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名为PPARγ的蛋白质结合的药物。过氧化物酶体增殖物激活受体γ是一种对脂肪细胞和骨细胞的产生和维持至关重要的转录因子，并影响免疫功能。一些PPARγ依赖性效应可能对糖尿病患者有益，而另一些则不然。目前的挑战是开发出结合PPARγ的药物，既能保持独特而强大的抗糖尿病作用，又能减少心力衰竭、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，有可能实现不想要的和想要的效果的分离。这一概念得到了以下事实的支持：动物研究表明，一些新的PPARγ药物激活了与目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PPARγ以产生这些药物特异性效应尚不清楚。当知道药物如何改变PPARγ以及这些变化如何产生功能变化（如基因表达变化）时，开发改进的抗糖尿病PPARγ药物的可能性更大。了解配体如何在PPARγ和密切相关的蛋白质中发挥作用是主要研究者（PI）的长期目标。这些知识将有助于开发针对整个PPARγ样蛋白（核受体）家族的更好药物，这些蛋白是约13%的FDA批准药物的靶点。最近，我们发现，在单独的溶液中或与疗效较低的药物结合时，大区域的PPARγ以至少两种构象存在，然而，当与诱导高转录的配体结合时，检测到一种PPAR构象（Hughes et al. 2012）。重要的是，这些数据是耗时和昂贵的获得，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PPARγ的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物的低成本、快速NMR方法和NMR线形分析程序，该程序详细揭示了在PPARγ的柔性区域内的一个位点处存在的构象范围（即构象系综）。这些方法揭示了使用其他NMR方法很难观察到的构象复杂性（手稿正在编写中），并允许表征足够数量的PPARγ药物，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统计学有意义的结论。在项目的独立阶段（目标2），NMR探针位置的数量将扩大（从单一的当前位置），以获得不同区域中PPARγ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）全长异二聚体复合物，其由PPARγ，RXRγ和DNA组成。这项工作将以前所未有的细节揭示配体如何影响组成PPARγ构象系综的相关结构的范围。然而，他们不会检测到配体诱导的PPARγ小的快速运动（即构象熵）的变化，这可能是配体如何在人体中产生作用的关键。为了研究这些运动，PI将接受使用分子动力学模拟的培训。在可能的情况下，将使用NMR对照实验检查这些模拟。这些数据将用于估计药物结合PPAR时发生的PPARγ（构象熵）的小的快速内部运动的平均区域变化。将检测所有这些药物诱导的PPARγ变化的测量值与功能结局的相关性，如与FL-RXRα的二聚化、辅调节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的进展，并建立最好的模型， 在分子动力学模拟中，为了训练PI，需要保护时间。包括Cheatham博士在内的咨询小组将在这一领域提供专家指导。在资助的指导部分（目标1），PI将继续接受合作者（Art Palmer博士和Mark Rance博士）在NMR和蛋白质分子动力学模拟方面的指导，此外还有PI的主要导师Kojetin博士。PI还将接受Griffin博士（共同导师）提供的关于PPARγ药物对细胞中靶基因表达影响的方法和分析的培训。位于佛罗里达的斯克里普斯研究所（TSRI）有七个研究核受体的小组（Nettles、Griffin、Kojetin、Kameneka、Solt、Spinch和Smith）。其中四个小组目前正在使用不同的方法和途径来回答有关PPARγ的重要问题。这使得合作自然，并提供了一个良好的环境，在其中接受必要的培训，以维持在这一领域的独立研究。PI拥有物理学和生物学学位，这使他能够在TSRI的3年培训期间快速获得蛋白质核磁共振（NMR）和其他几种生物物理学和生物学技术的许多领域的专业知识，并提供了广泛的培训，这对于将生物物理学和PPAR运动和结构的热力学与功能结果联系起来至关重要。

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.