A new method uses the cell’s garbage disposal system to rapidly and reversibly knock down protein levels, potentially facilitating development of novel therapies, according to a study published in Nature Neuroscience (1).
Shelly (Xuelai) Fan of the University of British Columbia and her colleagues developed the method using a targeting peptide with three domains: a cell membrane–penetrating domain that allows the peptide to bypass the blood-brain barrier and plasma membranes, a protein-binding domain that specifically binds to the protein of interest, and a motif that targets the peptide-protein pair to the lysosome for degradation.
The researchers demonstrated that their system specifically degraded active forms of proteins found in neurons in a dose- and time-dependent manner, allowing for easy control over the degree and duration of protein knockdown. They performed proof-of-concept experiments knocking down postsynaptic density protein 95, which plays a role in synaptic plasticity; α-synuclein, which is implicated in neurodegenerative disorders such as Parkinson’s disease; and death associated protein kinase 1 (DAPK1), which is activated by stroke. But according to the authors, the system could potentially be used to degrade any cytosolic protein.
When administered through intravenous injection in a rat model of stroke, the peptide-based system lowered DAPK1 levels specifically in stroke-damaged brain areas and reduced tissue death caused by obstruction of the blood supply. “This is the first time scientists have managed to only knock down a protein in disease-affected areas,” Fan said. “The trick is that the peptide only binds to DAPK1 after it's activated.”
This activity-dependent specificity cannot be achieved with DNA or mRNA targeting, the authors explained. The targeting peptide–based system is also much faster, producing results within two hours rather than days or weeks; thus it is expected to be less susceptible to molecular and cellular compensation, compared to genetic or siRNA-mediated protein knockdown. Unlike most strategies that degrade proteins through the proteasome, the new approach does not require genetic manipulation. Moreover, most of the previously reported protein knockdown methods require viral delivery in vivo, limiting their therapeutic potential.
“The method is one of the first knockdown methods that drug companies may eventually adopt for clinical use,” said Fan. “In fact, our lab is currently developing peptides to knock down proteins involved in Huntington's [disease] and prostate cancer.”
But Fan and her collaborators face several challenges in refining the technique before this method moves into the clinic. “The largest limitation is that the peptide is usually not very stable once it gets into the body, so in order to sustain knockdown, one would have to apply it repeatedly,” Fan said. “Another problem is we can't say for sure that a peptide will only bind to its intended target. However, compared to small molecules, peptide-protein binding is usually a lot more specific, and so far we haven't noticed any off-target effects.”
Fan X, Jin WY, Lu J, Wang J, Wang YT. (2014). Rapid and reversible knockdown of endogenous proteins by peptide-directed lysosomal degradation. Nat Neurosci 17(3):471-80. doi: 10.1038/nn.3637.