Identifying Chemical Modulators of CRF-Binding Protein and CRF Receptor Complexes

描述(由申请人提供)：有越来越多的科学证据表明，应激源会增强成瘾行为，是药物滥用复发的常见原因。促肾上腺皮质激素释放因子(CRF)是一种由41个氨基酸组成的多肽，可诱导与应激适应相关的多种行为改变。CRF系统，包括CRF结合蛋白(CRF-BP)和CRF受体，CRF-R1和CRF-R2，被认为对应激导致的生理适应起作用。也有研究表明，CRF与CRF-BP的相互作用可能正向调节CRF-R2的功能，而且当CRF与CRF-BP结合时，它调节CRF-R2信号，并有助于应激诱导的药物复发。这项应用的目的是鉴定一系列化学配体和化合物，这些配体和化合物破坏CRF-BP和CRF-R2之间的相互作用，以防止复发的药物寻找行为。非肽基化学抑制剂在细胞通透性、稳定性和体内药理方面均优于CRF多肽。在过去，这是困难的，因为无法开发一种合适的高通量方法来筛选CRF-BP。为了解决这个问题，我们开发了一种创新的基于荧光的钙分析方法，其中CRF-BP在与CRF-R2形成的异二聚体复合体中表达并连接在细胞表面。这极大地促进了我们找到在存在和不存在CRF-BP的情况下抑制CRF-R2激活的分子的能力。这一创新形成了我们为化学文库筛选而优化的高通量分析的基础。我们建议利用这一方法筛选一个有针对性的合成化合物文库，并鉴定CRF-BP-CRF-R2受体复合体介导的信号传导的化学抑制剂。两种类型的二次化验将独立确认任何命中。CRF-BP-CRF-R2受体复合体的原型抑制剂将进行结构活性关系(SAR)和HIT to Lead优化，从而实现探索性药理学和临床前开发。总之，这些努力将产生有效的化学探针，用于研究CRF-BP-CRF-R2在各种细胞和生理环境中的相互作用的生物学，以期开发治疗成瘾的新疗法。 公共卫生相关性：我们开发了一项多学科合作研究计划，以生产一系列化学配体和化合物，抑制CRF-BP-CRF-R2受体复合体，这是治疗成瘾的新靶点。我们建议开发研究工具来探索CRF-BP-CRF-R2受体复合体在成瘾发展中的功能。我们将结合新的基于细胞的分析、高通量筛选、结构活性关系分析、基于化学的“Hit to Lead”优化来生成一个新配体和化合物的靶向文库。这一应用将促进针对CRF-BP-CRF-R2受体复合体的药物的开发，用于治疗成瘾。

DESCRIPTION (provided by applicant): There is accumulating scientific evidence showing that stressors enhance addictive behaviors and are a common cause of relapse to substance abuse. Corticotrophin releasing factor (CRF) is a 41-aa peptide that has been shown to induce various behavioral changes related to adaptation to stress. The CRF system, including the CRF-binding protein (CRF-BP) and the CRF receptors, CRF-R1 and CRF-R2, are thought to contribute, to the physiological adaptations that result from stress. It has also been shown that CRF interaction with CRF-BP may positively modulate CRF-R2 function and, further that when CRF binds to the CRF-BP, it modulates CRF-R2 signaling and contributes to stress-induced relapse to drug seeking. The aim of this application is to identify a chemical series of ligands and compounds that disrupt the interaction between CRF- BP and CRF-R2 to prevent relapse to drug seeking behaviors. Non-peptidyl chemical inhibitors would have advantages over CRF peptides, in terms of cell permeability, stability, and in vivo pharmacology. In the past, this has been difficult due to an inability to develop a suitable high through-put assay for screening against CRF-BP. To address this problem, we have developed an innovative and novel fluorescence based calcium assay where CRF-BP is expressed and tethered at the cell surface in a heterodimeric complex with CRF-R2. This has greatly facilitated our ability to find molecules that inhibit CRF-R2 activation in the presence and absence of CRF-BP. This innovation forms the basis of the high-throughput assay that we have optimized for chemical library screening. We propose to screen a targeted synthetic compound library using this assay and identify chemical inhibitors of CRF-BP-CRF-R2 receptor complex-mediated signaling. Two types of secondary assays will independently confirm any hits. Structure Activity Relations (SAR) and hit to lead optimization will be performed for prototypical inhibitors of the CRF-BP-CRF-R2 receptor complex leading to exploratory pharmacology and preclinical development. Altogether, these efforts will result in validated chemical probes for studying the biology of CRF-BP-CRF-R2 interaction in a variety of cellular and physiological contexts, with the view to developing new therapeutics for treatment of addiction. PUBLIC HEALTH RELEVANCE: We have developed a multidisciplinary collaborative research program to produce a chemical series of ligands and compounds inhibit the CRF-BP-CRF-R2 receptor complex a novel therapeutic target for the treatment of addiction. We propose to generate research tools for probing the function of the CRF-BP-CRF-R2 receptor complex in the development of addiction. We will combine novel cell based assays, high throughput screening, structure activity relationship analysis, chemistry-based 'hit to lead' optimization to generate a targeted library of novel ligands and compounds. This application will facilitate the development of medications that target the CRF-BP-CRF-R2 receptor complex for the treatment of addiction.