Prion Disease Therapeutics

TSE的发病机制涉及到一种异常的错误折叠蛋白，称为PrPres，在受感染的宿主中积累。2014财年： 1)我们已经与外部研究人员合作，通过靶向有毒低聚物来识别设计的抑制淀粉样蛋白形成的片状多肽。以前的研究表明，不同淀粉样蛋白的毒性可溶低聚物具有共同的骨架构象，但这种低聚物的无定形性质阻碍了实验对其结构的表征。基于分子动力学模拟，我们提出不同淀粉样蛋白的有毒中间体采用一个共同的、非标准的二级结构，称为-Sheet。我们在实验中表征了设计成与模拟中观察到的-折叠构象互补的多肽。我们展示了两种不同的淀粉样蛋白系统--淀粉样多肽(A&946；)和转甲状腺素(TRAN)--的聚集抑制作用。当固定化时，与非聚集的、无毒的物种或成熟的纤维相比，薄片设计优先结合有毒构象中丰富的溶液中的物种。这些设计显示了区别于传统二级结构的特征光谱特征，支持将-Sheet作为参与淀粉样蛋白形成的有毒低聚体阶段的结构，并为新的治疗和诊断方法铺平了道路。 2)我们探索了一种体外/体内相结合的方法来筛选具有可接受的药效性和药代动力学参数的活性抗病毒化合物。在持续感染PrP(SC)(SCN2A)的神经母细胞瘤细胞中筛选出一组不同的芳香族化合物，以观察它们抑制PK耐药PrP(PrP(Res))积累的能力。从200个化合物中，47个化合物有效地减少了PrP(RES)在SCN2A细胞中的积聚。在硅胶中预测药代动力学和物理化学性质，使我们能够获得相对血脑屏障渗透性和致突变性的估计。四甲基偶氮唑盐还原实验表明，大部分活性化合物无细胞毒性。对从SCN2A细胞中清除PrP(RES)的化合物进行了研究，研究了它们抑制致淀粉样蛋白PrP多肽片段(PrP(109-149))聚集的能力，这些化合物在MTT法中是无毒的，并且具有良好的药代动力学特征。分子对接结果为PrP与最有希望的化合物之间的相互作用提供了结构模型和结合亲和力。综上所述，使用这种体外/电子相结合的方法，我们已经确定了新的小型有机抗瘙痒化合物，它们减少了PrP(RES)在SCN2A细胞中的积累，抑制了PrP多肽的聚集，并具有支持其药代动力学的特征。这些化合物是Pron疾病治疗的有吸引力的候选者。 3)研究了糖胺多糖(GAG)对PrP转化为PrP(SC)的影响。GAG表现出一种矛盾的效应，因为它们将PrP转化为抗蛋白酶PrP(PrP-RES)，但也发挥保护作用。我们通过应用不同的体外聚集方法，包括实时震动诱导转换(RT-QuIC)，比较了PrP和肝素-PrP复合体的稳定性和聚集倾向。小鼠和仓鼠脑匀浆中的TSE相关形式被用来接种RT-QuIC诱导的纤维化。在我们的研究中，肝素和细胞PrP(PrP(C))之间的相互作用增加了PrP的热稳定性，导致温度诱导的聚集减少了8倍。在RT-QuIC实验中，低分子肝素(LMWHEP)与PrP的N-末端或C-末端结构域的相互作用不仅影响PrP的纤化程度，而且影响PrP的动力学，使反应速率常数从1.0 4降至0.2 9 S(-1)，并使滞后期从12 h增加到19 h。我们的发现解释了肝素在不同模型的蛋白和蛋白样神经退行性疾病中的保护作用，并为基于GAGS的治疗策略的发展奠定了基础。

TSE pathogenesis involves the accumulation of an abnormal misfolded protein, called PrPres, in infected hosts. In FY 2014: 1) We have collaborated with extramural investigators to identify designed α-Sheet peptides that inhibit amyloid formation by targeting toxic oligomers. Previous studies suggest that the toxic soluble-oligomeric form of different amyloid proteins share a common backbone conformation, but the amorphous nature of this oligomer prevents its structural characterization by experiment. Based on molecular dynamics simulations we proposed that toxic intermediates of different amyloid proteins adopt a common, nonstandard secondary structure, called α-sheet. We experimentally characterized peptides designed to be complementary to the α-sheet conformation observed in the simulations. We demonstrated inhibition of aggregation in two different amyloid systems, β-amyloid peptide (Aβ) and transthyretin, by these designed α-sheet peptides. When immobilized the α-sheet designs preferentially bind species from solutions enriched in the toxic conformer compared with non-aggregated, nontoxic species or mature fibrils. The designs display characteristic spectroscopic signatures distinguishing them from conventional secondary structures, supporting α-sheet as a structure involved in the toxic oligomer stage of amyloid formation and paving the way for novel therapeutics and diagnostics. 2) We have explored a combined in vitro/in silico approach to screen for active anti-prion compounds presenting acceptable drugability and pharmacokinetic parameters. A diverse panel of aromatic compounds was screened in neuroblastoma cells persistently infected with PrP(Sc) (ScN2a) for their ability to inhibit PK-resistant PrP (PrP(Res)) accumulation. From ∼200 compounds, 47 were effective in decreasing the accumulation of PrP(Res) in ScN2a cells. Pharmacokinetic and physicochemical properties were predicted in silico, allowing us to obtain estimates of relative blood brain barrier permeation and mutagenicity. MTT reduction assays showed that most of the active compounds were non cytotoxic. Compounds that cleared PrP(Res) from ScN2a cells, were non-toxic in the MTT assay, and presented a good pharmacokinetic profile were investigated for their ability to inhibit aggregation of an amyloidogenic PrP peptide fragment (PrP(109-149)). Molecular docking results provided structural models and binding affinities for the interaction between PrP and the most promising compounds. In summary, using this combined in vitro/in silico approach we have identified new small organic anti-scrapie compounds that decrease the accumulation of PrP(Res) in ScN2a cells, inhibit the aggregation of a PrP peptide, and possess pharmacokinetic characteristics that support their drugability. These compounds are attractive candidates for prion disease therapy. 3) We have studied the effects of glycosaminoglycans (GAGs)on the conversion of PrP into scrapie PrP (PrP(Sc). GAGs exhibit a paradoxical effect, as they convert PrP into protease-resistant PrP (PrP-res) but also exert protective activity. We compared the stability and aggregation propensity of PrP and the heparin-PrP complex through the application of different in vitro aggregation approaches, including real-time quaking-induced conversion (RT-QuIC). TSE-associated forms from mouse and hamster brain homogenates were used to seed RT-QuIC-induced fibrillization. In our study, interaction between heparin and cellular PrP (PrP(C)) increased thermal PrP stability, leading to an 8-fold decrease in temperature-induced aggregation. The interaction of low-molecular-weight heparin (LMWHep) with the PrP N- or C-terminal domain affected not only the extent of PrP fibrillization but also its kinetics, lowering the reaction rate constant from 1.04 to 0.29 s(-1) and increasing the lag phase from 12 to 19 h in RT-QuIC experiments. Our findings explain the protective effect of heparin in different models of prion and prion-like neurodegenerative diseases and establish the groundwork for the development of therapeutic strategies based on GAGs.