An enzyme-linked immunosorbent assay to compare the affinity of chemical compounds for β-amyloid peptide as a monomer.
Aβ(1-42) is the proteolytic cleavage product of cleavage of the amyloid precursor protein by β- and γ-secretases. The aggregation of Aβ(1-42) plays a causative role in the development of Alzheimer's disease. To lock Aβ(1-42) in a homogenous state, we embedded the Aβ(1-42) sequence in an unstructured region of Bcl-x(L). Both the N-terminus and the C-terminus of Aβ(1-42) were constrained in the disordered region, whereas the conjunction did not introduce any folding to Aβ(1-42) but maintained the sequence as a monomer in solution. With Bcl-x(L)-Aβ(42), we developed an enzyme-linked immunosorbent assay to compare the affinity of compounds for monomeric Aβ(1-42). Bcl-x(L)-Aβ(42) was coated on a microplate and this was followed by incubation with different concentrations of compounds. Compounds binding to Leu17-Val24 of Aβ(1-42) inhibited the interaction between Bcl-x(L)-Aβ(42) and antibody 4G8. The method can not only reproduce the activities of the reported Aβ(1-42) inhibitors such as dopamine, tannin, and morin but can also differentiate decoy compounds that do not bind to Aβ(1-42). Remarkably, using this method, we discovered a new inhibitor that binds to monomeric Aβ(1-42) and inhibits Aβ(1-42) fibril formation. As the structure of Bcl-x(L)-Aβ(42) monomer is stable in solution, the assay could be adapted for high-throughput screening with a series of antibodies that bind the different epitopes of Aβ(1-42). In addition, the monomeric form of the Aβ(1-42) sequence in Bcl-x(L)-Aβ(42) would also facilitate the identification of Aβ(1-42) binding partners by coimmunoprecipitation, cocrystallization, surface plasmon resonance technology, or the assay as described here.
Department of Molecular Pharmacology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu-Chong-Zhi Road, Shanghai, 201203, China.
Chunyi
C
Jiang
Yu
Y
Feng
Xiaotong
X
Huang
Yechun
Y
Xu
Yaping
Y
Zhang
Naiming
N
Zhou
Xu
X
Shen
Kaixian
K
Chen
Hualiang
H
Jiang
Dongxiang
D
Liu
D016229:Amyloid beta-Peptides / Q000037:antagonists & inhibitors / Q000737:chemistry*; D001665:Binding Sites / Q000187:drug effects; D018360:Crystallography, X-Ray; D004298:Dopamine / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005419:Flavonoids / Q000494:pharmacology; D008958:Models, Molecular; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000737:chemistry*; D020156:Salicylic Acid / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013634:Tannins / Q000494:pharmacology; D051020:bcl-X Protein / Q000737:chemistry; D047309:Flavones
D016229:Amyloid beta-Peptides; D004298:Dopamine; D005419:Flavonoids; D010446:Peptide Fragments; D020156:Salicylic Acid; D013634:Tannins; D051020:bcl-X Protein; C075222:amyloid beta-protein (1-42); C008548:morin; D047309:Flavones