UNDERSTANDING AND IMPROVING SMALL MOLECULE INHIBITORS OF PRION REPLICATION

Prion病的特征是PrP(SC)蛋白的一种错误折叠的亚型聚集。目前还没有可用于阻止普恩病毒疾病进展的治疗方法，因此，临床上可行的治疗方法正在被寻求。有效的吡啶类化合物被设计成模拟显性负PrP，从而与参与PrP(C)到PrP(SC)构象转换的辅助分子结合。这些化合物已被证明可以抑制PrP(SC)复制，并已使用SAR技术进行了模拟。虽然最初的先导化合物的EC(50)S在15-30微摩尔范围内，但最有效的新类似物的活性在300纳摩尔浓度。独立地，我们已经在一项基于细胞的分析中确定了基于吖啶的化合物，即奎纳克林，是一种有效的抗病毒药物。 PrP(SC)复制(EC(50)=300纳米摩尔)，并开发出更有效的双喹啉类似物(EC(50)=30纳米摩尔)。鉴于双类似物活性的提高，后续的研究工作将集中在开发这类化合物上。利用合成文库和计算方法对具有生物活性的吡啶和双吖啶类似物进行精制，可以提供有效的无毒杂环化合物，这些化合物可能是治疗Pron病的可行候选者。正在研究以吡啶和吖啶为基础的化合物的细胞和分子作用机制，以更好地了解这些化合物如何在基于细胞的PrP(SC)疾病模型中减少PrP(SC)复制。PrP(C)和PrP(SC)在细胞间的转运将使用共聚焦显微镜的神经成像系统来确定。随后，荧光团标记的吡啶和吖啶类似物将被引入成像系统，以确定这些化合物是否通过干扰PrP(SC)蛋白的运输而对PrP(SC)复制产生影响。可光激活的标记类似物将被合成用于受体交联性研究。这些探针将用于识别杂环类化合物的分子靶标。一旦分子靶标被分离和表征，与靶标有关的结构和功能信息将被用于有效和选择性化合物的基于结构的设计，对PrP(SC)复制具有活性。

The prion diseases are characterized by aggregation of a mis-folded isoform of the prion protein, termed PrP (Sc). No treatment is available to halt progression of prion diseases, and as such, clinically viable therapies are being sought. Potent pyridine-based compounds have been designed to mimic dominant negative PrP and hence bind to auxiliary molecules involved in the conformational conversion of PrP(C) to PrP (Sc). These compounds have been shown to inhibit PrP (Sc) replication and have been analogued using SAR techniques. While the EC(50)s of the original lead compounds were in the 15-30 micromolar range, the most potent new analogues are active at 300 nanomolar concentrations. Independently, we have identified the acridine-based compound, quinacrine as a potent anti-prion agent in a cell-based assay of PrP(Sc) replication (EC(50)=300 nanomolar), and developed bis-quinacrine analogs that are even more potent (EC(50)=30 nanomolar). Given the improved activity of the bis- analogs, subsequent research efforts will focus on developing this class of compound. Refinement of bioactive pyridine- and bis-acridine-based analogs using synthetic libraries and computational methods could furnish potent non-toxic heterocyclic compounds that could be viable candidates for the treatment of prion disease. The cellular and molecular mechanism of action of the pyridine- and acridine-based compounds is being examined to better understand how these compounds reduce PrP(Sc) replication in a cell-based model of prion disease. The trafficking of PrP(C) and PrP(Sc) between cellular compartments will be determined using a neuronal imaging system employing confocal microscopy. Subsequently, fluorophore labeled pyridine- and acridine-based analogs will be introduced to the imaging system to determine if these compounds exert their effect on PrP(Sc) replication by interfering with the trafficking of the prion protein. Photoactivatable labeled analogs will be synthesized for receptor cross-linking studies. These probes will be used to identify the molecular targets of the heterocyclic-based compounds. Once the molecular targets have been isolated and characterized, the structural and functional information pertaining to the targets will be used for the structure-based design of potent and selective compounds, active against PrP(Sc) replication.