Photoactive chlorin e6 is a multifunctional modulator of amyloid-b aggregation and toxicity via specific interactions with its histidine residues† Guy Leshem,‡ a Michal Richman,‡ a Elvira Lisniansky, a Merav Antman-Passig, a Maram Habashi, a Astrid Gr ¨ aslund, b Sebastian K. T. S. W ¨ arml ¨ ander * b and Shai Rahimipour * a The self-assembly of Ab to b-sheet-rich neurotoxic oligomers is a main pathological event leading to Alzheimer's disease (AD). Selective targeting of Ab oligomers without affecting other functional proteins is therefore an attractive approach to prevent the disease and its progression. In this study, we report that photodynamic treatment of Ab in the presence of catalytic amounts of chlorin e6 can selectively damage Ab and inhibit its aggregation and toxicity. Chlorin e6 also reversed the amyloid aggregation process in the dark by binding its soluble and low molecular weight oligomers, as shown by thioflavin T (ThT) fluorescence and photoinduced cross-linking of unmodified protein (PICUP) methods. Using HSQC NMR spectroscopy, ThT assays, amino acid analysis, SDS/PAGE, and EPR spectroscopy, we show that catalytic amounts of photoexcited chlorin e6 selectively damage the Ab histidine residues H6, H13, and H14, and induce Ab cross-linking by generating singlet oxygen. In contrast, photoexcited chlorin e6 was unable to cross-link ubiquitin and a-synuclein, demonstrating its high selectivity for Ab. By binding to the Ab histidine residues, catalytic amounts of chlorin e6 can also inhibit the Cu 2+ -induced aggregation and toxicity in darkness, while at stoichiometric amounts it acts as a chelator to reduce the amount of free Cu 2+ . This study demonstrates the great potential of chlorin e6 as a multifunctional agent for treatment of AD, and shows that the three N-terminal Ab histidine residues are a suitable target for Ab-specific drugs. Introduction Misfolding of proteins and their subsequent aggregation to cross b-sheet conformation are the hallmark of more than thirty different devastating diseases, including many neurodegenerative and peripheral diseases, such as Alzheimer's, Parkinson's, and Huntington's diseases and type II diabetes. Among these diseases, Alzheimer's disease (AD) is the most prevalent neurodegenerative disease with approximately 35 million people diagnosed world- wide. The neuropathology of AD is characterized by two types of lesions – senile plaques and neurobrillary tangles (NFTs). The NFTs are composed of aberrantly phosphorylated tau, while the plaques consist mainly of aggregates of b-amyloid (Ab) – a 39–43- amino acid protein generated from enzymatic degradation of the amyloid precursor protein (APP). 1 The self-assembly and aggregation of Ab to form soluble oligomers and brils are strongly associated with the onset of the disease, where the soluble oligomers are believed to be the most toxic species to the neurons. 2 Although the exact mechanism by which Ab oligomers cause neurotoxicity is still under scientic debate, specic tar- geting of toxic Ab oligomers has shown promising therapeutic results. 3 During the past two decades, numerous approaches have been envisioned and adopted to reduce the amount of Ab in AD patients and to inhibit Ab aggregation and accumulation. 2b,4 These efforts include the development of b- and g-secretase inhibitors that target the production of Ab from APP, anti-Ab immunotherapy, chelation therapy, and b-sheet blockers that inhibit the oligomerization of soluble Ab. However, none of these approaches has been approved yet for clinical application, mainly due to nonspeciceffects of the agents on other important bio- logical targets. Since many misfolded proteins have important biological activities in their correctly folded conformations, selective tar- geting of the specic protein at its pathological site might reduce systemic toxicity and increase patient safety. Photody- namic therapy (PDT) has been used for decades in cancer therapy and different skin diseases to improve drug selectivity. 5 In PDT a combination of light, oxygen, and a photosensitizer is used to generate reactive oxygen species (ROS), mainly singlet a Department of Chemistry, Bar-Ilan University, Ramat-Gan 5290002, Israel. E-mail: rahimis@biu.ac.il b Department of Biochemistry and Biophysics, Arrhenius Laboratories, Stockholm University, S-106 91 Stockholm, Sweden. E-mail: sebastian.warmlander@dbb.su.se † Electronic supplementary information (ESI) available: General details on the materials and methods, and any associated references and supporting scheme, table and gures. See DOI: 10.1039/c8sc01992d ‡ Guy Leshem and Michal Richman contributed equally to this work. Cite this: DOI: 10.1039/c8sc01992d All publication charges for this article have been paid for by the Royal Society of Chemistry Received 3rd May 2018 Accepted 3rd October 2018 DOI: 10.1039/c8sc01992d rsc.li/chemical-science This journal is © The Royal Society of Chemistry 2018 Chem. Sci. Chemical Science EDGE ARTICLE Open Access Article. Published on 03 October 2018. Downloaded on 12/11/2018 12:19:51 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online View Journal