Abstract
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.
Highlights
Membrane proteins mediate cell-cell communication events that provide important drug targets
Using a centrifugation assay coupled to dot immunoblots, we found that amyloid β oligomers (AβOs) binding is saturable with high affinity (Kd = 160±28 nM, Aβ monomer equivalent; Bmax = 580±30 pmoles Aβ mg−1 synaptosomes) (Fig 1A)
We have developed a nanobiotechnology that allows a new approach to High-throughput screening (HTS) for membrane protein targets
Summary
Membrane proteins mediate cell-cell communication events that provide important drug targets. An appealing approach to preserving membrane protein integrity in a soluble state is to incorporate the proteins into Nanodiscs. Nanodiscs are self-assembling nanoscale phospholipid bilayers stabilized by engineered membrane scaffold proteins (MSP) [6,7,8]. SMPL Nanodiscs can incorporate full membrane proteomes isolated directly from a biological tissue as a heterogeneous mixture of individualized proteins. Because of the affinity tags engineered on the MSP, it is possible to combine SMPL Nanodiscs with components of HTS assays designed for soluble proteins. This combination yields a platform for carrying out unbiased biochemical HTS assays of unknown targets derived from a specified membrane proteome
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