Nanoscale Synaptic Membrane Mimetic Allows Unbiased High Throughput Screen That Targets Binding Sites for Alzheimer's-Associated Aβ Oligomers.
Despite their value as sources of therapeutic drug targets, membrane proteomes are largely inaccessible to high-throughput screening (HTS) tools designed for soluble proteins. An important example comprises the membrane proteins that bind amyloid β oligomers (AβOs). AβOs are neurotoxic ligands thought to instigate the synapse damage that leads to Alzheimer's dementia. At present, the identities of initial AβO binding sites are highly uncertain, largely because of extensive protein-protein interactions that occur following attachment of AβOs to surface membranes. Here, we show that AβO binding sites can be obtained in a state suitable for unbiased HTS by encapsulating the solubilized synaptic membrane proteome into nanoscale lipid bilayers (Nanodiscs). This method gives a soluble membrane protein library (SMPL)--a collection of individualized synaptic proteins in a soluble state. Proteins within SMPL Nanodiscs showed enzymatic and ligand binding activity consistent with conformational integrity. AβOs were found to bind SMPL Nanodiscs with high affinity and specificity, with binding dependent on intact synaptic membrane proteins, and selective for the higher molecular weight oligomers known to accumulate at synapses. Combining SMPL Nanodiscs with a mix-incubate-read chemiluminescence assay provided a solution-based HTS platform to discover antagonists of AβO binding. Screening a library of 2700 drug-like compounds and natural products yielded one compound that potently reduced AβO binding to SMPL Nanodiscs, synaptosomes, and synapses in nerve cell cultures. Although not a therapeutic candidate, this small molecule inhibitor of synaptic AβO binding will provide a useful experimental antagonist for future mechanistic studies of AβOs in Alzheimer's model systems. Overall, results provide proof of concept for using SMPLs in high throughput screening for AβO binding antagonists, and illustrate in general how a SMPL Nanodisc system can facilitate drug discovery for membrane protein targets.
Department of Neurobiology, Northwestern University, Evanston, IL, United States of America.
Kyle C
KC
Wilcox
Department of Neurobiology, Northwestern University, Evanston, IL, United States of America.
Matthew R
MR
Marunde
Department of Comparative Biosciences, University of Illinois-Urbana Champaign, Urbana, IL, United States of America; Department of Biochemistry, University of Illinois-Urbana Champaign, Urbana, IL, United States of America.
Aditi
A
Das
Department of Neurobiology, Northwestern University, Evanston, IL, United States of America.
Pauline T
PT
Velasco
Department of Neurobiology, Northwestern University, Evanston, IL, United States of America.
Benjamin D
BD
Kuhns
Department of Chemistry, University of Illinois-Urbana Champaign, Urbana, IL, United States of America.
Michael T
MT
Marty
Department of Neurobiology, Northwestern University, Evanston, IL, United States of America.
Haoming
H
Jiang
High Throughput Analysis Laboratory and Department of Molecular Biosciences, Northwestern University, Evanston, IL, United States of America.
Chi-Hao
CH
Luan
Department of Biochemistry, University of Illinois-Urbana Champaign, Urbana, IL, United States of America; Department of Chemistry, University of Illinois-Urbana Champaign, Urbana, IL, United States of America.
Stephen G
SG
Sligar
Department of Neurobiology, Northwestern University, Evanston, IL, United States of America.
William L
WL
Klein
D000544:Alzheimer Disease / Q000378:metabolism*; D016229:Amyloid beta-Peptides / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D066298:In Vitro Techniques; D011485:Protein Binding; D051381:Rats; D013570:Synaptic Membranes / Q000378:metabolism*; D013574:Synaptosomes / Q000378:metabolism
D016229:Amyloid beta-Peptides