Modulation and Characterization of Alzheimer's Disease Associated gamma-Secretase
Alzheimer’s disease (AD) is a devastating neurodegenerative disorder that causes the most common form of dementia, affecting approximately 5% of the population over the age of 65 years in Europe. Short-term memory impairment, disorientation, aphasia, and a general cognitive decline are common sympto...
| Summary: | Alzheimer’s disease (AD) is a devastating neurodegenerative disorder that causes the most common form of dementia, affecting approximately 5% of the population over the age of 65 years in Europe. Short-term memory impairment, disorientation, aphasia, and a general cognitive decline are common symptoms early in disease development. According to the World Health Organization, an estimated 37 million people worldwide currently have dementia; AD affects about 18 million of them. In spite of tremendous research efforts, there is no causal therapy for AD. Histopathological hallmarks of AD are the extracellular plaques consisting of the amyloid β-peptide (Aβ) and neurofibrillary tangles which are found in the brains of the AD patients. Aβ is heterogeneously produced by the sequential cleavages of amyloid precursor protein (APP) by the two aspartic proteases: β- and γ-secretase. Intramembrane cleavage by γ-secretase occurs with little sequence specificity, resulting in Aβ fragments of different length, predominantly Aβ40, Aβ42, and some Aβ38. Aβ42 being the most aggregatory, and is believed to trigger the amyloid cascade, a pathological series of neurotoxic events, which eventually leads to neurodegeneration and finally AD. γ-Secretase has been an attractive target in many ways for AD therapeutics since it catalyzes the final step in the release of Aβ. The present work describes the design, synthesis and biological evaluation of γ-secretase modulators, affinity probes and photoaffinity labels. Epidemiological studies have indicated a close association between prolonged use of nonsteroidal anti-inflammaroy drugs (NSAIDs), and reduced risk for AD. Therefore, cyclooxygenase (COX) inhibitors such as flurbiprofen, sulindac sulfoxide and meclofenamic acid were converted into amides and esters and anticipated that the modification of acid functionality would reduce the COX inhibitory activity while increasing the γ-secretase modulatory activity. However, the conversion of the acid moiety of COX inhibitors into their amides or esters resulted either in inverse modulation or inhibition. Further efforts were then focussed on carprofen, a COX-2 inhibitor which is approved for the use in dogs and cows. N-substitution of carprofen resulted in potent modulators of γ-secretase, and the compounds displayed little or no effect on γ-secretase cleavage at the ε-site. Knowing about the relevance of N-substitution of carprofen and the necessity of the free acid functionality for γ-secretase modulation, a series of N-substituted carbazolyloxy acetic acids was synthesized. As anticipated, they turned out to be effective modulators of γ-secretase and displayed little or no effect on γ-secretase cleavage at the ε-site. An analogous derivative of the LXR agonist TO-901317 was synthesized, but the hexafluorocarbinol moiety was replaced by an oxyacetic acid in order to transform it into a modulator of γ-secretase. As expected, the introduction of the acid moiety changed the mode of action from an inverse modulation to normal modulation of γ-secretase. A series of NSAIDs derived affinity labels was synthesized in order to identify and characterize the binding site by immunoprecipitation assay. Flurbiprofen derived photoaffinity label captured active γ-secretase complex in a dose dependant manner, but others either failed to provide a binding partner or resulted in unspecific binding. Flurbiprofen and DAPT derived photoaffnity labels were synthesized in order to investigate the binding site via covalent bond formation with the active site on the γ-secretase complex. The photaffnity labelling experiment carried out with a flurbiprofen derived photoaffnity probe revealed that it binds to the C-terminal fragment of presenilin (PS). Whereas, DAPT derived photoaffinity label displayed very weak binding to PS, the active site of γ-secretase. |
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