UNDERSTANDING AND IMPROVING SMALL MOLECULE INHIBITORS OF PRION REPLICATION

朊病毒疾病的特征在于朊病毒蛋白质的错误折叠同种型（称为PrP（Sc））的聚集。没有治疗方法可以阻止朊病毒疾病的进展，因此，正在寻求临床上可行的治疗方法。已经设计了有效的基于吡啶的化合物来模拟显性负性PrP，并因此与参与PrP（C）到PrP（Sc）的构象转化的辅助分子结合。这些化合物已显示出抑制PrP（Sc）复制，并已使用SAR技术进行模拟。虽然原始先导化合物的EC（50）在15 - 30微摩尔范围内，但最有效的新类似物在300纳摩尔浓度下具有活性。独立地，我们已经确定了吖啶为基础的化合物，奎纳克林作为一个有效的抗朊病毒剂在细胞为基础的测定， PrP（Sc）复制（EC（50）= 300纳摩尔），并开发了更有效的双奎纳克林类似物（EC（50）= 30纳摩尔）。鉴于双类似物的活性提高，后续的研究工作将集中在开发这类化合物上。使用合成文库和计算方法对生物活性的基于吡啶和双吖啶的类似物进行精制可以提供有效的无毒杂环化合物，其可以是用于治疗朊病毒疾病的可行候选物。正在研究吡啶和吖啶类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朊病毒疾病模型中减少PrP（Sc）复制。将使用采用共聚焦显微镜的神经元成像系统来确定PrP（C）和PrP（Sc）在细胞区室之间的运输。随后，将荧光团标记的吡啶和吖啶基类似物引入成像系统，以确定这些化合物是否通过干扰朊病毒蛋白的运输对PrP（Sc）复制产生影响。将合成可光活化的标记类似物用于受体交联研究。这些探针将用于鉴定基于杂环的化合物的分子靶标。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.