& Therapeutic Peptides Self-Assembled Cyclic d,l-a-Peptides as Generic Conformational Inhibitors of the a-Synuclein Aggregation and Toxicity: In Vitro and Mechanistic Studies Marina Chemerovski-Glikman, [a] Eva Rozentur-Shkop, [a] Michal Richman, [a] Asaf Grupi, [b] Asaf Getler, [b] Haim Y. Cohen, [b] Hadassa Shaked, [a] Cecilia Wallin, [c] Sebastian K. T. S. Wärmländer, [c] Elisha Haas, [b] Astrid Gräslund, [c] Jordan H. Chill, [a] and Shai Rahimipour* [a] Abstract: Many peptides and proteins with large sequences and structural differences self-assemble into disease-causing amyloids that share very similar biochemical and biophysical characteristics, which may contribute to their cross-interac- tion. Here, we demonstrate how the self-assembled, cyclic d,l-a-peptide CP-2, which has similar structural and func- tional properties to those of amyloids, acts as a generic in- hibitor of the Parkinson’s disease associated a-synuclein (a- syn) aggregation to toxic oligomers by an „off-pathway“ mechanism. We show that CP-2 interacts with the N-termi- nal and the non-amyloid-b component region of a-syn, which are responsible for a-syn’s membrane intercalation and self-assembly, thus changing the overall conformation of a-syn. CP-2 also remodels a-syn fibrils to nontoxic amor- phous species and permeates cells through endosomes/lyso- somes to reduce the accumulation and toxicity of intracellu- lar a-syn in neuronal cells overexpressing a-syn. Our studies suggest that targeting the common structural conformation of amyloids may be a promising approach for developing new therapeutics for amyloidogenic diseases. Introduction Protein misfolding and aggregation into soluble aggregates, amyloid fibrils, and plaques are the characteristic features of many degenerative illnesses, including Alzheimer’s, Parkinson’s, and Huntington’s disease, and type II diabetes, which affect either the central nervous system or a variety of peripheral tis- sues. [1] Unfortunately, despite large efforts invested over the last two decades into developing agents that target the etiolo- gy of these diseases, most therapeutic treatments remain symptomatic. It is well established that misfolded proteins do not fully retain their native biological activity and that they are under a dynamic equilibrium between their monomeric and oligo- meric forms. Soluble aggregates of different amyloidogenic proteins, which are rich in b-sheet conformations, are suspect- ed to be the most toxic species. Indeed, very recent clinical studies have shown that targeting the toxic oligomers of Alz- heimer’s disease (AD)-associated Ab (amyloid-b) by an anti- body dramatically reduced plaques and slowed down the cog- nitive and functional declines in patients. [2] However, the spe- cific nature and structure of the aggregates, and the exact mechanism(s) that lead to cellular toxicity are still not well known. [1, 3] Interestingly, despite large differences in sequence, length, and structures, proteins and peptides may aggregate to form fibrils that share common conformational and biochemical properties. [4] Indeed, many proteins aggregate to form cross-b- sheet-rich structures, and are recognized by the conformation- al antibody A11 that selectively binds different oligomeric structures, [5] which may imply that the aggregation process is an intrinsic property of any polypeptide. [6] The structural simi- larity between different amyloids is also most likely responsible for their cross-interaction and cross-seeding in vitro and in vivo. [7] For example, it has been shown that Ab occurs together with the Parkinson’s disease (PD)-associated a-synuclein pro- tein (a-syn) in dementia with Lewy bodies (DLB) in vivo and that their individual amyloid seeds enhance the aggregation of the other protein in vitro. [8] Many studies also suggest that amyloids are not only struc- turally similar, but also share common functional activities. Compelling evidence shows that aggregates of many proteins interact similarly with a variety of membranes and that they [a] Dr. M. Chemerovski-Glikman, E. Rozentur-Shkop, Dr. M. Richman, Dr. H. Shaked, Prof. J. H. Chill, Prof. S. Rahimipour Department of Chemistry Bar-Ilan University, Ramat-Gan 5290002 (Israel) E-mail : rahimis@biu.ac.il [b] Dr. A. Grupi, A. Getler, Prof. H. Y. Cohen, Prof. E. Haas Department of Life Sciences Bar-Ilan University, Ramat-Gan 5290002 (Israel) [c] C. Wallin, Dr. S. K. T. S. Wärmländer, Prof. A. Gräslund Department of Biochemistry and Biophysics, Arrhenius Laboratories Stockholm University, 10691 Stockholm (Sweden) Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/chem.201601830. Chem. Eur. J. 2016, 22, 14236 – 14246  2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 14236 Full Paper DOI: 10.1002/chem.201601830