Description:
Princeton Invention #
06-2246
Researchers in the
Chemistry Department at Princeton University have developed a method for
screening large libraries of small molecules to find those ¿hits¿ that block
Aβ42 aggregation. The development of this high throughput screen is based
on the finding that fusions of Aβ42 to green fluorescent protein ( GFP) prevent
the folding and fluorescence of GFP, whereas mutations in Aβ42 that disrupt
aggregation produce green fluorescent fusions.
It
has been demonstrated that the Aβ42-GFP fusion does not produce a fluorescent
signal. The reason for the lack of fluorescence is because Aβ42 aggregates
rapidly and drags the attached GFP into the aggregated state before GFP has an
opportunity to fold into its correct fluorescent structure. This observation
that the fluorescence of GFP is prevented by Aβ42 aggregation provides an
outstanding opportunity to screen for agents that restore fluorescence by
blocking Aβ42 aggregation. Two mechanisms are envisioned in which Aβ42
aggregation can be blocked, the first involved changes in Aβ42 itself and the
second involves molecules (drugs or leads) that inhibit aggregation. Proof of
Principle has been established in mutations of Aβ42 (see publications listed
below) and most recently the screen has been used to identify small
molecules that have been shown to block aggregation.
The brains of
Alzheimer¿s patients contain an abundance of insoluble plaque, the primary
component of this plaque is a peptide called the Alzheimer¿s Amyloid β peptide.
This 42 amino acid peptide, also called Aβ42, is generated in vivo by
proteolytic cleavage of the Alzheimer¿s Precursor Protein (APP). In healthy
people, Aβ42 is cleared before it aggregates into plaque. However in Alzheimer¿s
brains, Aβ accumulates and aggregates into insoluble fibrillar material called
¿Amyloid¿. A wide range of genetic and biochemical data suggest that either the
amyloid aggregates themselves, or partially aggregated precursors on the pathway
towards amyloid, play a causative role in Alzheimer¿s disease. Therefore,
prevention of Aβ42 aggregation is considered a promising strategy for the
treatment and/or prevention of Alzheimer¿s disease.
Several strategies for
preventing Aβ42 aggregation have been pursued in academic labs and at
pharmaceutical companies. Two frequently cited strategies are the design of
inhibitors to block the proteases (called secretases) responsible for generating
Aβ from APP (Alzheimer¿s Precursor Protein) and secondly immunization with Aβ42
with the hope that the resulting immune response will generate antibodies
capable of reacting with Aβ42 and clearing it from the brain. Although
both strategies initially generated promising results, both suffer from serious
drawbacks. Inhibitors that block secretase activity have been synthesized;
however, because the secretases have other functions in normal metabolism, these
inhibitors produce side effects and are not likely to be suitable as
anti-Alzheimer¿s drugs. On the other hand, immunization with Aβ42 initially
appeared to be a specific approach, unlikely to produce side effects.
Unfortunately, however, although immunization elicited the desired anti-Aβ
antibodies, clinical trials had to be halted when several subjects developed
life-threatening side effects. These findings highlight the urgency
of finding a small molecule that prevents Aβ aggregation.
Previous attempts to
develop a screen for small molecules that prevent Aβ aggregation are hampered by
the following limitations: (i) direct measurement of aggregation, such as
turbidity assays, are difficult to quantify and are not suitable for high
throughput processing; (ii) the Aβ peptide is difficult to synthesize and would
be expensive to use in screens involving many thousands of candidate molecules;
and (iii) synthetic Aβ peptide forms aggregates or `micro-seeds¿ (which
initiate aggregation), and screens relying on synthetic Aβ peptide might not be
able to find molecules that prevent the initial ¿nucleation¿ steps of
aggregation.
To
circumvent these limitations, we have developed a novel screen. Our
screen
(1)
does not rely on turbidity measurements;
(2)
does not require the use of synthetic Aβ peptide;
(3)
uses a rapid and readily quantifiable assay (fluorescence or
absorbance);
(4)
uses Aβ
expressed de novo from a synthetic gene
in the presence of the candidate drugs (as compared to the previous attempts in
which pre-synthesized Aβ peptide ¿with its pre-aggregated
seeds ¿ is presented to the candidate
drugs);
(5)
can be performed in cell-free systems, and thus is not complicated by issues
pertaining to cell permeability.
The new method will
facilitate screening of many thousands of candidate molecules for those that are
capable of blocking Aβ aggregation and would
offer a novel way to screen for potential therapeutic candidates.
Princeton is currently seeking
industrial collaborators to commercialize this technology. Patent protection is
pending.
References
Wurth,C.,Guimard,N.,Hecht,M.,
(2002), Mutations that Reduce Aggregation of the Alzheimers Aβ
Amyloidogensis, J Mol. Biol., 319,1279-1290
Kim,W.,Hecht,M., (2005), Sequence
Determinants of Enhanced Amyloidogenicity of Alzheimer Aβ42 Peptide Relative to
Aβ40, J Biological Chemistry, Vol. 280, # 41, pgs 35069-35076.
For more information on Princeton
University Invention # 06-2246 please contact:
Laurie Tzodikov
Office of Technology Licensing and Intellectual Property
Princeton University
4 New South Building
Princeton, NJ 08544-0036
(609) 258-7256
(609) 258-1159 fax
tzodikov@princeton.edu