Huw D. Lewis

A presenilin dimer at the core of the gamma-secretase enzyme: insights from parallel analysis of Notch 1 and APP proteolysis.

Notch receptors and the amyloid precursor protein are type I membrane proteins that are proteolytically cleaved within their transmembrane domains by a presenilin (PS)-dependent γ-secretase activity. In both proteins, two peptide bonds are hydrolyzed: one near the inner leaflet and the other in the middle of the transmembrane domain. Under saturating conditions the substrates compete with each other for proteolysis, but not for binding to PS. At least some Alzheimers disease-causing PS mutations reside in proteins possessing low catalytic activity. We demonstrate (i) that differentially tagged PS molecules coimmunoprecipitate, and (ii) that PS N-terminal fragment dimers exist by using a photoaffinity probe based on a transition state analog γ-secretase inhibitor. We propose that γ-secretase contains a PS dimer in its catalytic core, that binding of substrate is at a site separate from the active site, and that substrate is cleaved at the interface of two PS molecules.

Fenton chemistry and oxidative stress mediate the toxicity of the β-amyloid peptide in a Drosophila model of Alzheimer’s disease

The mechanism by which aggregates of the β‐amyloid peptide (Aβ) mediate their toxicity is uncertain. We show here that the expression of the 42‐amino‐acid isoform of Aβ (Aβ1–42) changes the expression of genes involved in oxidative stress in a Drosophila model of Alzheimer’s disease. A subsequent genetic screen confirmed the importance of oxidative stress and a molecular dissection of the steps in the cellular metabolism of reactive oxygen species revealed that the iron‐binding protein ferritin and the H2O2 scavenger catalase are the most potent suppressors of the toxicity of wild‐type and Arctic (E22G) Aβ1–42. Likewise, treatment with the iron‐binding compound clioquinol increased the lifespan of flies expressing Arctic Aβ1–42. The effect of iron appears to be mediated by oxidative stress as ferritin heavy chain co‐expression reduced carbonyl levels in Aβ1–42 flies by 65% and restored the survival and locomotion function to normal. This was achieved despite the presence of elevated levels of the Aβ1–42. Taken together, our data show that oxidative stress, probably mediated by the hydroxyl radical and generated by the Fenton reaction, is essential for Aβ1–42 toxicity in vivo and provide strong support for Alzheimer’s disease therapies based on metal chelation.

The Novel γ Secretase Inhibitor N-[cis-4-[(4-Chlorophenyl)sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]-1,1,1-trifluoromethanesulfonamide (MRK-560) Reduces Amyloid Plaque Deposition without Evidence of Notch-Related Pathology in the Tg2576 Mouse

There is a substantial body of evidence indicating that β-amyloid peptides (Aβ) are critical factors in the onset and development of Alzheimers disease (AD). One strategy for combating AD is to reduce or eliminate the production of Aβ through inhibition of the γ-secretase enzyme, which cleaves Aβ from the amyloid precursor protein (APP). We demonstrate here that chronic treatment for 3 months with 3 mg/kg of the potent, orally bioavailable and brain-penetrant γ-secretase inhibitor N-[cis-4-[(4-chlorophenyl)-sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]-1,1,1-trifluoromethanesulfonamide (MRK-560) attenuates the appearance of amyloid plaques in the Tg2576 mouse. These reductions in plaques were also accompanied by a decrease in the level of reactive gliosis. The morphometric and histological measures agreed with biochemical analysis of Aβ(40) and Aβ(42) in the cortex. Interestingly, the volume of the plaques across treatment groups did not change, indicating that reducing Aβ levels does not significantly alter deposit growth once initiated. Furthermore, we demonstrate that these beneficial effects can be achieved without causing histopathological changes in the ileum, spleen, or thymus as a consequence of blockade of the processing of alternative substrates, such as the Notch family of receptors. This indicates that in vivo a therapeutic window between these substrates seems possible—a key concern in the development of this approach to AD. An understanding of the mechanisms whereby MRK-560 shows differentiation between the APP and Notch proteolytic pathway of γ-secretase should provide the basis for the next generation of γ-secretase inhibitors.

A new series of potent benzodiazepine γ-secretase inhibitors

Abstract A new series of benzodiazepine-containing γ-secretase inhibitors with potential use in the treatment of Alzheimers disease is disclosed. Structure–activity relationships of the pendant hydrocinnamate side-chain which led to the preparation of highly potent inhibitors are described.

Intra- or Intercomplex Binding to the γ-Secretase Enzyme A MODEL TO DIFFERENTIATE INHIBITOR CLASSES

γ-Secretase is one of the critical enzymes required for the generation of amyloid-β peptides from the β-amyloid precursor protein. Because amyloid-β peptides are generally accepted to play a key role in Alzheimer disease, γ-secretase inhibition holds the promise for a disease-modifying therapy for this neurodegenerative condition. Although recent progress has enhanced the understanding of the biology and composition of the γ-secretase enzyme complex, less information is available on the actual interaction of various inhibitor classes with the enzyme. Here we show that the two principal classes of inhibitor described in the scientific and patent literature, aspartyl protease transition state analogue and small molecule non-transition state inhibitors, display fundamental differences in the way they interact with the enzyme. Taking advantage of a γ-secretase enzyme overexpressing cellular system and different radiolabeled γ-secretase inhibitors, we observed that the maximal binding of non-transition state γ-secretase inhibitors accounts only for half the number of catalytic sites of the recombinant enzyme complex. This characteristic stoichiometry can be best accommodated with a model whereby the non-transition state inhibitors bind to a unique site at the interface of a dimeric enzyme. Subsequent competition studies confirm that this site appears to be targeted by the main classes of small molecule γ-secretase inhibitor. In contrast, the non-steroidal anti-inflammatory drug γ-secretase modulator sulindac sulfide displayed noncompetitive antagonism for all types of inhibitor. This finding suggests that non-steroidal anti-inflammatory drug-type γ-secretase modulators target an alternative site on the enzyme, thereby changing the conformation of the binding sites for γ-secretase inhibitors.

Cerebrospinal fluid levels of beta-amyloid(42) in patients with Alzheimer's disease are related to the exon 2 polymorphism of the cathepsin D gene.

The intracellular aspartyl protease cathepsin D (catD) is involved in such Alzheimers disease (AD)-related processes as the activation of the endosomal/lysosomal system and the cleavage of the amyloid precursor protein into amyloidogenic components, which may initiate neurodegeneration. A non-synonymous polymorphism (exon 2, C to T exchange leading to ala→val substitution) of the gene encoding catD (CTSD) was previously associated with AD, in that the T allele increased the risk for AD. To investigate whether the T allele is associated with disease-related traits, we measured the concentration of the amyloid &bgr;-peptide 1-42 (A&bgr;(42)) and 1-40 (A&bgr;(40)) in patients and control subjects. The T allele of the CTSD genotype was associated with a 50% decrease in A&bgr;(42) levels in the cerebrospinal fluid. Thus, we demonstrate a significant impact of the CTSD genotype on A&bgr;(42) levels in the cerebrospinal fluid of AD patients and underpin the importance of the validation of susceptibility genes by examining their potential pathophysiological relevance.

A study of long-term potentiation in transgenic mice over-expressing mutant forms of both amyloid precursor protein and presenilin-1

Synaptic transmission and long-term potentiation (LTP) in the CA1 region of hippocampal slices have been studied during ageing of a double transgenic mouse strain relevant to early-onset familial Alzheimers disease (AD). This strain, which over-expresses both the 695 amino acid isoform of human amyloid precursor protein (APP) with K670N and M671L mutations and presenilin 1 with the A246E mutation, has accelerated amyloidosis and plaque formation. There was a decrease in synaptic transmission in both wildtype and transgenic mice between 2 and 9 months of age. However, preparing slices from 14 month old animals in kynurenic acid (1 mM) counteracted this age-related deficit. Basal transmission and paired-pulse facilitation was similar between the two groups at all ages (2, 6, 9 and 14 months) tested. Similarly, at all ages LTP, induced either by theta burst stimulation or by multiple tetani, was normal. These data show that a prolonged, substantially elevated level of Aβ are not sufficient to cause deficits in the induction or expression of LTP in the CA1 hippocampal region.

A comparative assessment of γ-secretase activity in transgenic and non-transgenic rodent brain

Abstract Amyloid-beta (Aβ) deposits are one of the hallmarks of the neuropathological degeneration observed in Alzheimers disease (AD) and Aβ concentrations have been reported to vary in different brain regions of AD patients. Aβ is produced by the sequential cleavage of amyloid precursor protein (APP) by β-secretase and γ-secretase, respectively. Previous studies have shown that over-expression of the γ-secretase complex leads to increased γ-secretase proteolytic activity increasing Aβ production. However, it is not known whether brain regions with highest Aβ concentration also express relatively higher levels of γ-secretase activity. Accordingly, the relationship between Aβ levels and γ-secretase activity across brain regions was investigated and correlated in the brains of transgenic and non-transgenic rodents commonly used in AD research. The data demonstrated that Aβ levels do vary in different brain regions in both transgenic and non-transgenic mice but are not correlated with regional γ-secretase activity. Furthermore, this study demonstrated that while mutations in the APP and PS1 sequences affect the absolute Aβ levels this is not reflected in an increase in γ-secretase proteolytic activity. The data in the current paper indicate that this assay is able to measure the level of γ-secretase activity in rodent species. Using this methodology will aid our understanding of physiological γ-secretase function.

Discovery of potent and selective Spleen Tyrosine Kinase inhibitors for the topical treatment of inflammatory skin disease

The discovery and lead optimisation of a novel series of SYK inhibitors is described. These were optimised for SYK potency and selectivity against Aurora B. Compounds were profiled in a human skin penetration study to identify a suitable candidate molecule for pre-clinical development. Compound 44 (GSK2646264) was selected for progression and is currently in Phase I clinical trials.