Connecting the functional effects of drugs to how they change PPAR gamma

描述(由申请人提供)：将药物的功能效应与它们如何改变PPAR联系起来？治疗和预防II型糖尿病最有效的药物是那些与一种名为PPAR？的蛋白质结合的药物。PPAR？是一种转录因子，对脂肪细胞和骨细胞的产生和维持至关重要，它影响免疫功能。一些PPAR？依赖效应对糖尿病患者可能是有益的，但有些人则不然。挑战是开发PPAR？结合药物，保留独特和强大的抗糖尿病作用，但减少心力衰竭、体重增加和骨质流失的副作用。目前的研究表明，有可能通过靶向PPAR来实现有害和有害影响的分离。用正确的药物。动物研究表明一些新的PPAR？这一事实支持了这一概念。药物激活了与当前处方药物不同的基因集。然而，不同的药物如何独特地改变PPAR？为了生产这些药物，具体效果尚不清楚。改良的抗糖尿病PPAR的开发？当知道药物如何改变PPAR时，药物更有可能发生变化？以及这些变化如何产生功能变化，如基因表达的变化。了解配体如何在PPAR中发挥作用？而密切相关的蛋白质是首席研究员(PI)的长期目标。这些知识将有助于为PPAR的整个家庭开发更好的药物？例如蛋白质(核受体)，它们是FDA批准的药物中约13%的靶标。最近我们发现了一大片PPAR？在溶液中单独存在或与效果较差的药物结合时，至少存在两种构象，但当与诱导高转录的配体结合时，检测到一种PPAR构象(Hughes等人。2012年)。重要的是，获得这些数据既耗时又昂贵，而且只表明内部运动的存在，几乎没有其他细节。为了更好地理解PPAR？S内部运动与其在细胞中的功能之间的联系，我们开发了可用于大型复合体的低成本、快速的核磁共振方法和一个核磁共振线形分析程序，该程序可以详细地揭示PPAR？柔性区域内一个位置上存在的构象范围(即构象系综)。这些方法揭示了使用其他核磁共振方法(手稿正在准备中)很难观察到的构象复杂性，并允许表征足够数量的PPAR？关于药物引起的PPAR变化之间的相关性，得出有统计学意义的结论？以及细胞中基因表达的变化。在项目的独立阶段(目标2)，核磁共振探针位置的数量将扩大(从单一的当前位置)，以获得不同地区的PPAR？‘S构象集合的更完整的图景。此外，我们还将研究PPAR？在身体中发现的两种主要形式1)全长PPAR？(FL-PPAR？)2)由PPAR？、RXR？组成的全长杂二聚体复合体。和DNA。这项工作将以前所未有的细节揭示配体如何影响组成PPAR构象系综的相关结构的范围。然而，他们不会检测到配体诱导的PPAR？的变化。S的小的快速运动(即构象熵)可能对配体如何在人体内产生影响至关重要。为了研究这些运动，PI将接受使用分子动力学模拟的训练。在可能的情况下，将使用核磁共振将这些模拟与实验进行核对。这些数据将被用来估计PPAR的小而快的内部运动的平均区域变化？(构象熵)当药物与PPAR结合时发生的。所有这些药物诱导的PPAR变化的测量？将测试与功能结果的相关性，如FL-RXR的二聚化、辅助调节多肽的募集和脂肪细胞中的基因表达。为了充分利用这些最新进展，并建立可能的最佳模式 PPAR的动力学和构象如何决定其功能，在分子动力学模拟中，PI需要有保护的时间进行训练。包括齐塔姆博士在内的顾问团队将在这一领域提供专家指导。在资助金的指导部分(目标1)，除主导人Kojetin博士外，PI还将继续接受合作者(Art Palmer博士和Mark Rance博士)在核磁共振和蛋白质分子动力学模拟方面的指导。PI还将接受PPAR方法和分析方面的培训。药物对格里芬博士(共同导师)细胞中靶基因表达的影响。佛罗里达州的斯克里普斯研究所(TSRI)有七个研究核受体的小组(Nettle，Griffin，Kojetin，Kameneka，Solt，Rousch和Smith)。其中四个小组目前正在使用不同的途径和方法来回答有关PPAR？的重要问题。这使得合作变得自然，并提供了一个良好的环境，以便接受维持这一领域独立研究所需的培训。PI拥有物理学和生物学学位，这使他在TSRI的3年培训期间迅速获得了蛋白质核磁共振(核磁共振)以及其他几种生物物理和生物技术方面的专业知识，并提供了将PPAR运动和结构的生物物理和热力学与功能结果联系起来所必需的广泛培训。

DESCRIPTION (provided by applicant): Connecting the functional effects of drugs to how they change PPAR? The most effective drugs for treating and preventing Type II diabetes are those that bind to a protein named PPAR?. PPAR? is a transcription factor critical for the production and maintenance of adipocytes and bone cells and it affects immune function. Some PPAR? dependent effects can be beneficial to people with diabetes and some are not. The challenge is to develop PPAR? binding drugs that retain the unique and robust anti-diabetic effects but reduce the side effects of heart failure, weight gain and bone loss. Current research indicates that it may be possible to achieve separation of unwanted and wanted effects by targeting PPAR? with the right drug. This concept is supported by the fact that animal studies show some new PPAR? drugs activate distinct gene sets from currently prescribed drugs. However, how different drugs uniquely change PPAR? in order to produce these drug specific effects are unknown. Development of improved anti-diabetic PPAR? drugs is more likely when it is known how drugs change PPAR? and how these changes produce functional changes such as changes in gene expression. Understanding how ligands produce function in PPAR? and closely related proteins is the long term goal of the principle investigator (PI). This knowledge will aid in development of better drugs for the whole family of PPAR? like proteins (nuclear receptors) which are the target of ~13% of FDA approved drugs. Recently we have discovered that a large region of PPAR? exists in at least two conformations in solution alone or when bound to less efficacious drugs, however one PPAR conformation is detected when bound to a ligand that induces high transcription (Hughes et al. 2012). Importantly, these data are time consuming and expensive to obtain and only indicate that internal movement exists with little other detail. To better understand the link between PPAR?s internal motion and its function in cells we have developed low- cost, rapid NMR methods that can be used on large complexes and a NMR line shape analysis program which reveal in detail the range of conformations present (i.e. the conformational ensemble) at one site within the flexible region of PPAR?. These methods reveal conformational complexity that would be very difficult to observe using other NMR methods (manuscript in preparation) and allow characterization of a sufficient number of PPAR? drugs to draw statistically meaningful conclusions about any correlation between drug induced changes to PPAR? and changes in gene expression in cells. During the independent phase (aim 2) of the project the amount of NMR probe locations will be expanded (from the single current location) to get a more complete picture of PPAR?'s conformational ensemble in different areas. Additionally we will study PPAR? in the two main forms that it is found in the body 1) full length PPAR? (FL-PPAR?) and 2) the full-length heterodimer complex, which consists of PPAR?, RXR? and DNA. This work will reveal in unprecedented detail how ligands affect the range of related structures that comprise the conformational ensemble of PPAR?. However, they will not detect ligand-induced changes in PPAR?'s small fast movements (i.e. conformational entropy) that may be critical to how ligands produce effects in humans. To study these movements the PI will be trained in using molecular dynamics simulations. These simulations will be checked against experiment where possible using NMR. These data will be used to estimate the average regional change in the small fast internal movement of PPAR? (conformational entropy) that occurs when a drug binds PPAR. All of these measurements of drug-induced changes in PPAR? will be tested for correlation with functional outcomes such as dimerization with FL-RXR¿, recruitment of coregulator peptides and gene expression in adipocytes. In order to fully utilize these recent advances and to build the best possible model of how PPAR dynamics and conformation leads to function the PI needs protected time for training in molecular dynamics simulation. The advisory team, which includes Dr. Cheatham, will provide expert guidance in this area. During the mentored portion of the grant (aim 1) the PI will continue to receive guidance in NMR and protein molecular dynamics simulations from collaborators (Drs. Art Palmer and Mark Rance), in addition to the PI's primary mentor Dr. Kojetin. The PI will also receive training in the methods and analysis of PPAR? drug effects on target gene expression in cells from Dr. Griffin (co-mentor). The Scripps Research Institute in Florida (TSRI) has seven groups (Nettles, Griffin, Kojetin, Kameneka, Solt, Rousch and Smith) that study nuclear receptors. Four of these groups are currently using different approaches and methods for answering important questions about PPAR?. This makes collaboration natural and provides an excellent environment in which to receive the training necessary to sustain independent research in this area. The PI has degrees in physics and biology which has allowed him to quickly acquire expertise in many areas of nuclear magnetic resonance (NMR) of proteins and several other biophysical and biology techniques during his 3 years of training at TSRI and provides a breadth of training that will be essential for connecting the biophysics and thermodynamics of PPAR movement and structure to functional outcomes.