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• W2138880839 abstract "Prion diseases are fatal neurodegenerative diseases caused by the accumulation of the misfolded isoform (PrPSc) of the prion protein (PrPC). Cell-based screens have identified several compounds that induce a reduction in PrPSc levels in infected cultured cells. However, the molecular targets of most antiprion compounds remain unknown. We undertook a large-scale, unbiased, cell-based screen for antiprion compounds and then investigated whether a representative subset of the active molecules had measurable affinity for PrP, increased the susceptibility of PrPSc to proteolysis, or altered the cellular localization or expression level of PrPC. None of the antiprion compounds showed in vitro affinity for PrP or had the ability to disaggregate PrPSc in infected brain homogenates. These observations suggest that most antiprion compounds identified in cell-based screens deploy their activity via non-PrP targets in the cell. Our findings indicate that in comparison to PrP conformers themselves, proteins that play auxiliary roles in prion propagation may be more effective targets for future drug discovery efforts. Prion diseases are fatal neurodegenerative diseases caused by the accumulation of the misfolded isoform (PrPSc) of the prion protein (PrPC). Cell-based screens have identified several compounds that induce a reduction in PrPSc levels in infected cultured cells. However, the molecular targets of most antiprion compounds remain unknown. We undertook a large-scale, unbiased, cell-based screen for antiprion compounds and then investigated whether a representative subset of the active molecules had measurable affinity for PrP, increased the susceptibility of PrPSc to proteolysis, or altered the cellular localization or expression level of PrPC. None of the antiprion compounds showed in vitro affinity for PrP or had the ability to disaggregate PrPSc in infected brain homogenates. These observations suggest that most antiprion compounds identified in cell-based screens deploy their activity via non-PrP targets in the cell. Our findings indicate that in comparison to PrP conformers themselves, proteins that play auxiliary roles in prion propagation may be more effective targets for future drug discovery efforts. Misfolding and aggregation of endogenously expressed proteins cause several neurodegenerative disorders (1Dobson C.M. Trends Biochem. Sci. 1999; 24: 329-332Abstract Full Text Full Text PDF PubMed Scopus (1698) Google Scholar, 2Rochet J.C. Lansbury Jr., P.T. Curr. Opin. Struct. Biol. 2000; 10: 60-68Crossref PubMed Scopus (996) Google Scholar, 3Prusiner S.B. N. Engl. J. Med. 2001; 344: 1516-1526Crossref PubMed Scopus (677) Google Scholar). Prion diseases belong to this class of proteinopathies and result from the misfolding of the α-helix-rich, cellular prion protein (PrPC) into a β-sheet-rich, disease-causing, infectious isoform termed PrPSc (3Prusiner S.B. N. Engl. J. Med. 2001; 344: 1516-1526Crossref PubMed Scopus (677) Google Scholar, 4Pan Y.T. Hori H. Saul R. Sanford B.A. Molyneux R.J. Elbein A.D. Biochemistry. 1983; 22: 3975-3984Crossref PubMed Scopus (212) Google Scholar, 5Basler K. Oesch B. Scott M. Westaway D. Wälchli M. Groth D.F. McKinley M.P. Prusiner S.B. Weissmann C. Cell. 1986; 46: 417-428Abstract Full Text PDF PubMed Scopus (643) Google Scholar, 6Prusiner S.B. Annu. Rev. Med. 1987; 38: 381-398Crossref PubMed Scopus (43) Google Scholar). Unlike other neurodegenerative disorders, prion diseases are readily transmissible to laboratory animals and cultured cells. The availability of laboratory models harboring the infectious aggregate has enabled the development of an empirical drug discovery strategy against prion diseases. Typically, prion-infected, neuronally derived cell lines that accumulate and stably propagate PrPSc (7Race R.E. Fadness L.H. Chesebro B. J. Gen. Virol. 1987; 68: 1391-1399Crossref PubMed Scopus (161) Google Scholar, 8Butler D.A. Scott M.R. Bockman J.M. Borchelt D.R. Taraboulos A. Hsiao K.K. Kingsbury D.T. Prusiner S.B. J. Virol. 1988; 62: 1558-1564Crossref PubMed Google Scholar) are used as a primary screen for the identification of compounds that reduce prion levels in culture. Subsequently, the in vivo efficacy of putative antiprion compounds is assessed by analyzing their ability to prolong disease incubation periods in prion-infected rodents. Using this approach, numerous antiprion compounds have been identified, including pentosan polysulfate, dextran sulfate, HPA-23, Congo red, suramin, dendritic polyamines, 2-aminothiazoles, and quinacrine (9Trevitt C.R. Collinge J. Brain. 2006; 129: 2241-2265Crossref PubMed Scopus (230) Google Scholar). However, none of these compounds have been shown to be effective against a variety of prion strains in animal models when administered at a late, post-symptomatic stage, and none have been shown to have significant disease-modifying properties in human clinical studies. Although measuring PrPSc levels in infected cultured cells can be used to assess antiprion activity, this method does not elucidate the molecular targets of active compounds. As a result, the mechanisms of action of most antiprion compounds remain unknown. In principle, a compound can reduce the prion load in a cell by interacting with a number of molecular targets. The most direct mechanism is through direct binding to PrPC and stabilization of its native conformation (10Nicoll A.J. Trevitt C.R. Tattum M.H. Risse E. Quarterman E. Ibarra A.A. Wright C. Jackson G.S. Sessions R.B. Farrow M. Waltho J.P. Clarke A.R. Collinge J. Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 17610-17615Crossref PubMed Scopus (62) Google Scholar, 11Tatzelt J. Prusiner S.B. Welch W.J. EMBO J. 1996; 15: 6363-6373Crossref PubMed Scopus (269) Google Scholar). Alternatively, a drug may directly interact with PrPSc, leading to its disaggregation (12Supattapone S. Nguyen H.O. Cohen F.E. Prusiner S.B. Scott M.R. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 14529-14534Crossref PubMed Scopus (251) Google Scholar), or may target auxiliary factors or proteins that play a role in PrPC expression, localization, or conversion to PrPSc (13Perrier V. Wallace A.C. Kaneko K. Safar J. Prusiner S.B. Cohen F.E. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 6073-6078Crossref PubMed Scopus (184) Google Scholar). To investigate whether antiprion compounds identified in prion-infected neuronal cell lines have a tendency to interact with PrPC, PrPSc, or other targets, we screened a library of 2,160 known drugs and natural products and identified 206 compounds that cleared PrPSc in neuroblastoma (N2a) cell lines at a concentration of less than 1 μm. Of these initial hits, we validated the activity of 16 compounds and assessed their ability to bind to recombinant PrP, directly disaggregate PrPSc, reduce the expression level of PrPC, and alter the localization of PrPC. Taken together, the results suggest that the antiprion activity of these compounds is mainly mediated by non-PrP targets. The chemical library of 2,160 compounds screened in both cell-based and direct-binding assays was obtained from the MicroSource Discovery System (MSDI, Gaylordsville, CT), and includes known drugs, bioactives, and natural products. Compounds were solubilized at 10 mm in dimethyl sulfoxide (DMSO) 2The abbreviations used are: DMSOdimethyl sulfoxideITCisothermal titration calorimetryDSFdifferential scanning fluorimetryTMAOtrimethylamine N-oxideGdnHClguanidine hydrochloride. and stored in a 96-well format by the Small Molecule Discovery Center at the University of California San Francisco. dimethyl sulfoxide isothermal titration calorimetry differential scanning fluorimetry trimethylamine N-oxide guanidine hydrochloride. A mouse neuroblastoma (N2a) cell line was infected with the Rocky Mountain Laboratory (RML) strain of scrapie prions to produce ScN2a cells (14Bosque P.J. Prusiner S.B. J. Virol. 2000; 74: 4377-4386Crossref PubMed Scopus (185) Google Scholar). Screening the chemical library for antiprion activity was performed in a high-throughput ELISA. Briefly, 4 × 104 ScN2a cells were treated with the compound of interest for 5 days at 1 μm final concentration. Untreated ScN2a cells were used as negative controls; ScN2a cells treated with quinacrine (1 μm) were used as positive controls (15Korth C. May B.C. Cohen F.E. Prusiner S.B. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 9836-9841Crossref PubMed Scopus (601) Google Scholar, 16May B.C. Fafarman A.T. Hong S.B. Rogers M. Deady L.W. Prusiner S.B. Cohen F.E. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 3416-3421Crossref PubMed Scopus (142) Google Scholar). A toxicity screen was conducted in parallel at the same compound concentration and time of exposure in a 96-well format using an acetomethoxy derivative of calcein (calcein-AM) assay. Untreated ScN2a cells were used as negative controls. Both of these methods have been described previously (17Ghaemmaghami S. Phuan P.W. Perkins B. Ullman J. May B.C. Cohen F.E. Prusiner S.B. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 17971-17976Crossref PubMed Scopus (75) Google Scholar, 18Ghaemmaghami S. May B.C. Renslo A.R. Prusiner S.B. J. Virol. 2010; 84: 3408-3412Crossref PubMed Scopus (118) Google Scholar). ScN2a cells (5 × 105) were propagated in a 10-cm plate and treated for 5 days with the compound of interest at 50, 20, 10, or 1 μm, depending on cellular toxicity. Negative controls were performed by treating cells with DMSO alone. As a positive control, cells were treated with 1 μm quinacrine. Cells were lysed with lysis buffer (0.5% Nonidet P-40, 0.5% deoxycholate, 10 mm Tris-HCl, pH 8, 100 mm NaCl) and protein concentration was normalized to 1 mg/ml using the BCA assay. Samples were incubated with 20 μg/ml of proteinase K for 1 h at 37 °C. Digestions were stopped with 2 mm phenylmethylsulfonyl fluoride (PMSF), and samples were centrifuged at 100,000 × g for 1 h at 4 °C. Supernatants were discarded, and pellets were resuspended in reducing SDS sample buffer for SDS-PAGE. Western blotting was performed according to standard procedures. PrP was detected by using D13 antibody Fab fragment conjugated (19Williamson R.A. Peretz D. Pinilla C. Ball H. Bastidas R.B. Rozenshteyn R. Houghten R.A. Prusiner S.B. Burton D.R. J. Virol. 1998; 72: 9413-9418Crossref PubMed Google Scholar) with horseradish peroxidase (HRP) (Rockland Immunochemicals Inc.). Truncated recombinant mouse (Mo) PrP(89–230) and full-length recombinant MoPrP(23–230) were overexpressed and purified as previously described (20Mehlhorn I. Groth D. Stöckel J. Moffat B. Reilly D. Yansura D. Willett W.S. Baldwin M. Fletterick R. Cohen F.E. Vandlen R. Henner D. Prusiner S.B. Biochemistry. 1996; 35: 5528-5537Crossref PubMed Scopus (194) Google Scholar, 21Legname G. Baskakov I.V. Nguyen H.O. Riesner D. Cohen F.E. DeArmond S.J. Prusiner S.B. Science. 2004; 305: 673-676Crossref PubMed Scopus (907) Google Scholar). Lyophilized pellets were dissolved in a denaturation buffer (6.4 m GdnHCl, room temperature, 30 min) at 1 or 5 mg/ml for truncated or full-length proteins, respectively. The protein was then diluted 4-fold with cold refolding buffer (25 mm Tris-HCl, pH 8.0, 5 mm EDTA) and dialyzed 2 times against the screening buffer (20 mm sodium acetate, pH 5.1, 150 mm NaCl) at 4 °C. ITC measurements were performed at 37 °C using the ITC200 Microcalorimeter (GE Healthcare). To counteract the backlash effect observed during the first injection (22Mizoue L.S. Tellinghuisen J. Anal. Biochem. 2004; 326: 125-127Crossref PubMed Scopus (69) Google Scholar), ITC titrations were carried out with one 0.2-μl injection of ligand (compound), followed by 10 consecutive injections of 3.8 μl of the compound with injection durations of 4- and 150-s intervals between injections. The amount of energy released was measured following each injection. Prior to each measurement, DMSO was added to the protein sample to reach a final concentration of 1%. The sample chamber was filled with 15 μm recombinant MoPrP(89–230), which had been dialyzed previously in the screening buffer (see protein preparation). Solution stocks of compounds were prepared at 20 mm in 100% DMSO. Solutions of titrants were freshly prepared by diluting them with the dialyzed buffer to reach a 200 μm final concentration (1% final DMSO). Titrant solutions were centrifuged for 5 min at 14,000 × g, and supernatants were loaded into the syringe. The final molar ratio of ligand:protein exceeded 2.5. Isotherm data were analyzed with Origin7 software (MicroCal) supplied by the manufacturer. CD spectra of both truncated and full-length recombinant MoPrP were recorded at 10 μm, with a 0.1-cm cuvette using a Jasco J-715 spectrometer with a Jasco PTC-348WI Peltier-effect temperature control device, where the temperature reported reflects that of the heating block. Scans were acquired at 50 nm/min, with a bandwidth of 2 nm and data spacing of 0.1 nm. Thermal unfolding curves were measured at 10 μm recombinant MoPrP(89–230) and MoPrP(23–230). All compound solutions were made fresh from 100 mm DMSO stock solutions. Prior to each run, samples were equilibrated with the compound of interest for 15 min at 20 °C, then a temperature ramp rate of 2 °C/min was applied from 20 to 90 °C. To attenuate DMSO noise background, a wavelength of 230 nm was used to monitor protein unfolding. The melting point was determined from the thermal unfolding curve fit by the two-state folding EXAM algorithm (23Morton A. Baase W.A. Matthews B.W. Biochemistry. 1995; 34: 8564-8575Crossref PubMed Scopus (159) Google Scholar, 24Beadle B.M. Shoichet B.K. J. Mol. Biol. 2002; 321: 285-296Crossref PubMed Scopus (187) Google Scholar). A Δu Cp value of 4.76 kJ mol−1 K−1 was set in the fitting of the van't Hoff equation to determine the melting temperature (Tm) and the ΔH of the transition. A significant thermal upshift was defined as ΔTm exceeding the standard deviation of the technique (σ = 0.4 °C) by a factor of three. The standard deviation was calculated based on three Tm measurements of native PrP. Protein stability was assessed in a 96-well format using an MxPro3005P qRT-PCR Detection System (Stratagene, Agilent Technologies). Sypro-Orange dye (Invitrogen) was used to monitor the fluorescence by applying ROX filter for the fluorescence emission (610 nm) and FAM filter for the fluorescence excitation (492 nm). Optimal conditions to perform the assay were determined by varying protein concentrations, Sypro-Orange dilutions, buffers, and thermal ramp parameters. To experimentally screen compounds, recombinant MoPrP(89–230) and Sypro-Orange dye were plated manually. Stock solutions of 10 mm compounds (100% DMSO) were freshly diluted in the screening buffer (1:10 ratio), then added to the protein by using a 96/384 pipettors robot (Apricot Designs) to achieve a final compound concentration of 1 mm (1% final DMSO). DSF spectra of 10 μm recombinant MoPrP(89–230) with a 1:2000 Sypro-Orange dilution in the screening buffer were recorded. Samples (150 μl final) were heated at 2 °C/min, from 40 to 90 °C, and the fluorescence values were recorded after every 1 °C increase. Approximations of the melting temperature (Tm) were assessed by using the maximum value of the first derivative generated by the qPCR software (MxPro QPCR software, Stratagene, Agilent Technologies). To evaluate changes in Tm, for each run, first derivatives of the melting curves were generated from the MxPro software, exported as a text file and imported in Mathematica software. The first derivative of the curves was fitted with a polynomial function. Tm was approximated by determining the maximum value of the fitted first derivative plot. To analyze the effects of the compounds on PrPC expression, uninfected N2a cells were incubated with the selected molecules for 5 days. Negative control cells were incubated with DMSO (0.5% final concentration). After treatment and cell lysis, normalized crude extracts were analyzed by Western immunoblotting using conjugated D13-HRP antibody to detect PrP. PrPC expression levels were normalized with respect to actin controls (Fig. 6). For dose-response analysis of amcinonide, N2a cells were treated for 3 days and quantified by Western immunoblotting as described above. Western blots were quantified using ImageJ software. PrPSc and PrPC levels in compound-treated cells were normalized against the levels in untreated control cells. Brain homogenates (10% w/v) were prepared from terminal CD-1 mice infected with RML prions, then diluted 10-fold using an acidic sodium acetate buffer supplemented by detergents (5 mm sodium acetate, pH 3.5, 1% Nonidet P-40). Ninety μl of 1% brain homogenates (0.6 mg/ml) were incubated with 10 μl of test compounds or polyamidoamine (PAMAM) generation 4.0 and 4.5 to reach final concentrations of 100 μm or 100 μg/ml, respectively. Samples were incubated for 2 h with constant shaking at 37 °C. After neutralization with 100 μl of a freshly prepared buffer containing 0.2 m HEPES, pH 7.5, 0.3 m NaCl, and 4% Sarkosyl, samples were subjected to PK digestion (20 μl/ml, 1 h of incubation at 37 °C). Proteolytic digestions were stopped by the addition of PMSF (2 mm final concentration), and samples were analyzed by Western immunoblotting using conjugated D13-HRP antibody to detect PrP. For lipid raft isolation, N2a cells were treated with the compounds for 3 days in 6-well plates. Plates were placed on ice, cells were rinsed twice with chilled PBS and incubated for 20 min with ice-cold Triton X-100 lysis buffer made with Mes-buffered saline (25 mm Mes, 150 mm NaCl, pH 6.5) containing 1% (v/v) Triton X-100. The lysates were then homogenized by passing through a Luer 22-gauge needle and centrifuged at 500 × g for 5 min at 4 °C to pellet cell debris. Cold supernatants were harvested, normalized to 0.5 mg/ml, and mixed with an equal volume of 80% (v/v) sucrose to obtain a 40% (v/v) sucrose solution. A 1-ml aliquot of the sample was then transferred to the bottom of a SW-60 centrifuge tube. Two ml of 30% sucrose was then added to the top, followed by the addition of 1 ml of 5% sucrose to create a discontinuous sucrose gradient. Tubes were centrifuged at 4 °C for 18 h at 140,000 × g in a SW-60 rotor (Beckman Instruments). Fractions (1–8; 500 μl) were collected from the top and analyzed by Western immunoblotting using D13-HRP antibody (see the immunoblotting section). Flotillin-1 (Sigma) was probed by immunoblot and used as a marker for detergent-resistant membranes. N2a cells (2.5 × 105) were plated on a coverslip (Fisher Scientific, Circles No. 1.5) placed in a 24-well plate format, and treated for 3 days with the selected compounds. Cells were washed with warm (37 °C) PBS and fixed with 4% paraformaldehyde solution for 20 min at room temperature. Cells were rinsed 3 times for 5 min with room temperature PBS, permeabilized with 0.3% Triton X-100 in PBS for 5 min, and then rinsed 3 times with PBS buffer. Cells were blocked with 10% normal goat serum in PBS for 30 min at room temperature, then incubated with primary D18 antibody (5 μg/ml) in 10% normal goat serum overnight at 4 °C. Cells were washed successively 3 times with PBS for 10 min, and incubated in the dark at room temperature for 2 h with a FITC-conjugated goat anti-human IgG (H&L) polyclonal antibody (5 μg/ml diluted in 10% normal goat serum, Jackson ImmunoResearch). Samples were rinsed 3 times with PBS for 10 min in the dark. Coverslips were rinsed briefly with water, then mounted on Superfrost Plus microscope slides (Electron Microscopy Sciences, Hatfield, PA) with Vectashield with the counterstain DAPI (Vector Laboratories), and sealed with Cytoseal. Slides were analyzed at the QB3-UCSF Nikon Imaging Center on a Nikon Eclipse Ti-E Motorized Inverted Microscope. Images represent individual Z-slices taken from the middle of the cell. Uninfected N2a cells were incubated with amcinonide at 50 μm or 0.5% DMSO (untreated control cells) for 6, 24, 48, or 72 h. Total RNA was isolated with TRIzol reagent. cDNA was synthesized from 1 g of total RNA using the SuperScript II First Strand Synthesis System for RT-PCR at 42 °C for 60 min (Invitrogen), and then diluted 10-fold in water. Two microliters were used in duplicate for quantitative PCR amplification of Prnp, and actin as an internal control, using the MxPro3005P qRT-PCR apparatus (Stratagene, Agilent Technologies). The following program was used: denaturation step at 95 °C for 10 min, 40 cycles of PCR (denaturation at 95 °C for 10 s, annealing at 55 °C for 8 s, elongation at 72 °C for 15 s). Primers were as follows: Actin, forward, gatcattgctcctcctgagc-5′; reverse, ctcatcgtactcctgcttgc-3′; Prnp: forward, cgagaccgatgtgaagatga-5′; reverse, atcccacgatcaggaagatg-3′. The curves of amplification were read with MxPro 3005P software using the comparative cycle threshold method. Relative quantifications of the target mRNAs were calculated after normalization of cycle thresholds with respect to actin levels. Values are expressed as fold-change compared with untreated cells (0.5% DMSO). N2a cells were preincubated with amcinonide at 20 μm or 0.5% DMSO (untreated control cells) for 3 days. Subsequently, 30 μg/ml of cycloheximide (Sigma) was added to the culture to inhibit protein synthesis, and cells were incubated at 37 °C for various durations. Cells were then lysed and residual PrPC levels were evaluated by Western immunoblotting. We screened a chemical library of 2,160 compounds containing known drugs and natural products (Microsource) for antiprion activity in ScN2a cells. The cell-based assay was carried out in a multiwell format and PrPSc levels were measured using an ELISA-based assay (18Ghaemmaghami S. May B.C. Renslo A.R. Prusiner S.B. J. Virol. 2010; 84: 3408-3412Crossref PubMed Scopus (118) Google Scholar). Cells treated with DMSO (carrier) and quinacrine (a known antiprion drug (15Korth C. May B.C. Cohen F.E. Prusiner S.B. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 9836-9841Crossref PubMed Scopus (601) Google Scholar)) were used as negative and positive controls, respectively. These measurements were used as reference to calculate the normalized percentage change in PrPSc levels following treatment with experimental compounds. ScN2a cells were treated with compounds for 5 days at a concentration of 1 μm. In parallel, we analyzed the cytotoxicity of all compounds at this concentration by employing the calcein AM assay for membrane integrity (25Lichtenfels R. Biddison W.E. Schulz H. Vogt A.B. Martin R. J. Immunol. Methods. 1994; 172: 227-239Crossref PubMed Scopus (229) Google Scholar). The level of cytotoxicity was normalized with respect to untreated cells. Based on the distribution of activity and cytotoxicity measurements (Fig. 1, A and B), we defined a “hit” as a compound that reduced PrPSc load by 40% with less than 20% cytotoxicity as measured by the calcein AM assay (Fig. 1C). Using these criteria, we identified 206 nontoxic compounds with antiprion activity. A previous screen of the MicroSource compound library also identified active compounds (26Kocisko D.A. Baron G.S. Rubenstein R. Chen J. Kuizon S. Caughey B. J. Virol. 2003; 77: 10288-10294Crossref PubMed Scopus (207) Google Scholar), most of which were included in our hit set as well (Fig. 1C, red dots). We randomly selected 40 hits for secondary validation. Initially, compounds were added to ScN2a cells at a concentration of 50 μm. For compounds that proved toxic at this concentration, the experiments were repeated at 20, 10, and 1 μm to assess the antiprion activity of the compound at the highest possible nontoxic concentration. Changes in PK-resistant PrPSc levels were analyzed by Western blots. We were able to confirm the antiprion activity of 16 of 40 compounds (Fig. 1D). Some of these 16 compounds belonged to chemical classes previously known to have antiprion properties (statins, flavones, resveratrol, chalcone, quercetin, phenothiazine, and corticosteroid (26Kocisko D.A. Baron G.S. Rubenstein R. Chen J. Kuizon S. Caughey B. J. Virol. 2003; 77: 10288-10294Crossref PubMed Scopus (207) Google Scholar, 27Kocisko D.A. Engel A.L. Harbuck K. Arnold K.M. Olsen E.A. Raymond L.D. Vilette D. Caughey B. Neurosci. Lett. 2005; 388: 106-111Crossref PubMed Scopus (44) Google Scholar)). Several were, as far as we are aware, novel: these include dehydrovariabilin, 3-deoxy-3β-hydroangolensic acid methyl ester, and glycosides (Table 1). Experiments conducted on prion-infected N2a cells in the presence of the 16 compounds revealed a proportional decrease of all three glycoforms (unglycosylated, monoglycosylated, and diglycosylated) of digested PrPSc (Fig. 1E), suggesting that the compounds neither alter PrP glycosylation nor depend on PrP glycosylation for their antiprion activity.TABLE 1Validated antiprion compoundsFor each compound, the reduction in PrPC and PrPSc levels in culture, interaction with recPrP detected by ITC, induced ΔTm in recPrP measured by CD, induced disaggregation of PrPSc in vitro, and the ability to disrupt lipid rafts are reported. Open table in a new tab For each compound, the reduction in PrPC and PrPSc levels in culture, interaction with recPrP detected by ITC, induced ΔTm in recPrP measured by CD, induced disaggregation of PrPSc in vitro, and the ability to disrupt lipid rafts are reported. Next, we assessed the ability of these 16 antiprion compounds to interact directly with recombinant MoPrP(89–230) containing the structured domain of PrP found in the proteinase-resistant core of PrPSc (28Peretz D. Williamson R.A. Matsunaga Y. Serban H. Pinilla C. Bastidas R.B. Rozenshteyn R. James T.L. Houghten R.A. Cohen F.E. Prusiner S.B. Burton D.R. J. Mol. Biol. 1997; 273: 614-622Crossref PubMed Scopus (319) Google Scholar). ITC was used to measure direct binding between the compounds and recombinant PrP. We first sought to establish the ITC protocol with a control compound that is known to interact with PrP. We used suramin as a positive control as it has been shown to interact nonspecifically with PrP, inducing its aggregation (29Gilch S. Winklhofer K.F. Groschup M.H. Nunziante M. Lucassen R. Spielhaupter C. Muranyi W. Riesner D. Tatzelt J. Schätzl H.M. EMBO J. 2001; 20: 3957-3966Crossref PubMed Scopus (156) Google Scholar, 30Nunziante M. Kehler C. Maas E. Kassack M.U. Groschup M. Schätzl H.M. J. Cell Sci. 2005; 118: 4959-4973Crossref PubMed Scopus (36) Google Scholar). We used ITC to analyze the interaction between suramin and PrP at concentrations of 200 and 15 μm, respectively. In the presence of suramin, we observed a substantial release of energy for the first injections (approximately −6.0 Kcal/mol of injectant), demonstrating an interaction between the partners (Fig. 2A). Using the same experimental parameters, we performed ITC with the 16 confirmed antiprion compounds to detect any significant release of energy upon injection. No substantial release of energy was detected for any of the 16 compounds (Fig. 2B), suggesting that none interacted with recombinant MoPrP(89–230). We next sought to measure the binding of compounds to PrP by a thermal-denaturation upshift assay. Ligand binding and protein folding are thermodynamically linked, and the binding of a ligand stabilizes proteins against denaturation (31Matulis D. Kranz J.K. Salemme F.R. Todd M.J. Biochemistry. 2005; 44: 5258-5266Crossref PubMed Scopus (390) Google Scholar). We therefore used thermal denaturation to quantify potential binding of antiprion compounds by detecting induced upshifts in the melting temperature of PrP. We initially assessed the reversibility of the thermal unfolding of recombinant truncated MoPrP(89–230). Circular dichroism (CD) spectra of truncated PrP were measured successively at 20 and 90 °C (supplemental Fig. S1A). As expected, CD spectra indicated a loss of secondary structure at 90 °C. On subsequent cooling, MoPrP(89–230) regained its α-helical signal, indicating that the thermal unfolding of MoPrP(89–230) is reversible. We then determined the melting temperature (Tm) of truncated recombinant PrP by monitoring the change in α-helical content of the protein as a function of temperature. The resulting melting curve was well fit by a two-state unfolding model. The Tm for MoPrP(89–230) was 65.3 ± 0.4 °C and the van't Hoff enthalpy of denaturation was 270.4 ± 15.7 kJ/mol. We investigated whether changes in protein stability could be robustly detected by this approach. The melting point of PrP was measured in the presence of stabilizing and destabilizing agents. In the presence of 1 m trimethylamine N-oxide (TMAO), a chemical chaperone (11Tatzelt J. Prusiner S.B. Welch W.J. EMBO J. 1996; 15: 6363-6373Crossref PubMed Scopus (269) Google Scholar), protein stability increased substantially (Tm = 70.5 ± 0.2 °C; ΔTm = +5.2 °C). Conversely, addition of 1 m of the denaturant GdnHCl decreased the melting temperature (Tm = 62.3 ± 0.4 °C; ΔTm = −3.0 °C) (supplemental Fig. S1B). Having validated the assay with known stabilizing and destabilizing agents, we proceeded to analyze the potential binding interactions of the 16 cell-active antiprion compounds with the structured domain of PrP. The compounds (100 μm) were added to 10 μm recombinant MoPrP(89–230). Denaturation of truncated PrP in the presence of DMSO alone was used as a negative control. In accordance with the ITC results, none of the 16 compounds significantly increased the Tm of MoPrP(89–230), suggesting that they neither stabilized nor interacted with the folded domain of the protein (Fig. 3, A and B). To discount the possibility of compounds binding to the N-terminal unstructured region of PrP, we repeated the thermal-denaturation upshift assays using recombinant full-length MoPrP(23–230). The unfolding of MoPrP(23–230) was reversible (supplemental Fig. S1C) and changes in protein stability in the presence of TMAO or GdnHCl could be detected (supplemental Fi" @default.
• W2138880839 created "2016-06-24" @default.
• W2138880839 creator A5000298654 @default.
• W2138880839 creator A5009419179 @default.
• W2138880839 creator A5020075720 @default.
• W2138880839 creator A5058339023 @default.
• W2138880839 creator A5064245536 @default.
• W2138880839 creator A5066991343 @default.
• W2138880839 date "2011-08-01" @default.
• W2138880839 modified "2023-10-12" @default.
• W2138880839 title "A Survey of Antiprion Compounds Reveals the Prevalence of Non-PrP Molecular Targets" @default.
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