Novel Antagonists of the N-terminal Domain of the CRF Receptor Type 1 for Alzheimer's Disease

项目摘要/摘要 CRF是与应激有关的主要神经肽激素，主要通过信号转导途径 通过CRF受体亚型1(CRFR1)在中枢神经系统表达。CRF信号与阿尔茨海默氏症有关 疾病(AD)由于CRF表达增加，CRF与CRFR1受体结合和上调 皮质、海马体和下丘脑似乎出现在疾病进展的早期，并且 易受阿尔茨海默病神经病理影响的区域有显著的神经化学变化。CRFR1的表达也 与Abeta(Aβ)和tau病理相交，这两种病理都是已知的AD的关键特征。曾经有过 学术界和制药公司为开发有效的小分子拮抗剂所做的大量努力 然而，即使是目前的第三代拮抗剂，CRFR1仍然针对C-末端区域 CRFR1受体的大部分仍然基于第二代化学支架及其固有的毒性 负债。该项目利用了CRFR1激活的分子模型，该模型通过广泛的 研究中，第一步是CRF与受体N端的特定配体结合基序的结合 区域和结合基序。我们已经开发并验证了一种新的均相tr-fret检测方法 测定生物素标记的CRF与麦芽糖N端胞外区(ECD)的直接结合 标记CRFR1的结合蛋白与高通量筛选兼容。我们已经开发出了这个 实验证明，未标记的CRF取代了生物素-CRF，并且几个已知的C末端CRFR1 拮抗剂不会取代CRFR1ECD中的CRF。在最近这些实验的激励下，我们 建议与以下机构合作，筛选抗CRFR1ECD的大型化合物文库 桑福德·伯纳姆·普雷比医学发现研究所的康拉德·普雷比化学基因组中心。 在目标1中，我们将全面实施这种初级的CRFR1 ECD分析，并完成HTS活动 鉴定和确认候选的N端CRFR1拮抗剂。AIM 2将验证这些命中的效力， 特异性、功能拮抗性和阐明紧急构效关系(SAR)以评估其 化学可控性。目标3将通过药物周期的点击到领先来推进经过验证的命中 通过2°关键路径分析进行效力和功能拮抗的化学和测试，然后 引导优化周期，此外，目标生物相关3°测试中的效力和有效性 接合(例如脑组织切片)。最好的2-3探针化合物(S)，将被放大以作为他们的基准 AD模型中ADME/T、PK/PD和脑外显率及体内概念验证研究。

Project Summary/Abstract CRF is the major neuropeptide hormone associated with stress and is mediated predominantly signaling through CRF receptor subtype 1 (CRFR1) in the CNS. CRF signaling has been implicated in Alzheimer’s disease (AD) as there is increased CRF expression, CRF binding to and upregulation of CRFR1 receptors in the cortex, hippocampus and hypothalamus appears to occur early in disease AD progression and are prominent neurochemical changes in areas vulnerable to AD neuropathology. CRFR1 expression also intersects with Abeta (Aβ) and tau pathologies, both known to be critical hallmarks of AD. There have been substantial efforts in academia and pharmaceutical companies to develop potent small molecule antagonists of CRFR1, however, even the current 3rd generation antagonists are still directed against the C- terminal regions of the CRFR1 receptor are still based on the 2nd generation chemical scaffolds along with their inherent toxicity liabilities. This project capitalizes on the molecular model of CRFR1 activation, established through extensive studies, where the first step is binding of CRF to specific ligand binding motifs in the receptor’s N-terminal region and binding motifs. We have developed and validated an novel homogeneous TR-FRET assay to measure the direct binding of biotin-labeled CRF to the N-terminal extracellular domain (ECD) of maltose binding protein labeled CRFR1 compatible with very high-throughput screening. We have developed this assay, verified that unlabeled CRF displaces biotin-CRF, and that several known C-terminal CRFR1 antagonists do not displace CRF from the N-term CRFR1 ECD. Motivated by these recent experiments, we propose to screen a large library of compounds against N-terminal CRFR1 ECD in collaboration with the Conrad Prebys Center for Chemical Genomics at Sanford Burnham Prebys Medical Discovery Institute. In Aim 1, we will fully implement this primary N- terminal CRFR1 ECD assay and complete an HTS campaign to identify and confirm candidate N-terminal CRFR1 antagonists. Aim 2 will validate these hits for potency, specificity, functional antagonism and elucidate emergent structure activity relationships (SAR) to assess their chemical tractability. Aim 3 will advance validated hits through hit-to-lead through cycles of medicinal chemistry and testing through 2° critical path assays for potency and functional antagonism, then additional lead optimization cycles where additionally, potency and efficacy in biologically relevant 3° assays for target engagement (e.g. brain tissue slices). The best 2-3 probe compound(s), will be scale-up to benchmark their ADME/T, PK/PD and brain penetrance and in vivo proof-of-concept studies in mouse AD models.