A High Throughput Screen for Molecules that Prevent the Alzheimer's Peptide Aß42 from Aggregating

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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.



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



Patent Information:
For Information, Contact:
Laurie Tzodikov
Licensing Associates
Princeton University
Michael Hecht
Woojin Kim
Christine Wurth
Young-Tae Chang