleavage by α-secretase. Endogenous HS immunoprecipitates with BACE1 and colocalizes with BACE1 in the Golgi complex and at the cell surface, two of its putative sites of action. Furthermore, inhibition of cellular HS synthesis results in enhanced BACE1 activity. Our findings identify HS as a natural regulator of BACE1 and suggest a novel mechanism for control of APP processing.

Second generation of BACE-1 inhibitors part 3: Towards non hydroxyethylamine transition state mimetics

Our first generation of hydroxyethylamine BACE-1 inhibitors proved unlikely to provide molecules that would lower amyloid in an animal model at low oral doses. This observation led us to the discovery of a second generation of inhibitors having nanomolar activity in a cell-based assay and with the potential for improved pharmacokinetic profiles. In this Letter, we describe our successful strategy for the optimization of oral bioavailability and also give insights into the design of compounds with the potential for improved brain penetration.

Bace-1 Inhibitors Part 3: Identification of Hydroxy Ethylamines (Heas) with Nanomolar Potency in Cells.

This article is focusing on further optimization of previously described hydroxy ethylamine (HEA) BACE-1 inhibitors obtained from a focused library with the support of X-ray crystallography. Optimization of the non-prime side of our inhibitors and introduction of a 6-membered sultam substituent binding to Asn-294 as well as a fluorine in the C-2 position led to derivatives with nanomolar potency in cell-based assays.

TIMP-3 Inhibition of ADAMTS-4 (Aggrecanase-1) Is Modulated by Interactions between Aggrecan and the C-terminal Domain of ADAMTS-4

ADAMTS-4 (aggrecanase-1) is a glutamyl endopeptidase capable of generating catabolic fragments of aggrecan analogous to those released from articular cartilage during degenerative joint diseases such as osteoarthritis. Efficient aggrecanase activity requires the presence of sulfated glycosaminoglycans attached to the aggrecan core protein, implying the contribution of substrate recognition/binding site(s) to ADAMTS-4 activity. In this study, we developed a sensitive fluorescence resonance energy transfer peptide assay with a Km in the 10 μm range and utilized this assay to demonstrate that inhibition of full-length ADAMTS-4 by full-length TIMP-3 (a physiological inhibitor of metalloproteinases) is enhanced in the presence of aggrecan. Our data indicate that this interaction is mediated largely through the binding of glycosaminoglycans (specifically chondroitin 6-sulfate) of aggrecan to binding sites in the thrombospondin type 1 motif and spacer domains of ADAMTS-4 to form a complex with an improved binding affinity for TIMP-3 over free ADAMTS-4. The results of this study therefore indicate that the cartilage environment can modulate the function of enzyme-inhibitor systems and could have relevance for therapeutic approaches to aggrecanase modulation.

Second generation of BACE-1 inhibitors. Part 1: The need for improved pharmacokinetics

Inhibition of the aspartyl protease BACE-1 has the potential to deliver a disease-modifying therapy for Alzheimers disease. We have recently disclosed a series of transition-state mimetic BACE-1 inhibitors showing nanomolar potency in cell-based assays. Amongst them, GSK188909 (compound 2) had favorable pharmacokinetics and was the first orally bioavailable inhibitor reported to demonstrate brain amyloid lowering in an animal model. In this Letter, we describe the reasons that led us to favor a second generation of inhibitors for further in vivo studies.

BACE-1 inhibitors part 1: identification of novel hydroxy ethylamines (HEAs).

Inhibition of the aspartyl protease BACE-1 has the potential to deliver a disease-modifying therapy for Alzheimers disease. Herein, is described the lead generation effort which resulted, with the support of X-ray crystallography, in the discovery of potent inhibitors based on a hydroxy ethylamine (HEA) transition-state mimetic. These inhibitors were capable of lowering amyloid production in a cell-based assay.

Second Generation of Bace-1 Inhibitors Part 2: Optimisation of the Non-Prime Side Substituent.

Our first generation of hydroxyethylamine transition-state mimetic BACE-1 inhibitors allowed us to validate BACE-1 as a key target for Alzheimers disease by demonstrating amyloid lowering in an animal model, albeit at rather high doses. Finding a molecule from this series which was active at lower oral doses proved elusive and demonstrated the need to find a novel series of inhibitors with improved pharmacokinetics. This Letter describes the discovery of such inhibitors.

Continuous real-time measurement of tumor necrosis factor- α converting enzyme activity on live cells

Tumor necrosis factor-α (TNF) converting enzyme (TACE) is responsible for shedding of various membrane proteins including proinflammatory cytokine TNF. In vivo regulation of TACE is poorly understood mainly due to lack of reliable methodology to measure TACE activity in cell-based assays. Here we report a novel enzyme assay that enables continuous real-time measurement of TACE activity on the surface of live cells. Cells were incubated with a new fluorescent resonance energy transfer peptide consisting of a TACE-sensitive TNF sequence and fluorescein–tetramethylrhodamine (FAM–TAMRA), and enzyme activity was monitored by the rate of increase in fluorescent signal due to peptide cleavage. Validation studies using resting as well as stimulated monocytic cells indicated that the assay was sensitive, reproducible and quantitative. Pharmacological studies with various inhibitors indicated that the observed enzyme activity could largely be ascribed to TACE. Thus, the FAM–TAMRA peptide provides a powerful tool for measurement of constitutive and inducible cellular TACE activity. The principles developed may be applied to analyses of enzyme activity of various sheddases on live cells.

Direct Measurement of Intracellular Compound Concentration by RapidFire Mass Spectrometry Offers Insights into Cell Permeability

One of the key challenges facing early stage drug discovery is understanding the commonly observed difference between the activity of compounds in biochemical assays and cellular assays. Traditionally, indirect or estimated cell permeability measurements such as estimations from logP or artificial membrane permeability are used to explain the differences. The missing link is a direct measurement of intracellular compound concentration in whole cells. This can, in some circumstances, be estimated from the cellular activity, but this may also be problematic if cellular activity is weak or absent. Advances in sensitivity and throughput of analytical techniques have enabled us to develop a high-throughput assay for the measurement of intracellular compound concentration for routine use to support lead optimization. The assay uses a RapidFire-MS based readout of compound concentration in HeLa cells following incubation of cells with test compound. The initial assay validation was performed by ultra-high performance liquid chromatography tandem mass spectrometry, and the assay was subsequently transferred to RapidFire tandem mass spectrometry. Further miniaturization and optimization were performed to streamline the process, increase sample throughput, and reduce cycle time. This optimization has delivered a semi-automated platform with the potential of production scale compound profiling up to 100 compounds per day.