Ladner-Keay Carol L, Ross Li, Perez-Pineiro Rolando, Zhang Lun, Bjorndahl Trent C, Cashman Neil, Wishart David S
a Department of Biological Sciences , University of Alberta , Edmonton , Canada.
c Brain Research Centre , University of British Columbia , Vancouver , Canada.
Prion. 2018;12(5-6):280-300. doi: 10.1080/19336896.2018.1525254. Epub 2018 Oct 9.
Prion diseases are caused by the conversion of normal cellular prion proteins (PrP) into lethal prion aggregates. These prion aggregates are composed of proteinase K (PK) resistant fibrils and comparatively PK-sensitive oligomers. Currently there are no anti-prion pharmaceuticals available to treat or prevent prion disease. Methods of discovering anti-prion molecules rely primarily on relatively complex cell-based, tissue slice or animal-model assays that measure the effects of small molecules on the formation of PK-resistant prion fibrils. These assays are difficult to perform and do not detect the compounds that directly inhibit oligomer formation or alter prion conversion kinetics. We have developed a simple cell-free method to characterize the impact of anti-prion fibril compounds on both the oligomer and fibril formation. In particular, this assay uses shaking-induced conversion (ShIC) of recombinant PrP in a 96-well format and resolution enhanced native acidic gel electrophoresis (RENAGE) to generate, assess and detect PrP fibrils in a high throughput fashion. The end-point PrP fibrils from this assay can be further characterized by PK analysis and negative stain transmission electron microscopy (TEM). This cell-free, gel-based assay generates metrics to assess anti-prion fibril efficacy and kinetics. To demonstrate its utility, we characterized the action of seven well-known anti-prion molecules: Congo red, curcumin, GN8, quinacrine, chloropromazine, tetracycline, and TUDCA (taurourspdeoxycholic acid), as well as four suspected anti-prion compounds: trans-resveratrol, rosmarinic acid, myricetin and ferulic acid. These findings suggest that this in vitro assay could be useful in identifying and comprehensively assessing novel anti-prion fibril compounds. Abbreviations: PrP, prion protein; PK, proteinase K; ShIC, shaking-induced conversion; RENAGE, resolution enhanced native acidic gel electrophoresis; TEM, transmission electron microscopy; TUDCA, taurourspdeoxycholic acid; BSE, bovine spongiform encephalopathy; CWD, chronic wasting disease; CJD, Creutzfeldt Jakob disease; GSS, Gerstmann-Sträussler-Scheinker syndrome; FFI, fatal familial insomnia; PrP, cellular prion protein; recPrP, recombinant monomeric prion protein; PrP, infectious particle of misfolded prion protein; RT-QuIC, real-time quaking-induced conversion; PMCA, Protein Misfolding Cyclic Amplification; LPS, lipopolysaccharide; EGCG, epigallocatechin gallate; GN8, 2-pyrrolidin-1-yl-N-[4-[4-(2-pyrrolidin-1-yl-acetylamino)-benzyl]-phenyl]-acetamide; DMSO, dimethyl sulfoxide; ScN2A, scrapie infected neuroblastoma cells; IC50, inhibitory concentration for 50% reduction; recMoPrP , recombinant full-length mouse prion protein residues 23-231; EDTA; PICUP, photo-induced cross-linking of unmodified protein; BSA, bovine serum albumin;; PMSF, phenylmethanesulfonyl fluoride.
朊病毒疾病是由正常细胞朊病毒蛋白（PrP）转变为致死性朊病毒聚集体所致。这些朊病毒聚集体由抗蛋白酶K（PK）的纤维和相对对PK敏感的寡聚体组成。目前尚无抗朊病毒药物可用于治疗或预防朊病毒疾病。发现抗朊病毒分子的方法主要依赖于相对复杂的基于细胞、组织切片或动物模型的检测方法，这些方法用于测量小分子对抗PK朊病毒纤维形成的影响。这些检测方法操作困难，且无法检测直接抑制寡聚体形成或改变朊病毒转化动力学的化合物。我们开发了一种简单的无细胞方法来表征抗朊病毒纤维化合物对寡聚体和纤维形成的影响。具体而言，该检测方法采用96孔板形式的重组PrP的振荡诱导转化（ShIC）和分辨率增强的天然酸性凝胶电泳（RENAGE），以高通量方式生成、评估和检测PrP纤维。该检测方法得到的终点PrP纤维可通过PK分析和负染透射电子显微镜（TEM）进一步表征。这种基于凝胶的无细胞检测方法可生成评估抗朊病毒纤维功效和动力学的指标。为证明其效用，我们表征了七种知名抗朊病毒分子的作用：刚果红、姜黄素GN8、喹吖因、氯丙嗪、四环素和牛磺熊去氧胆酸（TUDCA），以及四种疑似抗朊病毒化合物：反式白藜芦醇、迷迭香酸、杨梅素和阿魏酸。这些发现表明，这种体外检测方法可用于鉴定和全面评估新型抗朊病毒纤维化合物。缩写：PrP，朊病毒蛋白；PK，蛋白酶K；ShIC，振荡诱导转化；RENAGE，分辨率增强的天然酸性凝胶电泳；TEM，透射电子显微镜；TUDCA，牛磺熊去氧胆酸；BSE，牛海绵状脑病；CWD，慢性消耗性疾病；CJD，克雅氏病；GSS，格斯特曼-施特劳斯勒-谢inker综合征；FFI，致死性家族性失眠症；PrP，细胞朊病毒蛋白；recPrP，重组单体朊病毒蛋白；PrP，错误折叠的朊病毒蛋白感染性颗粒；RT-QuIC，实时振荡诱导转化；PMCA，蛋白质错误折叠循环扩增；LPS，脂多糖；EGCG，表没食子儿茶素没食子酸酯；GN8，2-吡咯烷-1-基-N-[4-[4-(2-吡咯烷-1-基-乙酰氨基)-苄基]-苯基]-乙酰胺；DMSO，二甲基亚砜；ScN2A，羊瘙痒症感染的神经母细胞瘤细胞；IC50，50%抑制浓度；recMoPrP，重组全长小鼠朊病毒蛋白残基23-231；EDTA；PICUP，未修饰蛋白质的光诱导交联；BSA，牛血清白蛋白；；PMSF，苯甲基磺酰氟。
