Introduction

Prion diseases are a group of lethal neurodegenerative diseases of humans and animals, including human Creutzfeldt-Jakob disease; bovine spongiform encephalopathy; scrapie in sheep, hamsters and mice; and chronic wasting diseases in deer1,2. There are three causes of prion disease: hereditary, sporadic and acquired by infection. All of these disease types are known to share the same pathogenic mechanism2,3.

The central event in prion disease pathogenesis is the conversion of the α-helix-rich cellular form of prion protein (PrPC) to a misfolded, β-sheet-rich, pathogenic and infectious conformational isoform (PrPSc), although the detailed structure of PrPSc is still not fully characterised1,4,5. This conversion initiates a chain replication reaction, where each newly converted PrPSc molecule interacts with more PrPC molecules, fueling the formation of additional PrPSc6,7. After this post-translational conversion, PrPSc aggregates and becomes the detergent-insoluble, partially protease-resistant protein fraction that serves as the marker for prion diseases8,9. Therefore, stabilization of the native PrPC conformation, without blocking the normal functions of PrPC, could reduce the rate of conversion to PrPSc or even prevent prion disease.

To date, screening has led to the identification of many anti-prion compounds10. Several large molecules (pentosanpolysulfate5, suramin11, amphotericin B12, congo red13 and dendritic polyamines14) and small molecules (bis-acridine15, polyphenol, phenothiazine, anti-histamine, statin and some anti-malarial agents including quinacrine16) have been reported to inhibit PrPSc formation or to reduce the level of PrPC. The tyrosine kinase inhibitor, STI571 (Gleevec), cured scrapie-infected cells in a concentration- and time-dependent manner with an IC50 below 1 μM, by inducing cellular clearance of PrPSc3. In addition, phenothiazine, statin and quinacrine provide attractive options because they have been approved by the United States Food and Drug Administration for use in other diseases7,9. However, these drugs were shown to be ineffective against prion disease in rodents10,17. The toxicity of anti-prion compounds and their inability to cross the blood-brain barrier has limited their successful application18.

In cell culture systems, anti-prion compounds are generally assessed by monitoring the levels of protease-resistant PrPSc using proteinase K (PK) digestion followed by western blotting. As this screening approach is fairly time-consuming and semi-quantitative, we employed a highly quantitative high-throughput misfolded protein detection assay (multimer detection system; MDS) to screen compounds for anti-prion efficacy. This competition assay uses a magnetic bead-conjugated capture antibody and a horseradish peroxidase (HRP)-conjugated detection antibody, with overlapping epitopes to achieve specific detection of multimers (such as PrPSc) and not monomers (such as PrPC). The T2 and 3E7 prion antibodies employed by the MDS recognize amino acids 147–152 and 140–160, respectively, of the PrP sequence19.

Although PrPC and PrPSc usually have identical primary amino acid sequences, it has been shown that the conversion from PrPC to PrPSc causes a substantial change in the secondary protein structure at various locations, including the factor X-binding site, the hotspot binding site and the unstructured N-terminal binding site20,21,22. Several computational and biophysical studies have targeted these significant regions and used well-known anti-prion compounds to demonstrate stabilization of the secondary structural changes23,24. Anti-prion compounds that have been identified by different research groups possess diverse scaffolds and similar inhibitory activities, highlighting the need for clarification of the structure-activity relationship (SAR).

The recent development of structure-based virtual screening supported by docking simulations has facilitated effective in silico screening of the interactions between chemical compounds and their target proteins, which can contribute to the identification of a desired activity from a large database of chemicals that are structurally different from known active compounds, reducing the time and cost of identifying chemical hits25,26. Using the structure of PrPC-GN8 (a known anti-prion compound), a 3D pharmacophore model was generated and compounds were docked into the prion hotspot to determine their potential binding mode, which enabled the selection of a small number of molecules for in vitro testing. In total, 37 compounds were assessed by MDS assay, in scrapie-infected mouse neuroblastoma N2a (ScN2a), in PrPC-overexpressing N2a (L2-2B1) cells and by surface plasmon resonance (SPR) direct-binding experiments.

Results

Virtual Screening

The overall discovery steps employed in the present study are shown in Fig. 1a. Ten pharmacophore models were generated using the receptor-ligand interaction protocols in Discovery Studio (DS) and the best was chosen using the Genetic Function Approximation (GFA) model (Fig. 1b). To generate the receptor-ligand interaction-based pharmacophore model, a well-defined anti-prion compound (GN8) was docked into the PrPC hotspot (1AG2), as shown in Supplementary Figure 1a27,28. Although several crystal and nuclear magnetic resonance (NMR) structures are available for PrPC, it should be noted that only the NMR of PrPC (1AG2) with GN8 structural details has been thoroughly characterised23. Because the pharmacophore model was based on the previously reported NMR structure of PrPC-GN8, we expected it to include two important hydrogen bonds from Glu196 and Asn159. As anticipated, the pharmacophore model included five features: the two key hydrogen bonds, two hydrophobic features (H), two hydrogen bond acceptors (HBAs) and one hydrogen bond donor (HBD) (Fig. 1b). To validate this pharmacophore model, we also mapped a previously reported anti-prion compound, GJP49.

Figure 1
figure 1

The compound discovery process.

(a) Work flow for the in silico screen and in vitro assays. (b) The proposed pharmacophore model, showing prion protein in the normal conformation (PrPC; pink ribbon representation) mapped with the active anti-prion agent, GN8 (yellow stick representation). The model consists of five features: two hydrophobic elements (cyan), two hydrogen bond acceptors (green) and a hydrogen bond donor (pink).

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Virtual screening of our in-house chemical database with the selected pharmacophore model yielded 1110 compounds. Cluster analysis was performed to filter these compounds and 682 diverse compounds were selected based on their structural similarities and differences. The compounds were further subjected to visual inspection, resulting in the selection of 37 drug-like compounds for further evaluation.

PrPC Compound Docking

The highest binding energy of the 37 hit compounds was measured using the AutoDock score. All compounds were docked into the PrPC hotspot in the same way, with a similar binding mode. Fifty docked conformations were obtained per compound and their best binding energy (kcal/mol) is listed in Table 1. To illustrate the interaction, the binding modes of the PrPC-compound interactions are shown in Fig. 2, which illustrates a compound anchored at the PrPC hotspot (Fig. 2a) and interacting with specific amino acids via multiple hydrogen bonds and hydrophobic interactions (Fig. 2b).

Table 1 Binding Parameters.
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Figure 2
figure 2

The binding mode and interaction between a compound and prion protein in the normal conformation (PrPC).

(a) The predicted mode of a compound on the surface of PrPC, colored to indicate hydrophobicity (blue for the most hydrophilic, to white, to orange-red for the most hydrophobic). (b) Close-up view of the interaction between the important PrPC residues (pink sticks) and the compound (cyan stick). Hydrogen bonds are shown as green dashed lines and hydrophobic contacts as pink dashed lines.

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MDS Assay of PrPSc Inhibition

Our primary in vitro screening test employed the MDS enzyme-linked immunosorbent assay (ELISA) to quantify PrPSc formation. Recombinant PrP was exposed to each of the 37 hit compounds or 1 μM of quinacrine as a positive control. As shown in Fig. 3, quinacrine significantly reduced the formation of PrPSc. The effects of the hit compounds in this assay were not always concentration-dependent. Some compounds actually increased PrPSc formation at a concentration of 20 μM (BMD42-01, 12, 19, 20 and 30). Twelve compounds (BMD42-03, 06, 07, 08, 10, 16, 23, 26, 28, 29, 31 and 35) exhibited >50% inhibition of PrPSc formation at both concentrations tested (5 μM and 20 μM). Based on this primary screening, we selected 7 compounds with statistically significant effects (BMD42-06, 23, 26, 29, 31, 33 and 35) for secondary cell culture screening. One of the selected compounds (BMD42-31) showed outstanding inhibitory effects in the MDS assay and even out-performed the positive control, quinacrine.

Figure 3
figure 3

Multimer detection assay.

(a) The principle of the multimer detection system (monomer: PrPC, multimer: PrPSc). (b) Luminescence (as a marker of scrapie prion protein [PrPSc] formation) is shown for each of the indicated treatments and concentrations. Values represent the mean of triplicate determinations. Error bars show the standard deviation. The effective reduction of PrPSc level is indicated as an asterisk. Student’s t-test was used and differences were considered statistically significant at a P value of < 0.05.

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Cytotoxicity Assay

We used a commercially available cytotoxicity assay to identify suitable treatment concentrations for each compound. Seven compounds were tested at six different concentrations between 0.5 μM and 1 mM (Fig. 4) in ScN2a and L2-2B1 cells. Most of the compounds caused severe cytotoxicity at concentrations above 200 μM. BMD42-06 showed the lowest cytotoxicity of the 7 compounds tested. Concentrations between 0.5 and 30 μM were considered optimal and concentrations of 5 and 20 μM were thus selected for further study. There were no observable differences in cell viability between the ScN2a and L2-2B1 cell lines. We observed an obvious cytotoxic effect of quinacrine at above 2 μM in both cell lines.

Figure 4
figure 4

Effect of the compounds on cell viability.

Cell viability was determined in the indicated cell lines (L2-2B1 and ScN2a) in the presence of the compounds indicated. The dotted line indicates 50% cytotoxicity. Values represent the mean of three independent experiments and the standard deviation is shown as the error bars.

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Compound Effects on PrPC and PrPSc Propagation in Cultured Cells

ScN2a cells are infected with the Rocky Mountain Laboratory scrapie prion strain and persistently produce PrPSc. This cell model is widely used to screen anti-prion candidate compounds. We first used a transfected cell line over-expressing PrPC to examine anti-prion effects. This cell line facilitated the observation of changes in the amount of PrPC, relative to a normal neuronal cell. Figure 5 shows representative immunoblots for PrPSc and PrPC or total PrP obtained from ScN2a and L2-2B1 cells exposed to different concentrations of the 7 test compounds, quinacrine (1 μM), or dimethyl sulfoxide (DMSO). Compounds with anti-prion activity would reduce the protein levels. The 7 compounds selected by MDS primary screening revealed a variety of inhibitory effects on PrPSc levels in ScN2a cells (Fig. 5a). The amount of PrPSc was concentration-independently reduced by BMD42-06 and 35. The extent of this reduction equaled the effect of quinacrine (Fig. 5a lane 1). BMD42-23, 29 and 33 also reduced PrPSc levels, whereas BMD42-26 and 31 only produced minor inhibitory effects. However, the reduced levels were not detected in L2-2B1 cells during this study, irrespective of whether the cells were exposed to hit compounds or controls (Fig. 5b). In ScN2a cells, total PrP levels were reduced in the presence of BMD42-35 but were unaffected by quinacrine, DMSO, or the other 6 hit compounds (Fig. 5c,g). In L2-2B1 cells, PrPC was inhibited by 6 compounds, but not by BMD42-29 (Fig 5d,h). Thus, had only ScN2a cells been used, inhibitory effects against PrPC may not have been found for other compounds. Among them, BMD42-35 showed the strongest inhibitory effects in the cell-based assay. Quinacrine completely inhibited the formation of PrPSc, but not PrPC, at 1 μM.

Figure 5
figure 5

Western immunoblots of proteinase K-digested (a, b) and undigested (c, d) cell lysates. ScN2a (a,c) and L2-2B1 (b,d) cells were treated with the indicated compounds for 6 days. Lane 1, lysates from cells treated with 1 μM quinacrine; lane 2, 0.1% DMSO; subsequent lanes, 20 or 5 μM of the indicated compounds. β-actin immunoblots are shown in ScN2a (e) and L2-2B1 (f) as protein loading controls. Relative units of densitometry for (b,d) indicate the volume intensity/mm2, relative to the β-actin signal in (g,h) respectively. Each value represents the mean ± standard deviation; P < 0.05. Three independent experiments were performed in duplicate and representative immunoblots are shown. Molecular mass markers are indicated on the right of the immunoblots.

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SPR Measurement of Compound-PrPC Binding Affinities

The 37 hit compounds identified by VS were tested by SPR to quantify their direct PrPC binding ability. Multiple strategies were explored for the immobilization of PrPC to the surface of a high-density sensor chip. Direct measurements of quinacrine binding were repeatedly performed to confirm the stability and function of the coated chips. SPR revealed that the majority of the compounds interacted directly with PrPC. The apparent affinities were determined for 23 compounds (Table 1, dissociation constant rate; KD). The remaining 14 compounds were tested, but did not bind. These compounds showed consistent results in the MDS assay. Sensorgram curves revealed that similar to previously reported GJP derivatives, our compounds showed single and specific binding to PrPC. In particular, BMD42-29 showed rapid association and slow dissociation rates (Fig. 6). This compound showed at least 2-fold tighter binding to PrPC than the other hit compounds. Notably, these findings correlated with those of the computational docking study, where BMD42-29 had the highest AutoDock binding energy (−7.87 kcal/mol) of the hit compounds tested (Table 1). BMD42-35 was found to be effective in the cell-based assay, but did not show binding in the SPR assay.

Figure 6
figure 6

Kinetic analysis of a compound-prion protein (PrPC) interaction using surface plasmon resonance.

(a) The steady state (equilibrium) response units (RU) after fitting are plotted against the concentration of BMD42-29. (b) Sensogram curve depicting the interaction between the indicated concentrations of BMD42-29 and sensorchip-immobilized PrPC. The equilibrium dissociation rate constant (KD) was determined to be 103 μM for this compound.

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Binding Mode and Selectivity

The predicted binding modes for the 37 hit compounds were analyzed to investigate the structural basis of their selectivity. We grouped the compounds by their inhibition efficacy to examine the SAR. Although BMD42-35 was one of the most active compounds in the cell-based assay, it showed no binding in the SPR assay. Several of the other hit compounds showed independent anti-prion activity in cell-based and SPR assays (BMD42-2, 23, 29 and 33). Comparison of the scaffolds of these 4 compounds revealed diverse structural features. Notably, these most active compounds included three sulfonamide compounds (BMD42-23, 29, 33), two thiazole compounds (BMD42-02 and 23) and one benoxazole compound (BMD42-29). The 2-amino thiazole scaffold present in BMD42-23 had previously been widely studied in the context of anti-prion drug discovery and had even been used in a clinical trial for prion disease29.

We next investigated the binding mode of each compound with PrPC, bearing in mind that two strong hydrogen bonds from Asn159 and Glu196 play important roles in inhibitory activity (Fig. 2). In addition to the high affinity compounds, 10 more compounds (BMD42-31, 26, 16, 03, 04, 10, 22, 07, 34 and 25) were scrutinized. Their chemical structures included amide (BMD42-04 and 22), sulfonamide (BMD42-07 and 34) and pyrazole (BMD42-26) scaffolds. Notably, BMD42-16 and 31 possessed both amide and sulfonamide scaffolds and showed partial efficacy in SPR and MDS assays. BMD42-03, 10 and 25 possessed diverse scaffolds and were less active in all screening tests. In addition, their binding modes predicted a single hydrogen bond and hydrophobic contacts with the hotspot residues of Glu196/Asn159/Lys194/Glu160/Leu130, which may contribute to stabilization of the PrPC structure. In addition, we grouped 7 moderately active compounds (BMD42-17, 30, 19, 24, 36, 14 and 12) and elucidated their binding with hotspot residues. The chemical structures of these compounds confirmed that the presence of sulfonamide and amide scaffolds (BMD42-24 and 14) produced potent anti-prion effects.

To confirm the above findings, we investigated two less active compounds, BMD42-01 and 05 (Supplementary Fig. 2). These compounds had no hydrogen bonding or hydrophobic interactions with key hotspot residues, as their structures differed from those of the most active compounds. In contrast, one of the most active compounds (BMD42-29) showed strong hydrogen bonding at the helix regions of Asn159 and Glu196 and hydrophobic interactions with Leu130 and Arg156. These findings emphasized the importance of these hydrogen bonds and hydrophobic interactions for efficient anti-prion effects. Compounds lacking interaction with Asn159 and Glu196 were unable to inhibit PrPSc formation (Supplementary Fig. 2).

Discussion

Research aiming to develop treatments for prion diseases has led to the identification of a range of compounds such as quinacrine and doxycycline that can selectively block conversion of PrPC to PrPSc30. However, these compounds have not provided useful treatments for patients. To discover and validate novel non-toxic and effective anti-prion drugs, we studied a structurally diverse series of compounds with anti-prion activity using an in silico approach. Use of the structures of PrPC-hit compound complexes enabled analysis of compound localization at the hotspot sites and interactions with key residues in the previously determined NMR PrPC-GN8 complex.

The three most effective strategies for discovering anti-prion compounds include screening for inhibitory effects on PrPSc accumulation, inactivation of endogenous PrPC (as the substrate for prion conversion) and the enhancement of PrPSc degradation31. Inhibition of in vitro PrPSc accumulation represents a primary target for prion disease therapy16,32.

Expression of PrPc in host neurons is required for PrPSc replication and disease progress. As such, persistently infected cell lines acting as a host for PrPSc are frequently used to study prion-related cellular processes and also to screen for effective anti-prion compounds33. ScN2a cells have been used extensively as a relevant model for the study of prion diseases34. In the present study, considerable reductions in PrPC levels were only observed in L2-2B1 cells, possibly because the cells had higher initial levels of PrPC. The L2-2B1 cell line was therefore very useful for analyzing this aspect of the compounds’ activities. Quinacrine was cytotoxic in both cell lines at concentrations above 2 μM. This result was consistent with a previous study in which cytotoxicity was tested in the presence of 0.02–200 μM quinacrine and the optimal concentration found to inhibit PrPSc formation was below 4 μM7.

We performed a highly quantitative and precise protein misfolding detection assay for the primary screening of anti-prion compound efficacy. The results of this MDS assay facilitated the selection of 7 potent hit compounds (BMD42-06, 23, 26, 29, 31, 33 and 35). Although BMD42-31 was more effective than quinacrine in the MDS assay, it was excluded during our secondary cell-based screening as it produced only a minor reduction in PrPSc level, although the PrPC level was reduced at 20 μM. Chemical modification of this compound may increase its efficacy.

The MDS assay results appeared to indicate that BMD42-06, 23, 26, 29, 31, 33 and 35 conformationally stabilized PrPC by reducing its aggregation. BMD42-06, 23, 26, 31, 33 and 35 also decreased the level of PrPC. The mechanisms underlying this effect may involve PrPC consumption as a substrate for aggregation; a reduction in the PrP mRNA level; or protein degradation caused by compound-PrPC binding. The results indicated that in ScN2a cells, BMD42-06, 23, 29, 33 and 35 reduced the amount of PrPSc, either by interfering with the conformational interaction between PrPC and PrPSc, or by interacting directly with PrPSc.

Recently, Kamatari and co-workers classified anti-prion compounds according to four potential molecular mechanisms of action: (i) specific conformational stabilization of PrPC; (ii) nonspecific stabilization, including interference with the conformational interaction between PrPC and PrPSc, in addition to hotspot binding; (iii) promotion of PrPC aggregation and precipitation, thus reducing the amount available for conversion to PrPSc; or (iv) interactions with molecules other than PrPC, such as PrPSc or membrane proteins35. The compounds identified in the present study showed multiple mechanisms of action, based on their effects on PrPC aggregation, reduction and PrPSc propagation, as well as their binding affinities. Placing our results in the context of Kamatari’s classification, BMD42-29 may act by specific stabilization of PrPC, as evidenced by its strong binding affinity. BMD42-06, 23, 29, 33 and 35 may act by nonspecific blockade of PrPC conversion to PrPSc, or by interacting with PrPSc, as evidenced by their weak binding affinities and the observed reduction in the level of PrPSc or PrPC aggregate. BMD42-06, 23, 26, 31, 33 and 35 may act by reducing the amount of PrPC by reducing PrP expression, or stimulating PrP degradation. Further mechanistic studies are required to investigate exactly how these compounds alter PrPC levels.

Our results suggest that BMD42-29 is an optimal compound, exhibiting PrPSc inhibition and a stronger binding affinity than other anti-prion compounds reported to date. However, it did not produce a marked reduction in PrPC levels, possibly indicating that it stabilized PrPC and inhibited its pathological conformation change to PrPSc. BMD42-35 produced the highest decrease in PrPSc and PrPC levels, although it showed a lower binding affinity, which suggests that BMD42-35 acts by either stabilizing or eliminating PrPC and may control PrPC non-specifically. This contradictory result may relate to problems with SPR assays reported by earlier studies, including the unlimited increase in ProteOnGLH sensor responses, low solubility of compounds and slow binding kinetics36.

It has been proposed that the hydrogen bonds from stand S1 and helix B may prevent the conversion of PrPC to PrPSc21. Collectively, our compounds showed strong hydrogen bonds at Asn159 (stand S1) and Glu196 (helix B) within PrPC. The most active compounds occupied the hydrophobic area highlighted in the pharmacophore model generated from the PrPC-GN8NMR structure. BMD42-29, which was active in both cell-based and SPR-based assays and BMD42-35, which was most active in the cell-based assay, both interacted with conserved PrPC hotspot residues, indicating the importance of the two hydrogen bonds and the hydrophobic environment; this was predicted by the pharmacophore model.

Further elucidation of the mechanisms of action of BMD42-29 and BMD42-35 will provide unique tools to study the mechanism of prion replication. The approach used in the present study may provide in vitro screening data that are more highly predictive of in vivo activity, contributing to the rational design of novel and effective anti-prion treatments.

Methods

Pharmacophore Development

The protein data bank NMR structure of PrPC (1AG2) was used as the template for pharmacophore generation37. DS software was used to map the active site of PrPC and critical residues were identified based on the PrPC-GN8 NMR data10. Subsequently, the receptor-ligand pharmacophore generation protocols were used to generate a quantitative model of PrPC. The protocols resulted in 10 3D pharmacophore hypotheses and the highest ranked of these was selected.

Virtual Screening

The 3D pharmacophore hypothesis was used to extract chemical compounds from our in-house database of 700,000 compounds. The screening processes were performed using the best flexible conformation search method in DS, with standard settings.

Molecular Docking

Molecular docking calculations were performed using the AutoDock tool. To validate the AutoDock, we docked the known anti-prion compounds, GN8 and GJP49, into the PrPC hotspot (1AG2). A grid of 63, 57 and 53 points in the x, y and z directions was constructed on the center of the PrPC hotspot mass. A default setting grid spacing of 0.375 Å and a distance-dependent function of the dielectric constant were used for the energetic map calculations. The docked compounds were subjected to 50 runs of the AutoDock Lamarckian genetic algorithm, with 500,000 steps of energy evaluation and default values for the other parameters. Cluster analysis was performed on the results using 1.0 Å. The docking pose analysis was conducted for the first pose of the most populated cluster in the AutoDock output.

Compounds and Cell lines

BMD42-01–06, 21–24, 27–30, 32–34, 36 and 37 compounds were purchased from Enamine. BMD42-07 and 08 were purchased from ASINEX. BMD42-09–11 were purchased from Life Chemicals. BMD42-12–15 were purchased from Chembridge. BMD42-16 and 26 were purchased from ChemDiv. BMD42-17 was purchased from Vista M labs. BMD42-18–19 and 35 were purchased from Princeton Biomolecular Research. BMD42-20 was purchased from Synthon-lab. BMD42-25 was purchased from Sigma Aldrich. BMD42-31 was purchased from Uorsy. The compounds were dissolved in DMSO, diluted to produce a 50 mM stock solution and stored at –20°C. ScN2a cells were derived from N2a cells obtained from the ATCC and were generously provided by Dr. Ryu, Hanyang University, Korea. The L2-2B1 subclone cell line overexpressing PrPC derived from N2a was established by Dr. Kim, Korean National Institute of Health.

MDS Assay

The MDS assay kit was supplied by People Bio Inc. and performed according to the instructions, with minor revision19. Briefly, each compound was pre-treated with the reaction buffer containing 50 ng of recombinant PrP, 1% Triton X-100, 10% Blockace and Tris-buffered saline containing 0.1% (vol/vol) Tween 20 (TBST) in 2 mL screw cap tubes. DMSO (0.1% vol/vol) was used as a negative control and quinacrine (1 μM) (Sigma Aldrich, Q3251) was the positive control. The mixture was incubated with continuous shaking for 3 h at 37 °C. The 3E7 PrP antibody (2 μg), conjugated to magnetic beads and the HRP-conjugated PrP T2 antibody (8 μg) were added to the pre-incubated mixture. After incubation for 1 h under the same conditions, the beads were separated and washed three times with TBST using a magnetic particle concentrator (Invitrogen, 120.20D). The assay signal was developed by adding Supersignal ELISA pico chemiluminescence substrate (Pierce, 37070) and quantified using a VICTOR3 microplate reader (Perkin Elmer, 1420-032).

Cell Culture and Treatments

ScN2a and L2-2B1 cells were seeded in six-well plates (2 × 105 cells/well) and incubated in Opti-MEM (Gibco, 31985) containing 10% fetal bovine serum, 1% penicillin-streptomycin (Gibco, 15140) and 2 mM l-glutamine (Gibco, 25030) with the indicated concentrations of the compounds in 5% CO2 at 37 °C. The stock compound solutions (50 mM) were diluted in Opti-MEM. Control cell cultures were treated with DMSO only (0.1% v/v). Each compound was tested in duplicate in three independent experiments.

Lysis and PK Digestion

Cells were rinsed once in phosphate-buffered saline (PBS) and then incubated with 0.1% (vol/vol) trypsin-EDTA (Gibco, 25300) for 1 min at room temperature. The detached cells were centrifuged at 4000 × g for 10 min at 4 °C and rinsed once with PBS. Cells were lysed with ice-cold lysis buffer (10 mM EDTA, 10 mM Tris, pH 8.0, 100 mM NaCl, 0.5% [wt/vol] Nonidet P-40 and 0.5% [wt/vol] sodium deoxycholate). Two freeze-thaw cycles in liquid nitrogen were performed, followed by sonication at an amplitude of 30%. The total protein concentration was adjusted to 1 mg/mL.

For PrPSc detection, cell lysates were digested with PK (20 μg/mL) (Merck, 70663) for 60 min at 37 °C. The reaction was stopped with 2 mM Pefabloc (Roche, 11429876001) and the lysates were centrifuged for 90 min at 20,000 × g at 4 °C. Pellets were resuspended in sodium dodecyl sulfate (SDS) sample buffer (125 mM Tris-HCl, pH 6.8, 5% [vol/vol] glycerol, 6 mM EDTA, 5% [wt/vol] SDS, 0.04% [vol/vol] bromophenol blue and 12.5% [vol/vol] β-mercaptoethanol). For PrPC detection, cell lysates were treated with 2 mM Pefabloc only to achieve the same conditions and then resuspended in SDS sample buffer.

Western Immunoblotting

Protein samples were separated by SDS-polyacrylaminde gel electrophoresis for 2 h at 150 V at 4 °C, using 12% gels. Proteins were then transferred to a polyvinylidenefluoride membrane using the i-Blot system (Invitrogen) for 7 min. The membrane was blocked for 1 h at room temperature in 5% (wt/vol) skim milk in PBS containing 0.1% (vol/vol) Tween 20 (PBST) and then incubated overnight at 4 °C with the PrP antibody, SAF-32 (1:200) (Cayman Chemicals, 189720) diluted in 10% (vol/vol) blocking buffer. After washing with PBST, the membrane was incubated for 1 h at room temperature with HRP-conjugated anti-mouse IgG (1:2,000) (DAKO, P0260). The signals were visualized using ECL (Elpis Biotech, EBP-1073) and the protein bands were scanned by Image Scanner III (GE). The relative band densities are shown as the volume intensity/mm2, relative to the β-actin band density. Blots used for PrPC detection were stripped and then re-probed using a β-actin antibody (1:5000) (Cell Signaling, 4970).

Cytotoxicity Assay

Cytotoxicity was evaluated using a commercial kit (CellTiter 96 Non-Radioactive Cell Proliferation Assay, Promega, G4001), following the manufacturer’s instructions. ScN2a and L2-2B1 cell suspensions (5,000 cells) were plated into each well of a 96-well plate. After adding the compounds at six different concentrations between 0.5 μM and 1 mM or the control, the cells were incubated for 6 days. Dye solution was then added to each well and incubated for up to 4 h. Solubilization solution/stop mix was then added and cell viability was measured using an ELISA plate reader (Bio-Rad) at a wavelength of 570 nm. Cytotoxicity was expressed as a percentage of the signal observed in untreated control cells.

SPR Analysis

SPR was conducted using the ProteOn XPR36 protein interaction array system (Bio-Rad Laboratories). PrP was amine-coupled on the ProteOn GLH sensor chip. ProteOn running buffer (PBS, pH 7.4, with 0.05% Tween 20) was used at a flow rate of 100 μL/min. The test compounds (200 μL) were injected at six different concentrations (0 μM, 12.5 μM, 25 μM, 50 μM, 100 μM and 200 μM). Data were analyzed by ProteOn Manager Software 2.0 using the standard Langmuir models for fitting kinetic data. A high-affinity interaction was characterized by a low KD, rapid recognition and binding (rapid ‘on rate’ or high Ka) and stable complex formation (slow ‘off rate’ or low Kd), in accordance with the equation, KD = Kd/Ka.

Statistical analysis

Each experiment was repeated a minimum of three times. The one-way analysis of variance with the Tukey-Kramer procedure was used. Differences were considered as significant at P < 0.05.

Additional Information

How to cite this article: Hyeon, J. W. et al. Discovery of Novel Anti-prion Compounds Using In Silico and In Vitro Approaches. Sci. Rep. 5, 14944; doi: 10.1038/srep14944 (2015).