Cleavage of amyloid precursor protein (APP) by the Alzheimer's 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.

Generation and deposition of the amyloid b (Ab) peptide following proteolytic processing of the amyloid precursor protein (APP) by BACE-1 and c-secretase is central to the aetiology of Alzheimer's disease. Consequently, inhibition of BACE-1, a rate-limiting enzyme in the production of Ab, is an attractive therapeutic approach for the treatment of Alzheimer's disease. We have designed a selective non-peptidic BACE-1 inhibitor, GSK188909, that potently inhibits b-cleavage of APP and reduces levels of secreted and intracellular Ab in SHSY5Y cells expressing APP. In addition, we demonstrate that this compound can effectively lower brain Ab in 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 b-cleavage of APP and results in a significant reduction in the level of Ab40 and Ab42 in the brain. Encouragingly, subchronic dosing of GSK188909 in the absence of a p-glycoprotein inhibitor also lowers brain Ab. This pivotal first report of central Ab lowering, following oral administration of a BACE-1 inhibitor, supports the development of BACE-1 inhibitors for the treatment of Alzheimer's disease.

Inadequate target exposure is a major cause of high attrition in drug discovery. Here, we show that a label-free method for quantifying the intracellular bioavailability (F ic ) of drug molecules predicts drug access to intracellular targets and hence, pharmacological effect. We determined F ic in multiple cellular assays and cell types representing different targets from a number of therapeutic areas, including cancer, inflammation, and dementia. Both cytosolic targets and targets localized in subcellular compartments were investigated. F ic gives insights on membrane-permeable compounds in terms of cellular potency and intracellular target engagement, compared with biochemical potency measurements alone. Knowledge of the amount of drug that is locally available to bind intracellular targets provides a powerful tool for compound selection in early drug discovery. intracellular drug bioavailability | drug exposure | target engagement | published kinase inhibitor set | MAPK14

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 K m in the 10 M 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. The disintegrin metalloproteinases with thrombospondin motifs (ADAMTS)3 are a novel family of extracellular proteases forming an integral part of the extracellular matrix itself. Along with serine proteases, matrix metalloproteinases, bone morphogenetic protein-1/tolloid metalloproteinases, and ADAM (a disintegrin and metalloproteinase) proteins, ADAMTS proteins play a pivotal role in the proteolytic processing and turnover of the component molecules of the extracellular matrix of a broad range of tissues and have potential roles in the turnover of cell-surface proteins. ADAMTS proteins share the characteristic protease, disintegrin-like, and cysteine-rich domains in common with ADAM proteins, but differ in being soluble rather than membrane-bound and by the presence of thrombospondin type 1 (TSP-1) repeats (1, 2). ADAMTS-2, -3, and -14 are potential procollagen N-proteinases, and ADAMTS-13 has been identified as a von Willebrand factor-cleaving protease. ADAMTS-4 is a member of the "angiogenesis/aggrecanase" group of ADAMTS proteases (which also includes ADAMTS-1, -5, -8, -9, and -15) and is unique among the currently known ADAMTS proteases in containing only a single TSP-1-like motif, located between its disintegrin-like and cysteine-rich domains, and lacking any C-terminal TSP-1-like repeats (see Fig. 1). Like the other members of the angiogenesis/ aggrecanase group and ADAMTS-9, ADAMTS-4 has been demonstrated to act as an aggrecanase in vitro. In common with other aggrecanases, ADAMTS-4 is able to cleave aggrecan at multiple sites (five in total) (see Fig. 1) (3); however, it is one of only four that cleave aggrecan at the Glu 373 -Ala 374 "interglobulin domain" cleavage site (the others being ADAMTS-1, -5, and -8, which cleave with varying affinities). Aggrecan...

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.