| dc.description.abstract |
<p>Alzheimer's disease (AD) is a progressive neurodegenerative disease that affects
over 5 million people in the United States alone. This number is predicted to triple
to by the year 2050 due to both increasing life expectancies and the absence of disease-attenuating
drugs. The etiology of AD remains unclear, and although there are multiple theories
implicating everything from oxidative stress to protein misfolding, misregulated metal
ions appear as a common thread in disease pathology. </p><p>Chelation therapy has
shown some effectiveness in clinical trials, but to date, there are no FDA-approved
metal chelators for the treatment of AD. One of the biggest problems with general
chelators is their inability to differentiate between the metal ions involved in disease
progression verses those involved in normal metabolic function. To address this problem,
we have developed a prochelator approach whereby the prochelator (SWH) does not bind
metals with significant biological affinity. However, once activated to the chelator
(CP) via enzymatic hydrolysis, the molecule is able to bind copper and reduce its
toxicity both in vitro and in a cellular model of Alzheimer's Disease. </p><p>Central
to this strategy is the site-specificity provided by enzymatic activation of the prochelator.
In our system, SWH to CP conversion is mediated by beta-secretase, an enzyme involved
in A-beta generation. However, in order to render SWH capable of hydrolysis in cells,
we modified the prochelator to contain a dihydrocholesterol membrane anchor attached
via a polyethylene glycol linker. From this construct, we created beta-MAP, which
is an SWH-based FRET probe to demonstrate beta-secretase-mediated conversion of SWH
to CP. beta-MAP was also used to confirm the efficacy of a known beta-secretase inhibitor
without the need to for mutated cells lines or expensive antibodies. beta;-MAP and
the associated microscopy method represent a significant advancement to the currently
available ELISA assays for beta-secretase activity.</p><p>While activation of the
prochelator by an enzyme in cells is encouraging, non-specific hydrolysis of the peptide
prevents significant accumulation of the chelator on the cell membrane. Furthermore,
attachment of the polyethylene glycol and sterol units induce cell toxicity not seen
with the native CP peptide. These drawbacks prevent the current prochelator from
effectively protecting cells from AD conditions. Structural modifications to overcome
these problems, including implementation of a new peptide sequence are planned for
future experiments.</p>
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