Discovery of Novel Anti-prion Compounds Using In Silico and In Vitro Approaches

Abstract

Prion diseases are associated with the conformational conversion of the physiological form of cellular prion protein (PrPC) to the pathogenic form, PrPSc. Compounds that inhibit this process by blocking conversion to the PrPSc could provide useful anti-prion therapies. However, no suitable drugs have been identified to date. To identify novel anti-prion compounds, we developed a combined structure- and ligand-based virtual screening system in silico. Virtual screening of a 700,000-compound database, followed by cluster analysis, identified 37 compounds with strong interactions with essential hotspot PrP residues identified in a previous study of PrPC interaction with a known anti-prion compound (GN8). These compounds were tested in vitro using a multimer detection system, cell-based assays, and surface plasmon resonance. Some compounds effectively reduced PrPSc levels and one of these compounds also showed a high binding affinity for PrPC. These results provide a promising starting point for the development of anti-prion compounds.

Anti-Prion Screening for Acridine, Dextran, and Tannic Acid using Real Time–Quaking Induced Conversion: A Comparison with PrPSc-Infected Cell Screening

“…Acridine was highly toxic at > 50 μM, tannic acid was non-toxic at < 20 μM, DSS and doxycycline permitted cell viability in excess of 90% at 100 μM. Quinacrine showed consistent toxicity (high toxicity at > 5 μM) as per a previous study [25]. Final concentrations between 1 and 20 μM were found to be optimal for treatment, and therefore, concentrations of 5 and 20 μM were selected for further study (Fig 1D).…”

“…This cell line was generously provided by Dr. Ryu at Hanyang University in Korea. Cell culture was performed as described previously [25]. Briefly, cells were seeded in a T25 cm 2 flask (TPP, Z707481) at 2 × 10 5 cells/flask and pre-incubated in Dulbecco’s Modified Eagle Medium (Gibco, 12430) containing 10% fetal bovine serum, 1% penicillin-streptomycin (Gibco, 15140), and 2 mM l -glutamine (Gibco, 25030) for 24 h under 5% CO 2 at 37°C.…”

“…A MTT-based cytotoxicity assay was performed as described previously [25]. Compounds applied at 5 different concentrations between 1 and 100 μM or DMSO vehicle were incubated with cells for 3 days.…”

“…The MDS assay is a dye-independent conversion assay that has been used previously to screen anti-prion compounds [25]. The MDS assay kit was supplied by People Bio Inc. and performed according to manufacturer specifications with minor modifications [26].…”

Prion disease: experimental models and reality

“…In silico computational modelling in combination with cell-based screening systems [71] can be the first-line experimental approach to identify potential anti-prion therapeutics. This is usually further validated in animal models, where prion infected mice are challenged with promising candidate drugs [63, 125].…”