LINGANDS BINDING TO PRP

我们的目标是发现能结合并稳定人朊蛋白可溶性亚型的配体 （HuPrPc）。这些配体中的一些还可以抑制HuPrPSc转化为寡聚体，引起疾病的蛋白质。 同种型（HuPrPSc）。很少或没有经过充分验证的分子与PrPc结合， 是我们探测PrPc功能和减弱与PrPSc相关的病理的能力中的关键差距。 使用虚拟筛选方法，大型有机分子库将对接到结构上 的PrPc。将测试高分化合物的生物药理学结合，控制非特异性结合。 抗淀粉样蛋白抑制剂易于产生的抑制。我们的长期目标是使用特定的PrP配体， 研究蛋白质的生物学功能，并作为药物发现的潜在线索;我们的直接目的是 去发现并充分描述它们。具体目标是： 目标1.基于结构的虚拟筛选，以发现稳定PrPc的配体。我们将对接一套10万 碎片样和一百万铅样分子对PrPc的结构;所有都是商业上可获得的 因此，可以快速测试预测。每种化合物都适合PrPc中的两个裂缝之一，并按 与蛋白质互补。此外，我们还将预测已知试剂在细胞中的作用靶点， 培养对抗朊病毒聚集，使用第二种计算方法。 目标二。在直接结合试验中测试高分对接命中的有效性。对接命中将在 最近开发的热变性上移测定法。我们还将开发新的检测方法， 结合使用等温滴定量热法和表面等离子体共振。这种分析将是至关重要的， 了解配体的机制，并从长远来看，提高它们的亲和力。 目标3.控制特异性抑制并研究胶体聚集体对朊病毒的作用。许多 淀粉样蛋白的特异性抑制剂不是通过直接结合的经典机制起作用，而是作为 混杂的胶体基抑制剂这些胶体隔离蛋白单体，防止淀粉样蛋白 阵我们将这种作用的初步研究扩展到其他众所周知的抗淀粉样蛋白抑制剂，并控制 因为它是我们自己在目标1和2中发现和测试的分子机制。

Our goal is to discover ligands that bind to and stabilize the soluble isoform of the human prion protein (HuPrPc). Some of these ligands may also inhibit the conversion of HuPrPSc into the oligomeric, diseasecausing isoform (HuPrPSc). There are few or no well-validated molecules that bind to PrPc and their absence is a key gap in our ability to probe the function of PrPc and attenuate the pathologies associated with PrPSc. Using a virtual screening approach, large libraries of organic molecules will be docked against the structure of PrPc. High-scoring compounds will be tested for binding biophysically, controlling for non-specific inhibition to which anti-amyloid inhibitors are prone. Our long term goal is to use specific PrP ligands to investigate the protein's biological function and as potential leads for drug discovery; our immediate intent is to discover and fully characterize them. The specific aims are: Aim 1. Structure-based virtual screens to discover ligands that stabilize PrPc. We will dock a set of 100,000 fragment-like and one Million lead-like molecules against the structure of PrPc; all are commercially available so the predictions may be rapidly tested. Each compound is fit into one of two clefts in PrPc and ranked by complementarity to the protein. Additionally, we will predict the targets of known reagents active in cell culture against prion aggregation, using a second computational approach. Aim 2. To test high-scoring docking hits for efficacy in direct binding assays. Docking hits will be tested in a recently developed thermal denaturation upshift assay. We will also develop new assays that measure binding using isothermal titration calorimetry and surface plasmon resonance. Such assays will be critical to understanding mechanism of the ligands and, in the longer term, improving their affinities. Aim 3. To control for specific inhibition and investigate the role of colloidal aggregates on prions. Many putatively specific inhibitors of amyloid act not by direct-binding, classical mechanisms, but rather as promiscuous, colloid-based inhibitors. These colloids sequester the protein monomers, preventing amyloid formation. We extend initial studies of this effect to other well-known anti-amyloid inhibitors, and also control for it as a mechanism in the molecules that we ourselves discover and test in Aims 1 and 2.