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

描述（由申请人提供）：越来越多的科学证据表明，压力源增强成瘾行为，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41-aa肽，已被证明可诱导与适应压力相关的各种行为变化。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有贡献。研究还表明，CRF与CRF- bp的相互作用可能正向调节CRF- r2功能，并且当CRF与CRF- bp结合时，它调节CRF- r2信号传导并有助于应激诱导的药物寻求复发。本应用程序的目的是确定一系列化学配体和化合物，破坏CRF- BP和CRF- r2之间的相互作用，以防止药物寻求行为的复发。在细胞渗透性、稳定性和体内药理学方面，非肽基化学抑制剂将比CRF肽具有优势。在过去，由于无法开发合适的高通量检测方法来筛选CRF-BP，这一直很困难。为了解决这个问题，我们开发了一种创新的基于荧光的钙检测方法，其中CRF-BP以异二聚体形式与CRF-R2在细胞表面表达并拴在一起。这极大地促进了我们在存在或不存在CRF-BP的情况下发现抑制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优化的高通量分析的基础。我们建议使用该方法筛选一个靶向合成化合物文库，并鉴定CRF-BP-CRF-R2受体复合物介导的信号传导的化学抑制剂。两种类型的二次分析将独立确认任何命中。将对CRF-BP-CRF-R2受体复合物的原型抑制剂进行结构活性关系（SAR）和hit to lead优化，从而进行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，用于研究各种细胞和生理背景下CRF-BP-CRF-R2相互作用的生物学，以期开发治疗成瘾的新疗法。

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.