Anal. Chem. 2008, 80, 3502-3506
Assay To Screen for Molecules That Associate with Alzheimer’s Related β-Amyloid Fibrils Petra Inbar, Mahealani R. Bautista, Stacy A. Takayama, and Jerry Yang*
Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0358
Small molecules that bind to aggregated forms of Aβ peptides show promise as potential in vivo labeling agents for the diagnosis and monitoring of Alzheimer’s disease. A major challenge in developing potential imaging agents that target Aβ is to rapidly identify and evaluate the association of molecules with insoluble deposits of aggregated Aβ peptides. This paper describes a simple, parallel method to rapidly screen libraries of molecules for their ability to associate with fibrils formed from synthetic Aβ peptides by monitoring their ability to inhibit the interaction of a monoclonal anti-Aβ IgG with these fibrils. We demonstrate that this assay can detect the association of small molecules with Aβ fibrils at concentrations of small molecule in the nanomolar to millimolar range. By comparing results from the screening of a small set of 30 compounds, we illustrated that this assay can rapidly analyze the relative affinity of small molecules for Aβ fibrils and identified eight compounds that can bind to Aβ fibrils at 60 47 >56
IC50d (µM)
max inhe (µM)
23 135 no inhibition 1.2 4.6 4.0 5.0 144 61 8.2 no inhibition no inhibition 0.39 64 885 19000 290 46 40 5.8 72 1.4 >6800 no inhibition 30 no inhibition >174 no inhibition no inhibition no inhibition
310 4000 3.0 67 410 80 3200 410 47 4.6 490 3100 100000 5700 830 780 830 550 20 >13000 170 >23000
a Molecules exhibiting e20% inhibition were considered to have “no inhibition”. bStructures and inhibition curves of all molecules screened in this study can be found in Figure S-3 in the Supporting Information. c Maximal percent of the IgGs inhibited from binding to the Aβ fibrils in the presence of the Aβ-binding molecules. d Concentration of Aβbinding molecules required to inhibit 50% of the IgGs from binding to the Aβ fibrils. e Concentration of Aβ-binding molecules required for maximal inhibition of the IgGs from binding to the Aβ fibrils. f Denotes inhibitors dissolved in 5% DMSO in PBS buffer (see the Experimental Section for a discussion on the use of cosolvents).
a maximum inhibition of 66 ( 10% of the IgG-Aβ fibril interactions12). We demonstrate that molecules previously reported by spectroscopic methods to bind to Aβ fibrils (i.e., molecules 1,7 6,3 7,16 18,17 19,17 and 2219) are observed as “hits” in this assay. Molecules reported not to bind to Aβ fibrils (i.e., 3020) accordingly do not show up as “hits” in this assay (we have also previously reported that 1-naphthol-4-sulfonatesa molecule that does not bind to Aβ fibrilssdoes not show up as a “hit” using this assay12). Although it is possible that this assay may “miss” potential Aβbinding molecules if the binding sites for the small molecules on Aβ fibrils do not overlap with the binding sites for the IgG, the results from these studies indicate that this assay can identify a structurally diverse range of Aβ-binding molecules (including molecules with non overlapping binding sites along the fibril axis such as 1 versus 74,17). The results (Table 1) indicate that 22 of the 30 molecules tested appear to associate with Aβ fibrils. The concentrations of molecules required to inhibit the binding of the anti-Aβ IgG to the Aβ fibrils ranged from the high nanomolar (e.g., (19) Agdeppa, E. D.; Kepe, V.; Liu, J.; Small, G. W.; Huang, S.-C.; Petric, A.; Satyamurthy, N.; Barrio, J. R. Mol. Imaging Biol. 2003, 5, 404-417. (20) Klunk, W. E.; Debnath, Pettegrew, M. L., J. W. Neurobiol. Aging 1994, 15, 691-698.
Figure 2. Graphs comparing the inhibition of IgG-Aβ fibril interactions with 5 members of the tetracycline class of molecules: (a) oxytetracycline; (b) chlortetracycline; (c) tetracycline; (d) doxycycline; (e) rolitetracycline. These graphs highlight that subtle differences in the structure of these molecules can dramatically affect their capability to associate with Ab fibrils.
tannic acid, 13) to millimolar (e.g., nicotine, 16), with eight of these molecules binding in a concentration range of
