Cyclic peptides (CPs) and their derivatives are potent bioactive compounds, and represent an underexplored, natural-product-like chemical space1,2
. Phage and RNA display have made it possible to screen large libraries of CPs, cyclized via disulfide bonds or other side chain linkages, to identify high-affinity ligands for nearly any in vitro
. By contrast, to date there are few methods for directly screening large libraries of CPs inside eukaryotic cells. Such methods would provide several advantages over in vitro
techniques, ensuring that hits are nontoxic, can bind their target(s) in the appropriate cellular environment, are not rapidly degraded by cellular proteases, and possess at least enough selectivity to function in living cells. In addition, in vivo
methods would enable phenotypic selections of CPs, providing a less expensive alternative to traditional high-throughput screening with a greater chance of identifying effectors with non-traditional modes of action such as inhibition of protein-protein interactions.
Recent reports describe a promising method of generating libraries of head-to-tail CPs in vivo
using a single genetic construct named SICLOPPS (split-intein-mediated circular ligation of proteins and peptides, )5–7
. This construct uses a cleverly arranged split intein that splices out a linker region as a CP post-translationally; the linker can be as small as four amino acids or as large as a whole protein5,8
. SICLOPPS-based CP libraries represent a powerful opportunity for rapid forward and reverse chemical genetics using in vivo
. Previous work has interfaced expressed CP libraries with bacterial two-hybrid selections, an elegant strategy for reverse chemical genetics7,9
. Phenotypic screening of CP libraries was also performed in bacteria10,11
. Despite these successes, to date there has been only one reported attempt to adapt SICLOPPS libraries to a eukaryotic system. A retroviral CP library was applied to a selection for inhibitors of interleukin-4 signaling in human B cells, yielding roughly a dozen CP pentamers with varying activity but no sequence consensus12
. The general utility of this library was hampered by its low actual diversity (2.7 × 105
members), a lack of quantitative quality assessment, and the several weeks required just to perform an initial round of selection. Building upon these earlier studies, we sought to apply expressed CP libraries to eukaryotic cells and perform phenotypic selections in cellular models of human disease in a rapid, efficient and generally applicable manner.
A cyclic peptide library that expresses and processes in yeast
We and others have demonstrated that, because protein misfolding often affects highly conserved biological pathways, complex diseases such as Parkinson’s disease (PD) can be modeled in simple organisms such as yeast13–18
. The human protein α-synuclein (α-syn) has been linked to PD via genetic evidence and its prominence in the PD-associated intracellular aggregates known as Lewy bodies19–21
. α-syn is a small lipid-binding protein that is prone to misfolding and aggregation, and in the yeast Saccharomyces cerevisiae
expression of human α-syn over a threshold level leads to ER stress, disruption of ER-Golgi vesicle trafficking, accumulation of lipid droplets, mitochondrial dysfunction, and ultimately cell death16–18
. This cellular pathology mirrors many aspects of dysfunction seen in neurons and glia of individuals with PD and other synucleinopathies21
. Genetic screening using our yeast synucleinopathy model has yielded suppressors of α-syn toxicity that are also effective in neuronal models17,18,22
. Moreover, genetic analyses in these models have directly linked α-syn toxicity to the function of PARK9
/ATP13A2, a protein whose mutations lead to an early-onset form of PD but otherwise had no known connection to α-syn22,23
. Thus, cellular models have been critical to our understanding of α-syn and its role in the selective degeneration of dopaminergic (DA) neurons in PD. However, despite these and other intensive efforts, there remains a paucity of proven pharmacological targets for PD and other synucleinopathies.
Taking advantage of our established yeast synucleinopathy model, we constructed the first yeast-compatible CP library and used rapid phenotypic selections to isolate CPs that specifically reduce α-syn toxicity. Further, we identified the CP motif responsible for activity and demonstrated that the selected CPs significantly reduce DA neuron loss in a C. elegans PD model. These advances establish in vivo CP selections as an immediately useful tool for a broad range of biologists.