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

将药物的功能效应与其改变 PPAR gamma 的方式联系起来

• 批准号: 9206156
• 负责人: Travis Shane Hughes
• 金额: $ 24.24万
• 依托单位: UNIVERSITY OF MONTANA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-01-15 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9206156
• 关键词: 3T3-L1 Cells; Acute; Adipocytes; Adverse effects; Advisory Committees; Affect; Affinity; Agonist; Animals; Antidiabetic Drugs; Area; Arts; Atherosclerosis; Binding; Biology; Biophysics; Calorimetry; Cell Line; Cells; Cluster Analysis; Collaborations; Complex; Crystallization; DNA; Data; Development; Diabetes Mellitus; Diabetes prevention; Dimerization; Drug Design; Drug Targeting; Drug effect disorder; Entropy; Environment; FDA approved; Family; Florida; Fluorine; Fracture; GTP-Binding Protein alpha Subunits, Gs; Gene Expression; Gene Targeting; Genes; Genetic Transcription; Glyburide; Goals; Grant; Heart failure; Human; Inflammation; Knowledge; Length; Ligand Binding Domain; Ligands; Light; Link; Location; Maintenance; Manuscripts; Maps; Measurement; Measures; Mentors; Metformin; Methods; Modeling; Molecular Conformation; Motion; Movement; Names; Non-Insulin-Dependent Diabetes Mellitus; Nuclear Magnetic Resonance; Nuclear Receptors; Osteogenesis; Output; PPAR gamma; PPARBP gene; Patients; Peptides; Peroxisome Proliferator-Activated Receptors; Pharmaceutical Preparations; Phase; Physics; Play; Prediabetes syndrome; Preparation; Production; Proteins; Recruitment Activity; Research; Research Institute; Research Personnel; Role; Sampling; Site; Structure; Surface; Techniques; Testing; Thermodynamics; Time; Titrations; Training; Weight Gain; Woman; Work; adipocyte differentiation; bone cell; bone loss; cost; diabetic; dimer; experimental study; flexibility; functional outcomes; immune function; immunoregulation; improved; insulin sensitizing drugs; lipid biosynthesis; molecular dynamics; peroxisome; prevent; programs; protein E; public health relevance; receptor; shape analysis; simulation; transcription factor

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.

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。PPARγ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，它影响免疫功能。一些PPARγ依赖效应可能对糖尿病患者有益，而另一些则不然。挑战在于开发出结合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，通过使用合适的药物靶向PPARv，有可能实现不想要的和想要的效果的分离。动物研究表明，一些新的PPARγ药物与目前的处方药不同，可以激活不同的基因组，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。当了解药物如何改变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PPARγ和密切相关的蛋白质中产生功能是主要研究者（PI）的长期目标。这些知识将有助于开发针对PPARγ样蛋白（核受体）整个家族的更好的药物，这些蛋白是FDA批准的药物中约13%的靶标。最近，我们发现PPARγ的一个大区域在单独溶液中或与不太有效的药物结合时至少以两种构象存在，然而，当与诱导高转录的配体结合时，可以检测到一种PPAR构象（Hughes et al. 2012）。重要的是，这些数据既耗时又昂贵，而且只能表明内部运动的存在，几乎没有其他细节。为了更好地了解PPARγ内部运动与其细胞功能之间的联系，我们开发了低成本，快速的NMR方法，可以用于大型配合物和NMR线形状分析程序，详细揭示了PPARγ柔性区域内一个位点的构象范围（即构象集合）。这些方法揭示了用其他核磁共振方法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，从而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上有意义的结论。在项目的独立阶段（目标2），将扩大核磁共振探针位置的数量（从单一电流位置），以获得不同区域PPARγ构象集合的更完整图像。此外，我们将研究PPARγ在体内的两种主要形式：1)全长PPARγ (FL-PPARγ)和2)全长异二聚体复合物，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构象集合的相关结构的范围。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，这可能对配体如何在人体中产生作用至关重要。为了研究这些运动，PI将接受分子动力学模拟的训练。这些模拟将在可能的情况下使用核磁共振进行实验检查。这些数据将用于估计药物结合PPAR时发生的PPARγ小而快速的内部运动（构象熵）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，如与FL-RXRα的二聚化、协同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