LINGANDS BINDING TO PRP

我们的目标是发现能结合并稳定可溶的人类蛋白亚型的配基。 (HuPrPc)。其中一些配体还可能抑制HuPrPSc向寡聚体的转化，从而导致疾病 异构体(HuPrPSc)。很少或没有得到充分验证的分子与PrPc结合，而且它们不存在 是我们探索PrPc功能和减弱与PrPSc相关的病理的能力的关键差距。 使用虚拟筛选方法，大型有机分子文库将与结构对接 PrPc.得分高的化合物将接受生物物理结合测试，控制非特异性 抗淀粉样蛋白抑制剂容易出现的抑制作用。我们的长期目标是使用特定的PrP配体来 研究蛋白质的生物学功能，并将其作为药物发现的潜在线索；我们的直接意图是 发现它们并充分描述它们的特征。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10万套 与PrPc结构相反的碎片样分子和100万个类铅分子；均可在商业上获得 因此，这些预测可能会很快得到检验。每种化合物都适合PrPC中的两个空隙之一，并按 对蛋白质的互补性。此外，我们还将预测细胞内已知的活性试剂的靶标。 使用第二种计算方法，培养出不同的蛋白聚集态。 目的2.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 最近发展起来的热变性上移法。我们还将开发新的检测方法，以测量 结合使用等温滴定量热法和表面等离子激元共振。这样的分析将是至关重要的 了解配体的机制，并从长远来看，改善它们的亲和力。 目的3.控制特异性抑制，并研究胶体聚集体对Pron的作用。许多 据推测，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 混杂的胶体类抑制剂。这些胶体隔离蛋白质单体，防止淀粉样蛋白 队形。我们将这一效应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和目标2中发现和测试的分子中的一种机制。

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.