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

描述(申请人提供)：将药物的功能效应与它们如何改变PPARγ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PPARγ的蛋白质结合的药物。PPARγ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，并影响免疫功能。一些PPARγ依赖效应可能对糖尿病患者有益，而另一些则不是。挑战是开发与PPARγ结合的药物，既保留独特而强大的抗糖尿病效果，又减少心力衰竭、体重增加和骨质流失的副作用。目前的研究表明，通过用正确的药物靶向PPARv，有可能实现有害和有害效果的分离。动物研究表明，一些新的PPARγ药物激活了与当前处方药物不同的基因集，这一事实支持了这一概念。然而，不同的药物如何独特地改变PPARγ以产生这些药物特异性作用尚不清楚。当药物如何改变PPARγ以及这些改变如何产生功能变化(如基因表达的变化)时，更有可能开发改进的抗糖尿病PPARγ药物。了解配体如何在PPAR、γ和密切相关的蛋白质中产生功能是首席研究员(PI)的长期目标。这一知识将有助于为整个家族开发更好的药物，γ样蛋白(核受体)是FDA批准的药物中约13%的靶标。最近我们发现，在溶液中或与效果较差的药物结合时，PPARγ的大区域至少存在两种构象，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。为了更好地了解PPARγS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核磁共振线形分析程序，该程序可以详细地揭示PPARγ柔性区域内一个位置存在的构象范围(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，并允许对足够数量的PPARγ药物进行表征，以得出关于药物诱导的PPARγ变化与细胞中基因表达变化之间的任何相关性的具有统计学意义的结论。在项目的独立阶段(目标2)，核磁共振探测点的数量将扩大(从单一的当前位置)，以获得不同地区PPARγ的S构象系综的更完整的图像。此外，我们还将研究在体内发现的两种主要形式的PPARγ：1)全长PPARγ(FL-PPARγ)和2)由PPARγ、RXRγ和DNA组成的全长异二聚体复合体。这项工作将以前所未有的细节揭示配体如何影响组成PPARγ构象系综的一系列相关结构。然而，他们不会检测到配体诱导的PPARγ的S小的快速运动(即构象熵)的变化，这可能是配体在人类中产生效应的关键。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当药物与PPAR结合时，PPARγ(构象熵)的小而快的内部移动的平均区域变化。所有这些药物诱导的PPARγ变化的测量都将被测试与功能结果的相关性，例如FL-RXRα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还将接受格里芬博士(共同导师)提供的关于PPARγ药物对细胞中目标基因表达影响的方法和分析的培训。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回答有关PPARγ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 PPARγ The most effective drugs for treating and preventing Type II diabetes are those that bind to a protein named PPARγ. PPARγ is a transcription factor critical for the production and maintenance of adipocytes and bone cells and it affects immune function. Some PPARγ dependent effects can be beneficial to people with diabetes and some are not. The challenge is to develop PPARγ 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 PPARv with the right drug. This concept is supported by the fact that animal studies show some new PPARγ drugs activate distinct gene sets from currently prescribed drugs. However, how different drugs uniquely change PPARγ in order to produce these drug specific effects are unknown. Development of improved anti-diabetic PPARγ drugs is more likely when it is known how drugs change PPARγ and how these changes produce functional changes such as changes in gene expression. Understanding how ligands produce function in PPARγ 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 PPARγ like proteins (nuclear receptors) which are the target of ~13% of FDA approved drugs. Recently we have discovered that a large region of PPARγ 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 PPARγs 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 PPARγ. 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 PPARγ drugs to draw statistically meaningful conclusions about any correlation between drug induced changes to PPARγ 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 PPARγ's conformational ensemble in different areas. Additionally we will study PPARγ in the two main forms that it is found in the body 1) full length PPARγ (FL-PPARγ) and 2) the full-length heterodimer complex, which consists of PPARγ, RXRγ and DNA. This work will reveal in unprecedented detail how ligands affect the range of related structures that comprise the conformational ensemble of PPARγ. However, they will not detect ligand-induced changes in PPARγ'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 PPARγ (conformational entropy) that occurs when a drug binds PPAR. All of these measurements of drug-induced changes in PPARγ will be tested for correlation with functional outcomes such as dimerization with FL-RXRα, 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 PPARγ 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 PPARγ. 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.