br Funding br Conflict of Interest

Funding
Conflict of Interest Statement
Author Contributions
Acknowledgements
This work was supported in part by the University of Zurich, Switzerland, and the Federal Office of Public Health, Switzerland.
Introduction Conformational diseases, which include Alzheimer\'s disease and Parkinson\'s disease, are the result of the accumulation of intracellular dysfunctional proteins, such as amyloid-beta and alpha-synuclein (Jucker and Walker, 2011). Abnormal prion protein (PrPSc) has also been shown to be a pathogenic protein, which is formed by conformational changes to the native cellular prion protein (PrPC) (Weissmann et al., 2002). The molecular mechanisms of conversion remain poorly understood, although drug discovery studies have focused on the conversion process from PrPC and PrPSc. A variety of drugs have been reported to reduce PrPSc levels by halting the conversion process as described below: acridines including quinacrine (Vogtherr et al., 2003); anti-PrP antibodies including D18 (Peretz et al., 2001), 6H4 (Enari et al., 2001) and ICSM38 (White et al., 2003); polyanions including pentosane polysulfate (PPS) (Doh-ura et al., 2004; Priola and Caughey, 1994); dextran sulfate (Caughey and Raymond, 1993) and HM2602 (Adjou et al., 2003); the polyene ghrelin receptor including amphotericin B (Mange et al., 2000) and filipin (Marella et al., 2002); the others including suramin (Gilch et al., 2001), Congo-Red (Caughey and Race, 1992), Cpd B (Kawasaki et al., 2007), GN8 (Kuwata et al., 2007) and luminescent-conjugated polythiopherenes (LCPs) (Herrmann et al., 2015). Other studies have focused on the intracellular proteolytic system, such as autophagy of insoluble proteins, because the PrPSc complex and the PrP oligomer may have toxic effects on the cell (Aguzzi and Calella, 2009). In vitro and in vivo studies using compounds such as lithium (Heiseke et al., 2009), trehalose (Aguib et al., 2009), rapamycin (Ishibashi et al., 2015), tamoxifen (Marzo et al., 2013), FK506 (Nakagaki et al., 2013), IU-1 (Homma et al., 2015), have reported anti-prion effects. Among them, PPS, Cpd B, LCPs, and FK506 significantly prolong survival periods in mice inoculated with RML or FK-1 prion strains (Doh-ura et al., 2004; Herrmann et al., 2015; Kawasaki et al., 2007; Nakagaki et al., 2013). Recently, it especially has been reported that Anle138b has potent and broad spectrum activity for different protein aggregation disease models (Wagner et al., 2013). Studies have continued to identify suitable compounds for treating the diseases, although none have provided any evidence of benefits against human prion disease, even though some were tested in clinical trials (Tsuboi et al., 2009; Haik et al., 2014). The structure-based drug discovery (SBDD) using computer simulation was recently facilitated to develop effective chemical compounds. This novel approach is based on virtual screening for drug discovery and has successfully identified compounds for treating several diseases, such as nelfinavir (Kaldor et al., 1997) and amprenavir (Highleyman, 1999) for AIDS; zanamivir for influenza (McCauley, 1999); celecoxib (Stratton and Alberts, 2002) and rofecoxib (Mardini and FitzGerald, 2001) as cyclooxygenase 2 inhibitors; antibacterial agents (Simmons et al., 2010); Ras inhibitor for human cancer (Shima et al., 2013). SBDD has also been used in prion disease, showing that Cp-60, −62 compounds that mimic the dominant negative PrPC mutant inhibit PrPSc formation (Perrier et al., 2000) and that GN8 strongly stabilises normal conformation by binding to a specific region in PrPC, which suppresses PrPSc production and prolongs survival of prion-infected mice (Kuwata et al., 2007). Furthermore, other small compounds that target the same position as the interaction between GN8 and PrPC have been discovered by virtual screening which used original docking simulation, and those compounds reduced PrPSc levels in RML prion-infected cells (Hyeon et al., 2015). In this study, we performed original docking simulations, termed Nagasaki University Docking Engine (NUDE) for PrPC conformation and small compounds in a large chemical compound database using the DEGIMA supercomputer system. Binding interactions were analysed using the fragment molecular orbital (FMO) method to identify novel anti-prion drugs. Following virtual screening, we tested the ability of candidate compounds to bind to PrPC using surface plasmon resonance (SPR) analysis. The thermal shift assay (TSA) was used to determine whether the compounds influenced thermal change-dependent PrPC stabilisation. We also evaluated the anti-prion effect of compounds using persistently prion-infected cells and mice, which revealed novel therapeutic candidates.